Advances in Spermatozoal
Phytogeny and Taxonomy
7£ o c ^
Edited by
Barrie G. M. JAMIESON
Juan A USIO
Jean-Lou JUSTINE
MEMOIRES DU MUSEUM NATIONAL D’HISTOIRE NATURELLE
- 3 JAN.
TOME 166
1995
MEMOIRES DU MUSEUM NATIONAL D'HISTOIRE NATURELLE
Redacteur en chef (Editor-in-ChieJ) : Jean-Lou Justine
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Source : MNHN. Paris
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Cover illustration:
The editors provided the artist, Pierre DlEGHI, with the illustrations of the book, with only two constraints: to observe the organisation of
the book into three chapters, and to draw some cladograms in order to depict the phylogenetic spirit of the book. Complete freedom was
otherwise given to him in the design of the illustrations.
Pierre is a bard of the Corsican culture. He has been seduced by the shape of some of the models, but makes it clear that it is the first time
he has painted spermatozoa!
Illustration de couverture :
Les coordinateurs ont found a l’ artiste un jeu des illustrations de ce volume, avec seulement deux contraintes : respecter l' organisation en
trois chapitres, ctfigurer des cladogrammes pour indiquer V orientation phylogenetique de iouvrage. Une totale liberie a ete laissee au peintre
pour le choix des illustrations, les dimensions et, bien sur, les couleurs.
Pierre DlEGHI est peintre et chantre de la culture Corse. II recommit avoir ete seduit par la forme de certains des modeles, mats precise
que e’est la premiere fois qu'il a peint des spermatozoides !
Source : MNHN , Paris
r\
\ i VqQ C /^
Advances in Spermatozoal Phytogeny and Taxonomy
BIBL. DU i
. MUSEUM J
PARIS i
if
Source : MNHN. Paris
ISBN : 2-85653-225-X
ISSN : 1243-4442
© Editions du Museum national d’Histoire naturelle, Paris, 1995
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MEMOIRES DU MUSEUM NATIONAL D'HISTOIRE NATURELLE
TOME 166
ZOOLOG m
Advances in Spermatozoal
Phytogeny and Taxonomy
edited by
Barrie G. JAMIESON *, Juan AUSIO *‘ & Jean-Lou JUSTINE ***
* Zoology Department
University of Queensland
Brisbane 4072, Queensland
Australia
Department of Biochemistry and Microbiology
University of Victoria
Victoria, British Columbia, V8W 3P6
Canada
Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie
Museum national d’Histoire naturelle
61, rue Buffon
F-75231 Paris Cedex 05
EDITIONS
DU MUSEUM
PARIS
1995
Source : MNHN. Paris
Source : MNHN , Paris
CONTENTS / SOMMAIRE
Pages
PREFACE . 1 1
INVERTEBRATES AND GENERAL
1 . Outer arm dynein of sperm flagella and cilia in the animal kingdom . 15
Hideo MOHRI, Miyoko KUBO-IRIE & Masaru IRIE
2. Amoeboid gametes and fertilization in Dictyostelium : gamete and pronuclear fusion
are mediated by calmodulin and its binding proteins . 2 3
Danton H. O’DAY, Keith E. LEWIS & Michael A. LYDAN
3. Sperm and spermiogenesis of the “Turbellaria” and implications for the phylogeny
of the Phylum Platyhelminthes . 3 7
Nikki A. WATSON & Klaus ROHDE
4. Spermatozoal ultrastructure and phylogeny in the parasitic Platyhelminthes . 5 5
Jean-Lou JUSTINE
5. Spermiogenesis, spermatozoa and phyletic affinities in the Cestoda . 8 7
Cheikh Tidiane BA & Bernard MARCHAND
6. Immunocytochemistry of tubulin in spermatozoa of Platyhelminthes . 9 7
Carlo IOMINI, Olga RAIKOVA, Nezha NOURY-SRAIR1 & Jean-Lou JUSTINE
7. Comparative spermatology of Gastrotricha . 105
Marco FERRAGUT1 & Maria BALSAMO
8. Centrioles with ten singlets in spermatozoa of the parasitic nematode
Heligrnosomoides polygyrus . 1 1 9
A'l'cha MANS1R & Jean-Lou JUSTINE
9. Ultrastructure of sperm and sperm-egg interaction in Aculifera: implications for
molluscan phylogeny . 129
John BUCKLAND-NICKS
10. Comparative spermatozoal ultrastructure and its taxonomic and phylogenetic
significance in the bivalve order Veneroida . 155
John M. HEALY
11. Spermatozoal morphology of Patellogastropoda and Vetigastropoda (Mollusca:
Prosobranchia) . 167
Alan N. HODGSON
12. Comparative silver staining of molluscan spermatozoa . 179
Mario SOUSA, Elsa OLIVEIRA, Joao CARVALHEIRO & Victor OLIVEIRA
13. The use of spermatozoal ultrastructure in phylogenetic studies of Tubificidae
(Oligochaeta) . 189
Christer ERSEUS & Marco FERRAGUTI
14. Comparative spermatology of Chelicerata: review and perspective
Gerd ALBERTI
203
8
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
15. Spermatozoal ultrastructure in Dendrobranchiata (Crustacea, Decapoda): taxonomic
and phylogenetic considerations . 231
Antonio MEDINA
16. The atypical sperm morphologies of Aristeus antennatus and Aristae omorpha
foliacea (Crustacea, Dendrobranchiata, Aristeidae) and their phylogenetic
significance . 243
Antonio MEDINA
17. Ultrastructure and phylogeny of the spermatozoa of the infraorders Thalassinidea
and Anomura (Decapoda, Crustacea) . 25 1
Christopher C. TUDGE
18. Phylogeny of the Brachyura (Crustacea, Decapoda): evidence from spermatozoal
ultrastructure . 265
Barrie G. M. JAMIESON, Daniele GUINOT & Bertrand RICHER DE FORGES
19. Nuclear alterations during spermiogenesis of Triatoma infestans (Hemiptera,
Reduviidae) . 285
Heidi DOLDER
20. Sperm ultrastructure of Xenos vesparum (Rossi) and its significance in the
taxonomy and phylogeny of Strepsiptera (Insecta) . 29 1
Marcella CARCUPINO, Giuseppe PROFILE Jeyaraney KATHIRITHAMBY & Massimo MAZZINI
21. Characteristics of the spermatozoon of Cosmopolites sordidus (Coleoptera:
Curculionidae) . 297
Jose LINO NETO & Heidi DOLDER
22. Phylogenetic significance of axonemal ultrastructure: examples from Diptera and
Trichoptera . 301
Romano DALLA1 & Bjorn A. AFZELIUS
VERTEBRATES
23. Comparative morphology of the sperm in chondrichthyan fishes . 3 1 3
Sho TANAKA, Hana KUROKAWA & Masako HARA
24. Comparative spermatology of anurans with special references to phylogeny . 321
Ae Sook KWON & Young Hwan LEE
25. Amphibian sperm: phylogeny and fertilization environment . 333
Gerhard VAN DER HORST, Brian WILSON & Alan CHANNING
26. Evolution of tetrapod spermatozoa with particular reference to amniotes . 343
Barrie G.M. JAMIESON
27. The ultrastructure of spermatozoa of the Squamata (Reptilia) with phylogenetic
considerations . 359
Barrie G. M. JAMIESON
28. Ultrastructure of spermatozoa of australian blindsnakes, Ramphotyphlops spp.
(Typhlopidae, Squamata): first observations on the mature spermatozoon of
scolecophidian snakes . 385
H. Ronnie HARDING, Ken P. APLIN & Maria MAZUR
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
9
29. Ultrastructural and light microscopic observations of mature epididymal
spermatozoa and sperm maturation of the Greater Bilby, Macrotis lagotis
(Metatheria, Mammalia) . 397
Stephen JOHNSTON, Lina DADDOW & Franck CARRICK
30. Variation in sperm head morphology of muroid rodents of Africa: phylogenetic
implications . 409
William G. BREED
31. Comparative sperm structure in bats (Chiroptera): some taxonomic and adaptive
implications . . . 42 1
Takayuki MORI
32. Molecular and ontogenic analysis of the human sperm tail fibrous sheath . 43 1
Ali JASSIM
33. Diversity of avian spermatozoa ultrastructure with emphasis on the members of the
order Passeriformes . 437
Lawrence D. KOEHLER
PROTEINS
34. Histone HI and the evolution of the nuclear sperm-specific proteins . 447
Juan AUSIO
35. Evolution and origins of sperm nuclear basic proteins . 463
Harold E. KASINSKY
36. Male germ line specific histones of sea urchins and sea stars . 475
Dominic POCCIA
37. The gene encoding the sperm-specific basic nuclear protein <\>0 from sea cucumber
. , . 491
Eva PRATS & Luis CORNUDELLA
38. Nuclear basic proteins from the sperm of tunicates, cephalochordates, agnathans and
fish . 501
Manel CHIVA, Nuria SAPERAS, Carme CACERES & Juan AUSIO
39. Heparin-binding proteins on bull, boar, stallion, and human spermatozoa . 515
Juan Jose CALVETE, Libia SANZ, Markus REINERT, Zuzana DOST ALOVA & Edda TOPFER- PETERSEN
40. Histone gene expression during mammalian spermatogenesis: structural and
functional aspects . 525
Detlef DOENECKE& Birgit DRABENT
41. Sequence, evolution and transcriptional regulation of avian-mammalian PI type
protamines . 537
Rafael OLIVA
INDEX
549
Source : MNHN, Paris
Preface / Preface
With the approach of the Seventh International
Symposium on Spermatology at Cairns, Australia, in
1994, one of us (BGMJ) conceived the idea of editing a
book which would cover recent advances in the use of
spermatozoal ultrastructure in phylogeny and taxonomy.
Contributions were invited not only from those attending
the section of the Symposium in this field but also from
non-participants. Such was the response to the
invitations that the aid of a further person (JLJ) active in
the field of spermiocladistics was sought in the exacting
task of editing the contributions. The need for some
treatment of advances in knowledge of sperm nuclear
proteins led to the involvement of a third editor (JA) and
raised the total number of chapters to 41, with 73 authors.
It was not to be expected that contributions on all
animal groups would be received nor could they have been
published in a single volume. Nevertheless, the authors
have provided a most impressive testimony to the vigour
of the burgeoning discipline of spermatozoon-based
taxonomy and of the great utility of spermatozoal
morphology and particularly ultrastructure as what has
been termed an "independent arbiter" of conflicting
hypotheses of phylogenetic relationship. The
contributions also constitute a contemporary
documentation of fine structure of the spermatozoon
which will prove invaluable for students of reproductive
biology. Several chapters deal also with the functional
significance of components of the sperm cell.
The scope of the volume extends beyond the confines
of the Animal Kingdom in a study of fertilization and
calmodulin-mediated events in the slime mould
Dictyostelium , valuable, inter alia, in exemplifying the
simplest, amoeboid level of the sperm cell which may
also have typified the earliest Metazoa. Variations in the
molecular structure of dyncin arms are demonstrated and
shown to be specific to higher metazoan taxa.
In the invertebrate section, the value of sperm
ultrastructure for phylogeny and taxonomy is
demonstrated in a suite of chapters on the Platyhelminthes
(providing synapomorphies for the majority of supra-
generic groups in this phylum), in the most complete
survey of chelicerate sperm to date, in reviews of
thalassinidean, anomuran and brachyuran sperm and in
those of dendrobranchiate shrimps; and in studies on
bivalve and prosobranch mollusc sperm. In contrast, a
parsimony analysis of tubificid sperm shows that
although higher oligochaete and other clitellate groups
are well defined spermatologically, high homoplasy
largely masked relationships within the Tubificidae. The
taxonomic significance of sperm structure in strepsipteran
and hemipteran insects is also investigated and hitherto
unknown taxonomic variation in axonemal structure in
dipteran and trichopteran sperm is revealed. An elegant
study of gastrotrieh sperm shows profound differences
between their two orders and a sperm morphology which
is arguably the most bizarre known. An investigation of
nematode sperm reveals the first centriole known to have
10 singlets. The remarkable similarity of the complex
sperm of some aculiferan molluscs and protodri lid
annelids is cited in support of relationships of their two
A I'approche du Septieme Symposium International de
Spermatologie d Cairns (Australie), I'un d'entre nous {BGMJ)
congut l’ idee de coordonner un ouvrage qui couvrirait les apports
recents de V utilisation de I’ ultrastructure des spermatozoides pour
la phylogenie et la taxonomie. Des invitations a contribuer furent
envoyees, non settlement aux participants du Symposium, mais
aussi a d 'autres chercheurs. Les reponses furent si nombreuses que
I'aide d une personne supplementaire (JIJ) active dans le domaine
de la spermatologie cladistique fut recherchee, en particulier pour
la tdche exigeante de la verification des contributions. La nicessite
de la couverture des apports concernant les proteines nucleates
des spermatozoides amena au recrutement d’un troisieme
coordinateur (JA) et augmenta fmalement le nombre d’ articles a
quarante et un, avec soixante-treize auteurs.
Nous n ’ avions jamais espere que des contributions concernant
tous les groupes animaux pussent etre regues ou me me etre reunies
en un volume unique. Neanmoins, les auteurs ont fourni un
temoignage impressionnant de la vigueur de cetle discipline qu est
la taxonomie basee sur les spermatozoides, et de la grande utilite de
la morphologic des spermatozoides, en particulier ultrastructurale.
pour fournir ce qui a ete appele un " arbitrage independant" quand
plusieurs hypotheses de relations phylogenetiques sont en conflit.
Les contributions constituent aussi une documentation actualisee sur
I' ultrastructure des spermatozoides. d’une valeur incomparable
pour les chercheurs en biologie de la reproduction. Plusieurs
chapitres concernent, de plus, la signification fonctionnelle des
organites des spermatozoides.
Le sujet de ce volume s 'etend au dela des confins du Regne
Animal, dans une etude de la fecondation et des evenements
modules par la calmoduline chez le Myxomycete Dictyostelium,
dont Pun des interets est de donner I'exemple du niveau le plus
simple et amiboide de spermatozoide, qui pourrait aussi etre typique
des premiers Metazoaires. Une autre etude demontre que les
variations de la structure moleculaire des bras de dyneine sont
specifiques des taxons superieurs des Metazoaires.
La section sur les Invertebres demontre la valeur de
I' ultrastructure des spermatozoides pour la phylogenie et la
taxonomie: dans une serie de chapitres concernant les
Plathelminthes et fournissant des synapomorphies pour la majorite
des groupes supra-generiques de cet embranchement; dans la
revision publiee la plus complete concernant les spermatozoides
des Chelicerates: dans les articles sur les spermatozoides de
Crustaces incluant des syntheses sur les Thalassinides. les
Anomoures et les Brachyoures. et un article sur les
Dendrobranchiata; dans des etudes sur les spermatozoides des
Mollusques Bivalves et Prosobranches. A 1' oppose, une analyse de
parcimonie sur les spermatozoides des Tubificidae montre que les
homoplasies masquent les relations dans cette famille, bien que la
spermatologie comparee permette de bien definir les grands
groupes d'Oligochetes et les autres Clitelles. Chez les Insectes. des
etudes concernent aussi la signification taxonomique de la structure
des spermatozoides chez les Strepsipteres et les Hemipteres. et des
variations inedites de /’ ultrastructure des axonemes sont decrites
chez les Dipteres et les Trichopteres. Une etude elegante des
spermatozoides des Gastrotriches montre des differences profondes
entre les deux ordres qui les constituent, et une morphologic du
spermatozoide que Ton pent considerer raisonnablement comme la
plus bizarre jamais decrite. Une etude sur les spermatozoides des
Nematodes revele /’ existence du premier centriole connu a dix
singulets. Dans un autre article, la similarity remarquable entre les
12
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
phyla, for which molecular evidence has recently emerged,
and in favour of an hypothesis of complex introsperm as
the primitive metazoan sperm.
As alternative techniques, silver staining of the
acrosomal vesicle and cytoskeletal elements reveals
species specific patterns of phylogenetic significance;
and immunocytochemical studies of tubulin in
platyhelminth sperm offer a new technique for elucidating
sperm structure. Refined tannic acid techniques are
provided for demonstrating details of axonemal structure.
In the vertebrate section, the remarkable diversity and
great taxonomic and phylogenetic utility of sperm
structure is demonstrated for chondrichthyan fish; for
anurans, and other lissamphibians, in which sperm
motility patterns are shown to be species specific and to
relate to fertilization biology; for squamate reptiles, in
which an hypothesis of relationship of eugongyloid, but
not sphenomorph skinks, with snakes and pygopods
merits consideration, and placement of typhlopods in the
Serpentes is upheld; for amniotes as a whole, in which
spermatozoal synapomorphies are found for most major
taxa; for African murid rodents; for Chiroptera; and for
passeriform birds. Spermatozoal ultrastructure of an
endangered marsupial, the Greater Bilby, endorses the
discreteness of its family Thalacomyidac. The function of
proteins from the fibrous sheath of human sperm is
discussed.
The third section, on sperm proteins, ranges widely
over the evolution and nature of nuclear proteins
throughout the Animal Kingdom; specific histones of the
male germ line in echinoderms; heparin-binding proteins
of mammalian sperm; genes encoding nuclear proteins;
histone gene expression in mammalian spermatogenesis;
and sequence, evolution and transcriptional regulation of
avian -mammalian protamines.
The work of preparing a book by three editors
separated by thousands of kilometres was not easy. After
preliminary meetings and discussion in Cairns, progress
was greatly aided by the generosity of the MNHN in
hosting BGMJ as Invited Professor for three months
during the summer of 95. JLJ carried out the work of
preparing the photoready copy. The editors acknowledge
an extensive use of the Internet between us, and with most
authors, without which this book could not have been
completed in reasonable time.
This volume is published in the series "Memoires du
Museum" which is known for major contributions
published in the field of systematics. Non-systematic
plenary papers of the Proceedings of the Cairns meeting
have been published by CSIRO in an independent volume.
We hope that this volume will set the stage for
quantum advances by the next symposium on
Spermatology, at Montreal in 1998.
spermatozoides des Mollusques Ac u lift res et des Annelides
Protodrilides fournit un argument en faveur de relations
phylogenetiques proches entre ces deux groupes. rec eminent
demontrees par des preuves moleculaires. et de Phypothese de la
presence d'un intro spermatozoide chez les Metazoaires primitifs.
Des techniques originates sont aussi decrites. telles que le
marquage a P argent des vesicules acrosomiennes et des elements
du cytosquelette. qui revele des structures specifiques d'interet
phylogenetique, et P immunocytochimie de la tubuline chez les
Plailielminthes. qui permet de mieux comprendre la structure des
spermatozoides. Les techniques raffinees de fixation a Pacide
tannique permettent aussi de montrer des details de la structure de
I 'axoneme.
La section sur les Vertebres decrit la diversite remarquable. et
P utilize indeniable, de la structure des spermatozoides pour la
taxonomie et la phylogenie. Les etudes concernent: les
Chondrichtiens; les Anoures et les Lissamphibiens, chez lesquels il
est montre que les types de motilite sont specifiques et en rapport
avec la biologic de la fecondation; les Reptiles Squamata. pour
lesquels une hypothese de relations des Eugongyloides, mais non
des Seine idae Sphenomorphes, avec les Serpents et les Pygopodes
merite consideration, et pour lesquels on maintient l ’inclusion des
Typhlopodes dans les Serpents; les Amniotes dans leurs ensemble,
chez lesquels des synapomorphies du spermatozoide existent pour
la plupart des taxons majeurs; les Rongeurs Muridae africains; les
Chiropteres. et les Oiseaux Passeriformes. L' ultrastructure du
spermatozoide chez une espece menacee, le Grand Lievre
marsupial, confirme le statut particulier de la famille
Thalocomyidae a laquelle elle appartient. La fonction des proteines
de la gaine fibreuse des spermatozoides humains est discutee.
La troisieme section, sur les proteines des spermatozoides,
concerne de maniere ties large revolution et la nature des
proteines nucleaires dans l' ensemble du Regne Animal, et aussi: les
histones specifiques de la lignee germinate male chez les
Echinodermes ; les proteines se liant a Pheparine dans les
spermatozoides des Mammiferes; les genes codant pour les
proteines nucleaires; I expression des genes des histones pendant
la spermatogenese des Mammiferes; et la sequence. 1‘evolution et
la regulation transcriptionnelle des protamines des Oiseaux et
Mammiferes.
II n'est pas facile de preparer un livre quand les trois
coordinateurs sont separes par des milliers de kilometres. Apres des
rencontres preliminaires a Cairns, cette tdche a pu etre menee a
bien grace a la generosite du MNHN qui a accueilli BGMJ comme
Professeur Associe pendant les trois mois de Pete 1995. JLJ a
effectue la mise en page en PAO. Les coordinateurs reconnaissent
I'aide apportee par P utilisation intensive d' Internet, entre eux, et
avec la plupart des auteurs, aide sans laquelle cet ouvrage n 'aurait
pas pu etre termine dans un temps raisonnable.
Cet ouvrage est publie dans la collection des " Memoires du
Museum", bien connue pour ses contributions majeures dans le
domaine de la Systematique. Les conferences plenieres du
Symposium de Cairns ne relevant pas de la Systematique sont
publiees par le CSIRO dans un volume independant.
Nous esperons que ce volume ouvrira la voie a des prog res
significatifs lors du prochain symposium de Spermatologie de 1998,
a Montreal.
Barrie Jamieson (Brisbane)
Juan Ausio (Victoria)
Jean-Lou Justine (Paris)
Source
Invertebrates and General
Source : MNHN, Paris
Source : MNHN , Paris
Outer Arm Dynein of Sperm Flagella and Cilia
in the Animal Kingdom
Hideo MOHRI * ***, Miyoko KUBO-IRIE * & Masaru IRIE **
* University of the Air, Wakaba 2-11, Mihama-ku, Chiba 261, Japan
** Department of Electrical Engineering, Waseda University, Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169, Japan
*** Present address: National Institute for Basic Biology, Myodaiji, Okazaki 444, Japan
ABSTRACT
It has been established that the outer arm dynein molecule of flagella and cilia in Protozoa such as Chlamydomonas and
Tetrahymena has a three-headed structure, whereas that of sperm flagella in sea urchins, fish and mammals exhibits a two-
headed structure. All the latter animals belong to Deuterostomia. The outer arm dynein from sperm flagella of the oyster, a
member of Protostomia, is also two-headed. Investigation has begun of the situation in other animals, especially in
Cnidaria and Porifera. In Chlamydomonas , there is a mutant whose outer arm dynein lacks one head. Electron microscopy
revealed that the outer arm of the wild type shows a pistol-like shape in the cross-section of the flagellar axoneme,
whereas that of the mutant exhibits a hook- or fist-like shape. This suggests that we may predict the number of heads of
outer arm dynein by observing the cross-sections of flagella or cilia. Examination of the cross-sections of sperm flagella
and cilia in various animals, including mammals, tunicates, echinoderms, molluscs, annelids, arthropods, flatworms and
sea anemones, indicated that their outer arms were hook- or fist-like, in contrast to the pistol-like shape in Paramecium
and Tetrahymena. The former was true with choanocyte flagella of the sponge and metazoan cilia. The reduction in the
number of heads of outer arm dynein molecule would have occurred during the evolution from Protozoa to Metazoa (and
Mesozoa). Alternatively, the outer arm dynein in Protozoans was specialized to support their peculiar behaviour.
RESUME
La dyneine du bras externe des flagelles des spermatozoides et des oils dans le Regne Animal
II a 6te 6tabli que la molecule de dyndine du bras externe des flagelles et des cils des Protozoaires, tels que
Chlamydomonas et Tetrahymena , a une structure & trois teles, alors que les flagelles des spermatozoides des Oursins,
Poissons et Mammiferes ont une structure & deux tetes. Tous les animaux pr6c6demment cites appartiennent aux
DeutSrostomiens. La dyneine du bras externe de l’huitre, un Protostomien, a aussi deux tetes. Les Etudes sur d'autres
animaux, sp£cialement des Cnidaires et des Porifiires, ont commence. Chez Chlamydomonas, il existe un mutant dont la
dyneine du bras externe est depourvue d’une des tetes. La microscopie 61ectronique montre que le bras externe de type
sauvage a une forme en pistolet sur les coupes transversales de 1’ axon&me du flagelle, alors que celui du mutant a une forme
en crochet ou en poing. Ceci sugg&re que nous pouvons predire le nombre de tetes de la dyn6ine du bras externe en
observant des coupes transversales de flagelles ou de cils. L’observation de coupes transversales de flagelles de
spermatozoides et de cils d’animaux divers, tels que des Mammiferes, Tuniciers, Echinodermes, Mollusques, Ann£lides,
Arthropodes, Plathelminthes et Anemones de Mer, montre que leur bras externe est en forme de crochet ou de poing, en
opposition avec la forme en pistolet de Paramecium et Tetrahymena. La mcme situation a ete rencontrce pour les flagelles
des choanocytes des Spongiaires et les cils des Metazoaires. La reduction du nombre de tetes de la molecule de dyneine du
bras externe a pu intervenir lors de 1’evolution depuis les Protozoaires vers les Metazoaires (et M6sozoaires). Une
alternative serait a’interpreter la dyneine du bras externe des Protozoaires comme spScialisee pour permettre leur
comportement particulier.
Mohri, H., Kubo-Irie, M., & IRIE, M., 1995. — Outer arm dynein of sperm flagella and cilia in the animal
kingdom. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy.
Mem. Mus. natn. Hist, nat., 166 : 15-22. Paris ISBN : 2-85653-225-X.
16
H. MOHRI, M. KUBO-IRIE & M. IRIE : DYNEIN ARMS IN THE ANIMAL KINGDOM
Dynein was first thought to be a globular molecule with molecular mass of 300 000-
600 000, representing either a single outer arm or an inner arm in the axoneme of flagella and
cilia [7, 16]. The outer arm dynein has three heads connected by stalks to a common base in
flagella or cilia of Protozoa such as Chlamydomonas [8, 26], Tetrahymena [11, 28] and
Paramecium [13], as revealed by either negatively by stained or quick-freeze deep-etch images
under the electron microscope. Biochemical analyses also indicated that there are three distinct
heavy chains with molecular masses of 400 000 - 500 000, which constitute the corresponding
heads, as well as a few intermediate chains and a few light chains. On the other hand, outer arm
dynein obtained from sperm flagella of sea urchin [24], rainbow trout [6] and bull [14] has two
heads and consists of the corresponding two heavy chains. Recent studies indicate that inner arm
dynein is different from outer arm dynein not only in molecular composition [19] but also in
function [4, 5], A couple of different inner arm dynein molecules are present in a single flagellum
and cilium, and they appear to be two-headed even in the case where outer arm dynein is three¬
headed [21, 25]. Phylogenetically, all the above-mentioned Metazoa belong to Deuterostomia.
We examined the outer arm dynein of sperm flagella in the oyster as a representative of
Protostomia [30]. The purified dynein contained two heavy chains, and its negative stained image
was two-headed under the electron microscope. However, the question arose of whether the two-
headed dynein obtained is really an intact molecule or the product of a three-headed molecule as
18S dynein in Chlamydomonas is [33], although the same extraction procedure was used as that
applied for outer arm dynein from sea urchin sperm flagella. It is desirable to check this point by
some other means. Furthermore, from a phylogenetic point of view, it is interesting to know
whether the outer arm dynein of flagella or cilia in other animals, especially Cnidaria, Porifera,
etc., is two-headed or three-headed.
Table 1. — List of species examined, (s): sperm, (f): flagella, (c): cilia.
In Chlamydomonas , there is a flagellar mutant (oda-1 1) missing the a- heavy chain of outer
arm dynein but retaining the |3- and y-chains [22]. In other words, the outer arm dynein molecule
in this mutant is two-headed. Examination of the cross-sections of axonemes under the electron
microscope indicates that the image of the outer arm in wild-type Chlamydomonas is pistol-like
Source MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
17
(see Fig. la), whereas that in the mutant is hook- or fist-like (see Fig. lb). This fact should
provide us a useful tool for predicting the number of heads of outer arm dynein molecules in situ.
In fact, the image of the outer arm, consisting of two-headed dynein, in the cross-section electron
micrograph of the flagellar axoneme of sea urchin sperm resembles that of the flagellar axoneme
of the mutant Chlamydomonas (see Fig. lc). Since the starting materials are often insufficient in
amount for biochemical analysis of a purified dynein molecule, such an examination of the image
of the outer arm on the cross-sections of flagella or cilia seem to be effective in comparatively
analyzing the number of heads of the outer arm dynein molecule in different animal species. In the
present study, the outer arms of sperm flagella or cilia were examined in the species listed in Table
1 . In the case of Porifera, choanocyte flagella were used as material instead of sperm flagella.
MATERIALS AND METHODS
Flagellar axonemes of the wild type and oda 1 1 mutant of Chlamydomonas reinhardtii isolated by the method of
Witman [32] and ciliary axonemes of Tetrahymena pyriformis isolated by the method of Tanaka & Miki-Noumura [27]
were gifts from Dr. R. Kamiya and Dr. T. Miki-Noumura, respectively. Sperm flagella and axonemes of the sea urchins
Hemicentrotus pulcherrimus, Psudocenirotus depressus and Antliocidaris crassispina, of the molluscs Crassosirea gigas
and Mytilus edulis, and of the sea anemone Anlhopleura midori, were prepared as previously described [10], Spermatozoa
of the mouse Mas musculus, of the tunicate Ascidia sydneiensis semea. of the cirripede Balanus uliginosus , of the annelid
Neanihes diversicolor , and of the flatworm Planoceros reticulata were directly treated with a demembranation solution
consisting of 0.1-0.4 % Triton X-100, 0.15 M KC1, 1 mM MgCl2, 1 mM EGTA, 0.1 mM DTT and 20 mM Tris-HCl, pH
8.0. Intact Paramecium caudatum. a piece of mouse trachea and pieces of the gill of bivalves, Crassostrea gigas. Meretrix
lusoria and Tapes japonica, were subjected to the demembranation solution. The sponge Halichondria japonica was minced
with scissors, filtered through a mesh to remove spicules and then treated with the demembranation solution.
Dcmembranated axonemes of flagella and cilia (intact flagella in the cases of insect sperm) were fixed with a
mixture of 2.5 % glutaraldehyde and 1 % tannic acid in 0.2 M sodium cacodylate, followed by either post-fixation with 1 %
Os04 or block-staining with uranyl acetate. Specimens were dehydrated and embedded in Quetol 812 (Epon 812 in the case
of Chlamydomonas flagella). Thin sections were made with a Sorvall ultra-microtome MT-2 and observed under a JEOL
1200A electron microscope. To examine the shape of the outer arms more thoroughly, images of the axonemes on cross-
section electron micrographs were superimposed and averaged using an IBAS image analyzer.
RESULTS
Protozoan flagella and cilia
As mentioned in the Introduction, the outer arm of the wild-type Chlamydomonas flagella
had a pistol-like shape on the cross-section electron micrograph (Fig. la). The image corresponds
to a three-headed dynein molecule. On the other hand, that of the mutant (oda 11) flagella, in
which the a-heavy chain of outer arm dynein molecule is missing, showed a hook- or fist-like
image (Fig. lb). Comparison of the averaged image of the outer arms between the wild type and
the mutant clearly indicated that the distal portion of the outer arm is lost in the mutant flagella, as
described by SAKAKIBARA et al. [22]. One head corresponding to the a-heavy chain must reside
in this domain of the outer arm. As is shown in Fig. 2a, the image of the outer arm on the cross-
section of Tetrahymena cilia was quite similar to that in the wild Chlamydomonas flagella. The
same was true of Paramecium cilia (Fig. 2b). The results are consistent with the fact that isolated
outer arm dynein molecules in these organisms are three-headed.
Metazoan flagella
Figure lc shows the cross-section of the sea urchin sperm flagellum ( Hemicentrotus ) and
the averaged image of the outer arm. It is clear that the image is hook- or fist-like, as in the case of
the outer arm in the mutant Chlamydomonas flagellum. Figures 2c and 2e show the cross-sections
of sperm flagella in the mouse and the oyster Crassostrea, whose outer arm dynein must be two-
headed. In both cases the image of the outer arm was similar to that in the mutant
Chlamydomonas. Thus the two-headed structure of outer arm dynein must be reflected in an
image of the outer arm. The outer arm of sperm flagella in the tunicate Ascidia, a member of
Deuterostomia as mammal and sea urchin, was also the two-headed type (Fig. 2d).
18
H. MOHRI. M. KUBO-IRIE & M. IRIE : DYNEIN ARMS IN THE ANIMAL KINGDOM
Fig. 1. — Cross-sections of axonemes with averaged outer doublet images, a: Flagellum of wild-type Chlamyclomonas ;
b: Flagellum of a mutant (oda 11) Chlamydomonas, c: Sperm flagellum of the sea urchin Hemicentrotus
pulcherrimus .
The results obtained with sperm flagella of other members of Protostomia are shown in Fig.
2g, h, i and j. They were the annelid Neanthes, the insect Callophrys, the cirripede Balanus and
the flatworm Planoceros. The images of the outer arms in these species were also hook- or fist¬
like, indicating the two-headed molecule of outer arm dynein. Although not shown here, sperm
flagella of many butterflies other than Callophrys were examined with the same result. In
arthropods such as insects and cirripedes, the morphology of the outer arm was somewhat
different from that in other animals, taking rather an axe-like shape (see also [1]). However, the
image corresponding to the distal domain of the protozoan outer arm could not be found. It should
be noted that the axonemc of flatworm sperm is a 9+“l” type.
Cnidaria is likely to be the phylogenetic ancestor of both Protostomia and Deuterostomia. A
cross-section of sperm flagellum of the sea anemone, Anthopleura , is shown in Fig. 2k. The
shape of the outer arm in this species was not different from that of sperm flagella in other
metazoan animals. A preliminary biochemical analysis of purified outer arm dynein preparation in
the sea anemone indicated the presence of two heavy chains on SDS polyacrylamide gel (data not
shown). Furthermore, the outer arm of choanocyte flagella in the sponge Halichondria was not of
the protozoan type (Fig. 21).
Metazoan cilia
Since cilia were examined in Tetrahymena and Paramecium, and furthermore
Chlamydomonas flagella beat like metazoan cilia in forward movement, metazoan cilia may
possess outer arm dynein somewhat different from that of metazoan flagella. As can be seen from
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
19
Fig.
2. — Cross-sections of flagellar and ciliary axonemes in various animals, a: Cilium of Tetrahymena pyriformis ;
b: Cilium of Paramecium caudatunr, c: Sperm flagellum of the mouse Mus musculus\ d: Sperm flagellum of the
tunicate Ascidia sydneiensis semea\ e: Sperm flagellum of the oyster Crassostrea gigas ; f: Gill cilium of the
oyster; g: Sperm flagellum of the annelid Neanthes diversicolor: ; h: Sperm flagellum of the butterfly Callophrys
ferrea\ i: Sperm flagellum of the cirripede Balanus uliginosus; j: Sperm flagellum of the flatworm Planoceros
reticulata ; k: Sperm flagellum of the sea anemone Anthopleura midori ; 1: Choanocyte flagellum of the sponge
Halichondria japonica. Arrows indicate the outer arms.
Source MNHN , Paris
20
H. MOHRI. M. KUBO-IR1E & M. IRIE : DYNEIN ARMS IN THE ANIMAL KINGDOM
Fig. 2f, the outer arm of gill cilia in the oyster shows the same image as that of sperm flagella of
the same species. The results were the same with gill cilia of other bivalves, Meretrix and Tapes ,
and with tracheal cilia of the mouse.
DISCUSSION
Morphology of the outer arm
The presence of several dynein species with somewhat different functions has been
disclosed within a single flagellum or cilium [20, 29]. According to a recent review by BROKAW
[4], the main function of outer arm dynein in flagella and cilia appears to be generation of power
to overcome viscous resistance, in contrast to the functions of inner arm dyneins, which appear to
be bend initiation and maintenance of the angle of propagating bend. Outer arm dynein molecules
so far purified from protozoan flagella and cilia possess three heads corresponding to three
different heavy chains, a, p and y, obtained by SDS-PAGE [8, 11, 13, 26, 28], On the other
hand, purified outer arm dynein molecules of sperm flagella in Metazoa such as mammals, fish,
sea urchins and molluscs, consist of only two heads and two heavy chains, a and p [10, 13, 24,
30].
Fig. 3. — Schematic cross-section of an outer doublet microtubule with the outer arm. a: protozoan axoneme; b: metazoan
axoneme.
Recent studies on Chlamydomonas flagellar mutants by KAMIYA and his colleagues [22,
23] clearly showed that one head corresponding to the a-heavy chain is situated at the distal
domain of the outer arm, which looks like a pistol on the axoneme cross-section, the other two
heads compose the body domain of the arm, one head corresponding to the y-heavy chain
localized at the innermost domain. Only the body domain of the outer arm is found in a flagellar
mutant, oda 11, of Chlamydomonas, which lacks the a-heavy chain, resulting in a hook-like
image of the outer arm on the cross-section electron micrograph. The present results indicate that
in the metazoan sperm flagella from which two-headed outer arm dynein molecules have been
isolated, the outer arms show the hook-like image representing the body domain of the outer arm
in protozoan axonemes. In other words, a- and p-heavy chains of metazoan outer arm dynein
would correspond to p- and y-heavy chains of protozoan outer arm dynein. This fact also proves
that the outer arm dynein molecule of oyster sperm flagella is certainly two-headed in situ, as
indicated by biochemical analysis and the negative stained image of isolated dynein [30],
Most schematic cross-sections of flagellar and ciliary axonemes are based on electron
micrographs obtained with either Tetrahymena cilia [2] or molluscan gill cilia [31], and have been
accepted as common to all kinds of flagella and cilia [e.g: 3, 15]. Little attention has been paid to
the image of the outer arm, in spite of the above-mentioned difference in the number of heads of
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
21
outer arm dynein. In the most recent diagram depicting the Chlamydomonas axoneme, the outer
arm is drawn as it is in Fig. la in this paper ([12]; see also computer-generated cross-section of
the axoneme in [9]). Therefore, a different image should be drawn for the metazoan axoneme. On
the basis of the present findings, we propose here an interpretative diagram of the outer arms in
protozoan and metazoan axonemes as represented in Fig. 3.
Phylogenetic aspects
Phylogenetically, sperm flagella of metazoan animals belonging to Protostomia and
Deuterostomia exhibit the same morphology of the outer arm, which incorporates the two-headed
structure of outer arm dynein. The outer arm of flatworm sperm flagella which have a 9+“l”
axoneme and show three-dimensional movement is also not exceptional. No difference is
observed with ciliary axonemes of these metazoan species, and outer arm dynein from embryonic
cilia of the sea urchin has been reported to contain only two heavy chains [18]. Thus motility
pattern does not seem to reflect on the structure of outer arm dynein among Metazoa.
An axe-like image of the outer arm in the cross-section electron micrographs of arthropod
sperm flagella (Fig. 2h, i) would be caused by a slight shift of the two heads inward as compared
with the arrangement of these heads in other metazoan axonemes. Down the phylogenetic tree to
Cnidaria and Porifera, the common ancestors of both Protostomia and Deuterostomia, outer arm
dynein molecules are considered to be two-headed. Furthermore, the image of the outer arm in the
cross-section of the ciliary axoneme of Mesozoa, Dicyema misakiensis, is also hook- or fist-like,
suggesting the two-headed structure of constituent dynein (Y. YAMAKAWA & M. OKUNO,
personal communication). Although more thorough examination would be needed, so far only
protozoan flagella and cilia have outer arm dynein with a three-headed structure.
Hence the reduction in the number of heads of outer arm dynein molecule would have
occurred during evolution from Protozoa to Mesozoa and Metazoa, as an adaptation to simplify
and make more efficient the motile machinery of the latter's flagella and cilia. Alternatively, the
outer arm dynein in protozoan flagella and cilia was rather specialized. The addition of an extra
head would facilitate complex movements such as the cilia-like motion in forward swimming and
flagella-like motion in backward swimming of Chlamydomonas as each of the heads of outer arm
dynein appears to exhibit a somewhat different function from the others [17].
ACKNOWLEDGEMENTS
We wish to thank Drs. Ritsu Kamiya and Taiko Miki-NOUMURA for the gifts of Chlamydomonas axonemes and
Tetrahymena axonemes. We are grateful to the director and staff of the Misaki Marine Biological Station for supplying
marine invertebrates. Thanks are also due to Misses J. Shuna, A. Takahashi and M. Ikuta for their technical assistance.
This work was supported by Grants-in Aid from the Ministry of Education, Science and Culture of Japan.
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Source MNHN, Paris
Amoeboid Gametes and Fertilization in Dictyostelium:
Gamete and Pronuclear Fusion are Mediated
by Calmodulin and its Binding Proteins
Danton H. O’DAY, Keith E. LEWIS * & Michael A. LYDAN
Department of Zoology,
University of Toronto, Erindale College, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
* Current address: Department of Chemistry,
University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada
ABSTRACT
The gametes of Dictyostelium discoideum are tiny amoeboid cells which contain a small condensed nucleus. Gamete
formation is calcium-independent and inhibited by calmodulin function. Accumulated data suggests that the gametes are a
source of sexual pheromone that promotes sexual cell fusion. Time-lapse videomicroscopy has revealed that gametes are
highly motile compared to non-gametic cells and, when two gametes contact, fusion results in the formation of a
binucleatc cell. Within each binucleate cell, pronuclear migration, swelling and fusion occur as the cytoplasmic volume of
the cell increases dramatically producing a zygote giant cell. Gamete fusion is calcium-dependent and involves at least one
membrane-bound, GlycNAc-containing glycoprotein (gp 1 38). Fertilization is mediated by the dual signal transduction
pathway involving calcium and protein kinase C. Of particular importance is the downstream role of calmodulin (CaM) and
its binding proteins (CaMBPs). A putative CaM Kinase III activity and two, as yet unidentified CaMBPs (i.e. CaMBP-48,
CAMBP-91) are developmental^ regulated and temporally associated with the events of cell and pronuclear fusion in D.
discoideum. Fertilization is terminated by a feed-back mechanism involving an autoinhibitor that is secreted by the
zygote giant cells. This low molecular weight, hydrophobic, heat-stable autoinhibitor inhibits both cell and pronuclear
fusion by preventing the interaction ot CaM with its binding proteins. These results are discussed in terms of fertilization
and signal transduction involving calmodulin and its binding proteins in higher animals.
RESUME
Gametes amiboides et fecondation chez Dictyostelium: la calmoduline et ses proteines liees sont
les mediateurs de la fusion des gametes et des pronucleus
Les gametes de Dictyostelium discoideum sont des petites cellules amiboides qui contienncnt un petit noyau condense.
La formation des gambles est ind6pendante du Calcium et est inhibde par un systdme a calmoduline. Une abondance de
donnees sugg^re que les gametes sont une source de pheromone sexuelle qui declenche la fusion sexuelle des gametes. La
videomicroscopie a montre que les gametes sont hautement mobiles en comparison avec les cellules non gametes, et que,
quand deux cellules entrent en contact, la fusion a pour resultat la formation d'une cellule binucl66e. Dans chaque cellule
binucleee, la migration, le gonflement et la fusion des pronucteus interviennent alors que le volume cytoplasmique de la
cellule augmente de maniere importante en produisant une cellule zygote geante. La fusion des gametes est dependante du
Calcium et implique au moins une glycoprotdine li£e £ la membrane contenant GlycNac (gp 1 38). La fecondation fait
intervene le systfcme de transduction double impliquant le Calcium et la prot6ine kinase C. Le role en aval de la
O Day, D. A. H., Lewis, K. E., & Lydan, M. A., 1995. — Amoeboid gametes and fertilization in Dictyostelium:
gamete and pronuclear fusion arc mediated by calmodulin and its binding proteins. In: Jamieson, B. G. M., Ausio, J., &
Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. ncitn. Hist, not 166 23-36
Paris ISBN : 2-85653-225-X.
24
D. H. O'DAY, K. E. LEWIS & M. A. LYDAN : CALMODULIN IN DICTYOSTELIUM
calmoduline (CaM) et dc ses proteines li<§es (CaMBPs) est d'une importance particulidre. Une activite CaM Kinase III
supposee et deux CaMBPs jusqu’ici non identifiees (CaMBP-48 et CaMBP-91) sont r^gulees au cours du developpement ct
associees temporellement avec les evenements de fusion des cellules et des pronuclcus chez D. discoideum. La fecondation
est achevee par un mecanisme en retour impliquant un autoinhibiteur qui est sdcrete par les cellules zygotes geantes, cet
autoinhibileur, qui est de petit poids moleculaire, hydrophobe, stable & la temperature inhibe & la fois la lusion des cellules
et des pronuclcus en empechant Linteraction de la CaM avec ses proteines liees. Ces resultats sont discutes en
comparaison avec la fecondation et la transduction de signal impliquant la calmoduline et ses proteines liees dans les
animaux superieurs.
The importance of signal transduction during fertilization and other developmental processes
has only recently come to light. In evolutionary terms, gamete fusion undoubtedly represents the
first regulated type of cellular fusion wherein the cell membranes of two different cell types
contact, bind and amalgamate to form a new cell type of unique genetic composition (i.e., a
zygote). How do cells that have contacted relay their compatibility? How do they set in motion the
sequence of events that will lead to their coalescence? These simple questions lie at the heart of
fertilization of all organisms from simple eukaryotic microbes such as Dictyoselium to mammals
such as mouse and humans.
While the probing of the sex life of Dictyostelium has only occurred during the last twenty
years or so, research is facilitated by the extensive literature that exists and the powerful cellular,
genetic and molecular methodologies that have been developed during studies on asexual
development of this social amoeba. For these reasons, advances in understanding fertilization and
sexual development in this model organism should progress rapidly in the future. Fertilization in
Dictyostelium discoideum is comparatively simple (Fig. 1). Tiny, highly motile, amoeboid
gametes appear when cells are cultured in the dark [57]. When two compatible gametes make
contact they fuse to produce a binucleate cell. Pronuclear swelling, migration and fusion occur
concomitantly with a large increase in cytoplasmic volume producing a zygote giant cell [43, 70],
The zygote giant cells takes over control of subsequent development in sexual cultures using
several secreted molecules to regulate the behaviour of non-zygotic cells in the culture [1, 13, 24,
52, 64, 68]. In addition to inhibiting continued gamete cell fusion, this is reflected by each giant
cell chemoattracting hundreds of other cells and then ingesting them in an act of cannibalistic
phagocytosis [29, 30, 31, 32, 52],
As will be detailed in the work presented here, the events of fertilization in Dictyostelium are
dependent on the functioning of calmodulin (CaM) and its binding proteins (CaMBPs).
Calmodulin is present in the sperm and eggs of all animal species that have been studied and its
function is essential to many of the component events of fertilization. For example, calmodulin
mediates such diverse sperm functions as activation, motility, capacitation, the acrosome reaction
as well as sperm-egg fusion (Table 1). As in other systems, calmodulin carries out its functions
by regulating the activity of other proteins, often enzymes. A number of these calmodulin binding
proteins have been isolated from spermatozoa from various species and in some cases specific
roles have been attributed to them (Table 1). To date, comparatively little work has been done on
calmodulin and its binding proteins in amoeboid spermatozoa.
The fusion of amoeboid gametes represents one of the simplest forms of eukaryotic
fertilization. In spite of this, the fundamental events of species-specific cellular recognition,
adhesion and cell fusion followed by pronuclear fusion that are common to all types of
fertilization also occur [e.g. 79]. On the other hand, the absence of complex surface structures
(e.g. egg coats) and other accessories (e.g. acrosome, acrosomal process, etc.) likely leaves us
with a stripped-down system made up solely of the essential elements that are fundamental to
regulated cell coalescence in all organisms. With this in mind, the aim of this article is to examine
the state of knowledge of gamete formation and fertilization in D. discoideum and other slime
mould species while keeping a general, comparative eye on some related work that has been done
on fertilization in animals.
Source :
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
25
Table 1. — Localizations and roles of calmodulin and calmodulin binding proteins in various spermatozoa 1
I. Calmodulin
II. Calmodulin Binding Proteins
1 This is a summary of selected relerences and is not meant to be a complete or comprehensive review of the literature or of
all proposed functions of calmodulin and its binding proteins.
The location of these molecules can vary with developmental stage, physiological state or experimental treatment.
26
D. H. O'DAY, K. E. LEWIS & M. A. LYDAN : CALMODULIN IN DICTYOSTELIUM
RESULTS AND DISCUSSION
Gamete development
After mixed mating type strains (NC4 x V12) of Dictyostelium discoideum are grown
together in the dark, a new tiny amoeboid cell type appears about 6 hours after the culture is
started (Fig. 1) [57], These cells have 1/4 of the cytoplasmic volume and 1/5 the nuclear volume
of typical vegetative amoebas. Under phase contrast microscopy the cells are more birefringent
than vegetative amoebae (Fig. 2a-c). When stained with Hoechst 33258, a fluorescent nuclear
stain, these cells are seen to possess tiny nuclei that fluoresce brightly (Fig. 2d, e). Time-lapse
videomicrography has shown that these tiny amoebae move much more quickly than vegetative
cells. In their movements they make contacts with other cells, pause, then move on. Upon contact
with another small amoeba, two cells of opposite mating type can fuse (Fig. 20- In a series of
elegant experiments, URISHIHARA & YANAGISAWA [73, 74] used cell ghosts to show that fusion
occured between cells of opposite mating type. In cultures containing 1 mM calcium chloride,
fusion is inititated around 8 hours after cultures are started. Fusion is initiated after contact of
extended filopodia (Fig. 2f). The fusion of these tiny cells coincides with a concomitant increase
in binucleates (fusion products) as the population of tiny cells decreases. These, and an extensive
amount of other kinetic data, indicate that the tiny amoebae represent the gametes of Dictyostelium
discoideum.
G B ZGC
I - 1
Fig. 1 . — Fertilization in Dictyostelium discoideum. Shortly after spore germination in dark grown cultures, tiny gametes
(G) appear. These actively moving cells make frequent contacts with other cells and upon contacting other gametes
they can fuse (f) to form a small binucleate. As the cytoplasm of the binucleate increases in volume, the pronuclei
swell (1), migrate together (2) and fuse (3) to form the zygote giant cell (ZGC).
Gametes with essentially identical structure and behaviour, as well as similar developmental
kinetics, have been identified in several other genera and species of cellular slime mould
including: D. giganteum, D. purpureum , D. mucoroides and Polysphondylium pallidum ([26, 27,
28, 52], O’Day, unpublished results) as well has homothallic strains of D. discoideum [RAMA &
O’ DAY, unpublished]. Thus the gametes of all cellular slime moulds studied to date appear as tiny
amoeboid cells possessing tiny nuclei. Preliminary DNA quantification in D. discoideum indicates
that these cells are haploid and appear to be arrested in G1 of the cell cycle [O’ Day & RIVERA,
unpublished results].
Gamete differentiation is strain and calcium-independent
In mated sexual cultures of D. discoideum cultivated in the presence of 1.0 mM calcium
chloride, the developmental kinetics of gamete differentiation and subsequent fertilization have
been well defined [34, 35, 42, 70]. After appearing about 8 hours after culture initiation, the
gamete numbers peak at between 10-12 hours after which they steadily decrease in number. The
decrease in binucleates coincides with the appearance of their binucleate fusion products which
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
27
Fig. 2. — Gametes of D. discoideum in vivo and after fixation and staining with the nuclear fluorescent dye Hoecsht
33258. a-c, examples of living gametes (arrows) as compared to typical non-sexual amoebae under phase
microscopy. Alter lixation and staining and observation under simultaneous phase and fluorescence microscopy
(d-f), the amoebae are seen to contain a small bright nucleus (d, e). Pairs of amoebae (f) also have been caught in
the process of fusing.
Fig. 3. — Ultrastructure of pronuclear fusion in D. discoideum. Pronuclear fusion (a-c) involves and initial contact (b) by
protrusions of the swollen pronuclei followed by the fusion of the nuclear envelopes (c) at their points of contact
in a manner that is reminiscent of the sea urchin type of fertilization.
then increase in number until about 18 hours. The binucleates are converted to zygotes by events
of pronuclear swelling, migration and fusion which are exemplified by the “sea urchin-type of
fertilization” [e.g. 33, 51, 69, 70].
In mated cultures grown without the addition of calcium, gametes appear and increase in
number to a peak of approximately 30% by 18 hours [42]. However, they do not decrease in
number and binucleates do not appear in significant numbers. When strains NC4 or V12 are
28
D. H. O'DAY. K. E. LEWIS & M. A. LYDAN : CALMODULIN IN DICTY OSTELIUM
grown alone in either the presence or absence of calcium ions, gametes also appear and reach a
plateau but do not fuse to form binucleates. If calcium is subsequently added to mixed mating type
cultures grown in the absence of added calcium, fertilization is spontaneous and rapid, often
resulting in the formation of extremely large, multinucleated cells. The same occurs when strains
that have been cultured separately are subsequently mixed together [63, 64], Gamete formation,
then, is independent of an interaction between cells of the opposite mating type. These results
reveal that gamete formation occurs in the absence of calcium and that the gametes that form are
fertilization-competent. The simple addition of calcium is sufficient to trigger their fusion and all
subsequent events of fertilization [42]. What is more, like animal fertilization, the over-abundance
of fertilization competent cells leads to polyspermy [42, 55].
Fertilization is calcium dependent and involves signal transduction
While gametes can form in the absence of calcium, they cannot fuse. Calcium is the trigger
for fertilization, as well as many other types of biomembrane fusion [53]. During animal
fertilization, the events leading to the amalgamation of the sperm and egg are mediated by signal
transduction involving calcium ions, calmodulin and its binding proteins (for review see [4, 5,
22]). Various aspects of sperm function in the events leading to fertilization are dependent upon
calmodulin function (Table 1). Contact between the sperm and egg, involving a G protein,
receptor-mediated process, leads to inositol- 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG)
production within the egg [e.g. 48]. IP3 formed intracellularly mediates the release of calcium
ions from membrane bound stores, most notably the calciosomes (e.g. 5, 46). The resulting
increase in intracellular calcium concentration leads to an activation of calmodulin followed by the
modulation of the activity of several calmodulin binding proteins including CaM-kinase.
Simultaneously, DAG is activating protein kinase C (PKC). These downstream events lead to the
phosphorylation of certain key proteins as well as other essential, but as yet unclarified, events of
fertilization.
Extensive studies during fertilization in D. discoideum have shown that the dual calcium
signalling pathway mediated by G proteins and involving IP3 and DAG also mediates cell and
pronuclear fusion [9, 35]. IP3 augments cell fusion while inhibitors of calcium release from
intracellular stores prevent both cell and pronuclear fusion [35], As in other organisms from
Chlamydomonas to vertebrates [e.g. 15, 22], inhibitors of calmodulin also prevent gamete cell
and pronuclear fusion in Dictyostelium (Fig. 4) [36]. In keeping with this, analysis of calmodulin
binding proteins (CaMBPs), using a highly sensitive method, revealed over 25 CaMBPs during
sexual development of which some developmentally regulated CaMBPs showed developmental
kinetics linking them to the events of fertilzation (Fig. 5) [38, 40]. While their identities remain a
mystery, two developmental calmodulin binding proteins (CaMBP91 and CaMBP48) are under
further analysis. Specific CaM kinase (CaM-Kinase III) and CaM phosphatase activities have also
been linked to the events of fertilization in D. discoideum (e.g. Fig. 6) [39]. Various studies have
shown that calmodulin activity and CaM-dependent protein kinase is involved in nuclear envelope
breakdown suggesting a role for this activity in pronuclear fusion [3, 12, 44, 45], Similarly,
pharmacological experments have shown that PKC activity is essential for fertilization and
substrates for phosphorylation by this enzyme have been identified [60]. On the other hand,
protein tyrosine kinase activity does not appear to be essential [60],
Gamete formation is inhibited by calmodulin
Of particular interest were some unexpected results from these experiments. The addition of
the CaM inhibitors, trifluoperazine (TFP) and calmidazolium (R24571) not only inhibited cell and
pronuclear fusion but they also led to a dramatic increase in gamete formation (Fig. 4b) [36]. The
results were reminiscent of cultures treated with an endogenous regulator of sexual development
Source
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
29
Fig. 4. — Differential effects of calmodulin antagonists
on gamete cell fusion and pronclear fusion in
sexual cultures of D. discoideum. When added at 10
hours, 1.0 fiM R24571 yields similar numbers of
binucleates (a) as control cultures as seen at 20
hours but 5.0 pM R24571 added at 10 hours
completely inhibits gamete fusion (b) and also
induces gamete differentiation within the same
time period. When added at 18 hours both 1.0 pM
TFP (c) and 5.0 pM R24571 (d) inhibit pronuclear
fusion as observed in cultures 6 hours later (24
hour old cultures).
called the autoinhibitor [59, 68]. The autoinhibitor is produced and secreted by zygotes apparently
as a shut-down mechanism to prevent further fertilization in older sexual cultures. The
autohinhibitor, like the inhibitors of calmodulin, inhibited both cell and pronuclear fusion when
added to early culture and it similarly augmented gamete differentiation. The autoinhibitor is a
small molecular weight (about 500 Daltons), heat stable, hydrophobic molecule [68J. Subsequent
studies showed that partially purified autoinhibitor specifically inhibits CaM-dependent
phosphodiesterase in a dose-dependent manner [37]. The accumulated information thus indicates
that the autoinhibitor functions to inhibit fertilization by inhibiting calmodulin function. It also
supports the contention that gamete formation is negatively regulated by calmodulin. Work on
C. elegans similarly indicates that calmodulin inhibits the onset of spermatogenesis. This may
reflect a fundamental role of calmodulin in regulating gametogenesis (Table 1).
Pheromone production by gametes
While several different developmental regulators operate during sexual development of
Dictyostelium discoideum , at least one other is relevant here. Early work showed that extracellular
medium from cultures of NC4 induced V12 to undergo sexual development alone [54]. Continued
work showed that a low molecular weight, volatile sexual pheromone was produced by NC4
which induced V12 [27, 41], On the other hand, strain V 12 did not produce detectable levels of
sex pheromone under the conditions used. Other species produce sexual pheromones as well and
the level of production (i.e., ability to induce other strains of the same species to undergo sexual
development) by each strain was directly related to the gamete levels formed by that strain [58],
30
D. A. H. O'DAY. K. E. LEWIS & M. A. LYDAN : CALMODULIN IN DICTYOSTELIUM
6 8 10 12141618202224
Developmental Age (hours)
G B ZGC
Fig. 5. — Calmodulin-binding proteins during gamete fusion, pronuclear fusion and zygote differentiation in D.
discoideum . Cells were isolated at the times indicated and subjected to SDS-PAGE followed by the detection of
calmodulin (CaM) binding proteins using the 35S-VU-CaM overlay procedure [29]. More than 25 CaMBPs are
present but only seven of these are expressed during sexual development (arrows). A diagrammatic depiction of
fertilization is presented for orientation.
Thus four strains of D. giganteum which exhibit a hierarchy of strain interactions produce
pheromonal activity which is directly related to the number of gametes each strain produces. Three
strains of D. purpureum which interact in a non-hierarchical manner show the same direct
correlation between the production of gametes and the level of pheromone activity. Finally, in D.
discoideum only NC4 produces pheromone while simultaneously producing the largest number of
gametes. Thus the results from several different species reveal a direct relationship between the
gametes produced and the level of sexual pheromone activity produced by the strain. This
suggests that the gametes are the source of the pheromonal activity. While the pheromone
stimulates fusion, no evidence exists as to whether it serves to direct cells of opposite sex together
via chemotaxis in an analogous manner to eggs chemoattracting sperm as occurs, for example, in
Source
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
31
*
31-
21 -
6 10 14 18 22 6 10 14 18 22
+C a -Ca
-CaM
t
6 10 14 18 22 6 10 14 18 22
+ Ca -Ca
Deve lopmen fa 1 Age (hrs)
F|G- 6- — Calmodulin (CaM) dcpcndeni protein kinase activity during fertilization in D. discoideum. While at least 10
different proteins are phosphorylated only two of these (arrows) appear to be preferentially phosphorylated by a
CaM dependent protein kinase activity. A high molecular weight protein (upper large arrow) is phosphorylated
very little in the absence of CaM while a lower molecular weight protein (lower small arrow) is enhanced to a
greater degree in the presence of CaM.
sea urchins via resact [76], So far, the gametes of Dictyostelium have proven difficult to purify
but their purification could lead to the ability to produce large amounts of pheromone for further
investigation which could answer some of the remaining questions about its structure and mode of
action. Furthermore, purified gametes would permit the direct analysis of gamete-specific
molecules which would allow further analyses of gamete differentiation and its regulation. In spite
of this, some information about gamete specific components can be inferred from other
experimental approaches.
Glycoprotein , g proteins and the initiation of fertilization
By definition, the function of gametes is to amalgamate in the process of fertilization to
generate a new genotype. By extension, the molecules that mediate gamete fusion should be
restricted to the gamete cells themselves. Much of the work reported so far is based upon
populational studies with cell-type specific macromolecules being identified on the basis of their
temporal association with cellular kinetics and cellular or developmental function. In this regard,
several glycoproteins have been identified as critical to fertilization and by extension to being
components of gametes. Early work showed that certain N-acetylglucosamine-containing
glycoconjugates localised at the cell surface were essential for fertilization [8, 56, 62, 67],
Subsequently a glycoprotein of about 130 kDa was identified with the appropriate developmental
kinetics, calcium dependence and behaviour after certain treatments (e.g. with tunicamycin [6]).
32
D. A. H. O'DAY, K. E. LEWIS & M. A. LYDAN : CALMODULIN IN DICTYOSTELIUM
Cell & Pronuclear Fusion
Fig. 7. — Signal transduction during fertilization in Diciyostelium discoideum. Cell fusion involves cells of opposite
mating type and extensive work has shown that at least one glycoprotein (gpl38) mediates the fusion process.
Gamete cell fusion involves a G protein mediated process leading to the intracellular increase in the second
messenger inositol-1,4, 5-trisphosphate (IP3). IP3 leads to an intracellular increase of calcium ions (Ca2+) which
then leads to an increase in protein kinase C (PKC) activity and resultant protein phosphorylation.
Simultaneously, the Ca2+ binds to calmodulin (CaM) and activates it (Ca2+~ CaM). This leads to the activation of
at least a CaM-dependent Protein Kinase and Phosphatase. Once cell and pronuclear fusion has produced some
zygote giant cells, those cells secrete a low molecular weight autoinhibitor (A) that inhibits CaM and leads to the
inhibition of cell and pronuclear fusion.
Almost simultaneously a similar glycoprotein (138 kDa) plus others were identified by Kaichiro
YANAGlSAWA’s group at Tsukuba [65, 67, 75], YANAGlSAWA’s group pursued this avenue,
leading to the cloning of a gene for gp 138 which when used to generate antisense mutants
revealed that this glycoprotein was essential for gamete fusion [16], On the other hand, since it
was not strain-specific, their data suggest that while involved in fusion, this gp 138 is not a cell
type receptor that initiates the fertilization process. Other work indicates that such receptors must
exist and awaits further investigation.
While identification of the receptor that initiates the events leading to 1P3 and DAG
production for fertilization awaits elucidation, other work has identified intermediary components.
Membrane-bound heterotrimeric GTPases mediate the interaction between the receptor and its
ligand and specific membrane effectors (e.g., phospholipase C leading to IP3 accumulation).
Developmental analyses have revealed specific GTPases present during the phases of gamete
differentiation and fertilization. Specifically, developmentally regulated, calcium-dependent
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
33
GTPases of 52kDa and 45kDA predominate at the time of fertilization [7, 9]. Interference with
GTPase function in general by GTP and GDP analogues and of G proteins specifically with
aluminum fluoride, argues for the impoitance of the GTPases in fertilization. Additional inhibitor
studies plus probing of western blots with specific antibodies has revealed that a G protein of
52kDa (i.e. dGas52) is important during sexual phagocytosis but not in fertilization [7].
Conclusions
Our extensive work on early sexual development of Dictyostelium discoideum has resulted
in the formulation of a working model for the signal transduction events that initiate and terminate
cell and pronuclear fusion (Fig. 7). While our studies to date have allowed us to generate an
integrated picture of fertilization in D. discoideum versus that in animals, many questions remain
to be answered. The purification of specific cells types has helped us localize some elements of
the signalling process to zygotes but the purification of gametes is essential. Experiments are now
underway to generate mutants defective in calmodulin function and to produce knockout mutants
tor specific CaMBPs [LYDAN & O'Day, unpublished]. The characterization and sub-cellular
localization of specific CaMBPs is currently underway. Several CaMBP genes have been isolated
by us from a cDNA library from asexual development. Our goal is to use the sequence
information from these CaMBPs to make knockout mutants in homothallic strains of
Dictyostelium to define which CaMBPs function during fertilization. Continued work should
permit us to understand the fundamental events of fertilization in a comparatively simple organism
that exhibits the fundamental communication and signal transduction events that occur during
fertilization in animals.
ACKNOWLEDGEMENTS
This work was funded by a Research Grant from the Natural Sciences and Engineering Research Council of Canada.
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Source : MNHN . Paris
Sperm and Spermiogenesis of the “Turbellaria”
and Implications for the Phylogeny
of the Phylum Platyhelminthes
Nikki A. WATSON & Klaus ROHDE
Department of Zoology,
University of New England, Armidale, N.S.W. 2351, Australia.
ABSTRACT
This chapter reviews recent ullrastructural investigations of sperm and spermiogenesis in turbellarian platyhelminths
(Catenulida, Nemcrtodermatida, Acoela. Macrostomida and Trepaxonemata including Polycladida, Lecithoepitheliata,
Prolecithophora, Proseriata, Tricladida, and Rhabdocoela, including Typhloplanida, Kalyptorhynchia, Dalyelliida,
Temnocephalida and Fecampiida). Some distinctive characteristics of the differentiating spermatid especially in the zone
of difterentiation may be useful for phylogenetic considerations. Spermiogenetic and mature sperm features including
axonemal or flagellar characteristics, dense bodies, nuclear components, mitochondria and microtubule arrangements are
documented for each of the major taxa. It is concluded that more investigations are needed and they must be comprehensive
to build a solid data base for a cladistic analysis of the phylum based on sperm and spermiogenetic characteristics.
RESUME
Spermatozoides et spermiogenese des “Turbellaria” et implications pour la phylogenie de
l’Embranchement des Plathelminthes
Les resultats recents sur les spermatozoides et la spermiogenese des Plathelminthes Turbellaries (Catenulida,
Nemertodermatida, Acoela, Macrostomida et Trepaxonemata y compris Polycladida, Lecithoepitheliata, Prolecithophora,
Proseriata, Tricladida et Rhabdocoela, y compris Typhloplanida, Kalyptorhynchia, Dalyelliida, Temnocephalida et
Fecampiida) sont synthetisds. Certaines caracteristiques distinctives de la spermatide en cours devolution, specialement
dans la zone de differenciation, peuvent etre utiles pour des considerations phylogdnetiques. Les caracteres suivants de la
spermiogenese et du spermatozoide sont ddcrits pour chacun des taxons majeurs: caractdristiques des axonemes et des
flagelles, corps denses, composants du noyau, mitochondries et arrangement des microtubules. La conclusion est que des
recherches suppldmentaires exhaustives sont necessaires pour construire une base de donnees destinee a une analyse
cladistique de I'embranchement basee sur les caracteres des spermatozoides et de la spermiogenese.
“Turbellaria” is used throughout this review as a convenient term for all the non¬
neodermatan platyhelminths, i.e. the groups other than Trematoda, Monogenea and Cestoda; it
does not imply monophyly. Early studies of spermatogenesis and spermatozoa in
Platyhelminthes, based on light microscopy (LM), contained only limited phylogenetic
information. Electron microscopy (EM) has proved of greater use. For a recent bibliography of
EM of turbellarians, see [74] (also [47, 51]). The last comprehensive review of turbellarian
Watson, N. A., & Rohde, K., 1995. — Sperm and spermiogenesis of the ‘Turbellaria" and implications for the
phylogeny of the Phylum Platyhelminthes. In: Jamieson, B. G. M, Ausio, J., & Justine, J.-L. (eds). Advances in
Spermatozoal Phylogeny and Taxonomy. Mem. Mus. nain. Hist, nat., 166 : 37-54. Paris ISBN : 2-85653-225-X.
38
N. A. WATSON & K. ROHDE : " TJRBELLAR1A " ( PLATYHELMINTHES )
spermiogenesis and spermatozoa was published by HENDELBERG in 1983 [25] with further
discussion of their phylogenetic significance in 1986 [26]. Turbellarian spermatozoa are
considered aberrant since they differ markedly from the supposedly primitive and modified forms
found in many other animal groups in association with the primitive mode of sperm transfer, viz.
external fertilisation [1, 20, 25, references therein]. They are generally elongate, without distinct
head, middle piece and tail, and may be without axonemes, have free flagella (usually two) or
axonemes incorporated in the sperm body. In this review we summarise the information currently
available from each of the major turbellarian taxa and discuss implications for the phytogeny of
the phylum. The classification follows CANNON, 1986 [10], except for the Fecampiida (removed
from Rhabdocoela on the basis of ultrastructural and DNA evidence [53]).
OBSERVATIONS
Within each taxon, data on sperm and spermiogenesis will be summarised with reference to
the following characteristics:
— Sperm: presence, number, location of axonemes or flagella; presence, arrangement of
longitudinal microtubules; nature of nucleus; nature, arrangement of mitochondria; presence,
nature of dense bodies.
— Spermiogenesis of flagellate or axonemal sperm: structures in the zone of differentiation
(microtubules, rootlets, intercentriolar body, dense plates); changes during elongation (movement
of basal bodies, rotation of flagella, fusion of flagella with shaft, etc).
Catenulida
SCHUCHERT & RIEGER [61] described spermiogenesis in Retronectes atypica by EM [see
also 50]. Ciliary rudiments are present during maturation of sperm (also in R. sterreri [15]),
associated with conspicuous lamellar bodies. Specialised cell protrusions, interpreted as
intercellular bridges, were seen during a restricted early period of spermatogenesis, and at a
slightly later stage groups of microvillar protrusions occur along the plasma membrane of the
spermatocyte. The spermatozoon has a more condensed nucleus and numerous lamellar bodies,
no longer with ciliary rudiments. There are frequent infoldings from the cell membrane and thin
branched mitochondria. There appear to be no dense bodies of any kind resembling those in other
turbellarian orders.
Nemertodermatida
Only mature sperm of two genera have been examined by EM, Meara and Nemertoderma
[24, 72, 73]. Although modified into an elongate form, the spermatozoa resemble the primitive
metazoan type with a distinct head containing the nucleus, middle region where a single axoneme
is surrounded by a coiled mitochondrial derivative, and tail where the axoneme extends as a free
flagellum. These attributes clearly distinguish Nemertodermatida from all other platyhelminth taxa
including the Acoela. The axoneme has the 9+2 microtubule arrangement as in normal body cilia
and in the axonemes of many acoel sperm.
Acoela
RAIKOVA & JUSTINE [48] recently summarised data on sperm ultrastructure of 30 species
from 1 1 families, including three investigated in that paper. There are two incorporated axonemes,
extending from the proximal end up to, and sometimes into the nuclear region, and a considerable
volume of cytoplasm. Sometimes each axoneme lies along the outer edge of a cytoplasmic
extension, forming an undulating membrane along each side of the sperm. There are possibly four
different arrangements of microtubules in the axonemes of acoel sperm: 9+2, 9+0, 9+1 and
9+‘T”. The most common configuration is 9+2/9+0, i.e. part of the axoneme has the normal 9+2,
with some region(s) lacking the central pair of microtubules (i.e. 9+0) (Fig. 1). Some species
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
39
appear to have 9+0 throughout the axonemes. In some species, an axonemal central element
consisting ol a hollow tube (microtubule?) surrounded by an electron-dense halo has been
descubed, denoted 9+1 [24, 57], It does not have the double helical arrangement found in the
dense central element of Trepaxonemata (see below). A dense central element possibly identical
with Trepaxonemata sperm axonemes (9+‘T”), was described in Childia groenlandica [24] and
illustrated in Mecynostomum auritum [5]; it is not known whether this element has a double
helical structure. Raikov A & JUSTINE [48] suggested that a misidentification cannot be excluded
lor the report of Mecynostomum ” with a 9+“l” structure. We have recently found a central dense
element with a hollow centre and without double helical structure in M. auritum (personal
observation). r
Individual mitochondria, membrane-bound electron dense bodies of one or two kinds, and
cortical and/or central microtubules are present in most species. In Amphiscolops [57] spermatids
have some peripheral but no internal microtubules, while in the maturing sperm only internal ones
were seen (Fig. 1). Raikova & JUSTINE [48] suggest that these two groups of microtubules may
be mutually exclusive, and that the internal ones derive from the external ones by an infolding of a
longitudinal groove. Several species have been described without any microtubule arrays in the
sperm. A fringe of terminal filaments has been observed by LM in two species [22, 27],
Spermiogenesis has been studied by EM in only a few acoel species [e.g. 6, 8, 48, 57],
ROHDE et al. [57] observed two centrioles, initially at right angles to each other in the early
spermatid. Two tree axonemes grow from the spermatid and fuse with the elongating shaft in a
distal to proximal direction. The basal body region thus becomes distal. Nucleus, dense bodies
and mitochondria migrate into the shaft, the elongated nucleus remaining in the proximal region.
1 here is no mtercentriolar body (see below) nor rootlets associated with flagellar basal bodies.
Macrostomida
Most species examined are from Macrostomidae, one from Dolichomacrostomidae
{Paramacrostomum tricladoides) [22] and two from Microstomidae ( Microstomum spp. [22]). No
appendages are reported in the sperm of the last two families, nor of some species of
Macrostomum [22] and Bradynectes sterreri [49] (both Macrostomidae). A pair of stiff bristles
has been described from several species of Macrostomum [e.g. 6, 44 55] and from
Promacrostomum gieysztori [6], They are anchored in tapering dense structures in the mid-region
of the sperm with a dense, banded region between the base of the rod and the anchor [55], In
cross section, bristles interpreted as modified flagella, consist of a large central dense rod
surrounded by irregular smaller dense rods [55] (Fig. 2). The proximal end of M. tubum sperm is
split into a fringe of microvijli [44, 55] and there are from 3-6 dense chromatin granules in a row
just behind this proximal region (Fig. 3). The remainder of the sperm body is rich in mitochondria
and elongate dense bodies with fewer, large dense bodies in close contact with the cell membrane.
Two symmetrical, contralateral rows of peripheral microtubules extend along most of the shaft
(Fig. 2), each bristle arising from the edge of one of these rows.
Spermatogenesis has been studied by EM only in M. tubum [55], Cells containing
numerous sections through chromatin were interpreted as spermatocytes. These gave rise, without
further division, to bristles and the lateral rows of microtubules, thus spermatozoa formed directly
from spermatocytes. The presence of up to six discrete chromatin granules in the sperm may
indicate belated nuclear division or nuclear fragmentation.
Trepaxonemata
The remaining platyhelminth taxa form a monophylum, distinguished by an autapomorphic
feature of the sperm axonemes (when present). Instead of the 9+2 arrangement of microtubules
found in primitive sperm, the 9 pairs of peripheral microtubules surround a unique central
complex (Fig. 6), made up of an electron dense core, a lighter zone around the core and an outer
40
N. A. WATSON & K. ROHDE : "TUKBELLARIA " ( PLATYHELM1NTHES )
electron dense zone which, in longitudinal sections, has a double helical structure (Fig. 4). This is
known as the 9+‘T’ arrangement and is unique to this monophylum of the platyhelminths. Many
of these taxa exhibit common structures and events during spermiogenesis, resulting in
similarities in mature sperm. We will thus describe these basic events, present individual taxa data
in tabulated form (Table 1), and note variations or noteworthy aspects separately in the text.
General description of spermiogenesis and spermatozoa in Trepaxonemata. Following
spermatogonial and spermatocyte divisions accompanied by incomplete cytokinesis, spermatids
usually remain joined in clusters by cytoplasmic bridges or cytophores. The spermatid nucleus
moves towards the apical plasma membrane, initially with scattered clumps of chromatin that then
become more dispersed and filamentous. The apical region becomes a zone of differentiation (ZD)
in which two centrioles develop into the basal bodies of free flagella growing out in opposite
directions from one another. A banded structure known as an intercentriolar body (ICB) develops
which, when seen between the basal bodies, usually consists of a dense central band with a series
of lighter, thinner bands on either side (e.g. Fig. 5). In cross section, the plates are circular, i.e.
the structure in three dimensions is a cylinder with discs of various densities. Rootlets, either well
developed and sometimes multipartite, or small and indistinct, usually attach to the sides of the
basal bodies and extend in the direction of the apex of the nucleus or along the sides of it.
Microtubules line the plasma membrane in the ZD. An apical projection, often with electron dense
material within it, develops distal to the basal bodies. The main spermatid body elongates,
carrying the distal projection and flagella distally (except in Kronborgia and Neodermata - see
below). The nucleus, with increasingly condensed chromatin, moves into the shaft, along with
mitochondria or a single fused mitochondrion, and dense bodies of one or more kinds. Flagella
may bend proximally to lie alongside or fuse with the spermatid shaft. In some taxa the flagella
also rotate around the shaft so that they emerge adjacent to each other. Aflagellate sperm develop
by elongation of the spermatid, lined by microtubules. The nucleus elongates, chromatin
condenses and dense bodies and mitochondria become distributed within the shaft.
Table 1. — Characteristics of sperm and spermiogenesis of Trepaxonemata (minus Neodermata)
ICB: intercentriolar body; 1 = present; 0 = absent.
R: rootlets or striated structures attached to the proximal sides of basal bodies; 1 = present (D divided); 0 = absent.
DP: dense plates located on the distal sides of basal bodies (may not be homologous in triclads and proseriates;
1 = present; 0 = absent.
ST: axoneme insertion; 1 = axoneme insertion becomes sub-terminal as a result of formation of a distal process,
± = slightly sub-terminal, 0 = no distal process, 1-0 = distal process present but disappears.
BBD: Basal body; 1 = basal body region moves distal during spermiogenesis; 0 = remains proximal.
AN: axoneme number; 2-1 = initially 2 but only one in mature sperm.
AS: axonemes state; F = free, A = adhering; I = incorporated in shaft, I-F = most of axoneme incorporated, short free
region.
AP: axonemal placement; A = adjacent to one another; O = on opposite sides of the shaft to one another.
SF: split tips of flagella; 1 = present.
M: mitochondria; M = multiple; R = in rows; D = altered, membranous derivative; F = fused into 1, 2 or 3+ long
mitochondria.
NN: nature of nucleus; L = lobed; 2 = 2 components coiled around one another; Rods = dense chromatin rods;
E = membranous elaborations; Env = nucleus partially envelops a string of mitochondria.
DB: dense bodies present in main shaft; 1 = present; 0 = absent; 2 = 2 kinds noted.
PM: peripheral microtubules; 1 = present, ISp = full peripheral row plus inner spiral group (see text); + = some
additional internal microtubules present in some parts; few = widely spaced; inc = incomplete outer row where
axoneme(s) are adjacent to the plasma membrane and/or remain outside the ring of microtubules.
G: granules (approximately 25 nm diameter) in conspicuous longitudinal rows beneath the cortical microtubules of the
shaft.
Wherever two or more states are separated by a stroke (/), different states were found in different species.
- = structure not present because sperm are aflagellate.
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
41
Characteristics of spermiogenesis Characteristics of spermatozoon
Source : MNHN , Paris
42
N. A. WATSON & K. ROHDE : "TURBELLARtA " ( PLATYHELMINTHES )
Polycladida
There are many LM studies of Polycladida [e.g. 21, 22], but only one EM study of
spermiogenesis ( Notoplana japonica ) [38] and few EMgraphs of sperm [e.g. 27]. Flagella remain
free in Cotylea but superficially united with the shaft in Acotylea, sometimes pail of an undulating
membrane but never wholly incorporated beneath the plasma membrane. Flagella arise from and
remain on opposite sides of the shaft very close to the distal tip of the spermatozoon [22], There
are multiple dense bodies [e.g. 27] and multiple mitochondria, some apparently fused into larger
aggregates [38]. Longitudinal microtubules lie in a complete ring beneath the plasma membrane,
apparently for the whole length of the spermatozoon.
Spermiogenesis in Notoplana japonica [38] is basically as described above for the
Trepaxonemata. KUBO-IRIE & ISHIKAWA [38] described additional proximal centrioles attached at
right angles to the main ones during spermiogenesis, but the micrographs are not clear. There has
been no other report of four centrioles in the ZD of a platyhelminth spermatid.
Lecithoepitheliata
There is one EM study of sperm and spermiogenesis of Prorhynchus sp. [79] (Table 1).
Sperm have an irregular contour, and flagella lie in grooves of wide lateral extensions of the shaft
(Fig. 13). The testes of some Baikal endemic Geocentrophora species ( G . inter sticialis, G. wagini
and G. wasiliewi) were examined [7] and sperm found to have two free flagella with 9+“l”
axonemes, longitudinal microtubules and 1-5 longitudinal folds. No members of Gnosonesimidae
have been investigated.
Prolecithophora
Mature sperm have been examined in several species [e. g. 11, 14, 16, 22, 43, 60] and
spermatid development in Multipeniata [60]. Sperm are aflagellate, have a row of cortical
microtubules, lack dense bodies (except for Multipeniata) and have a complex intraspermial
membranous system (mitochondrial derivative?) closely associated with the lobed nucleus in a
spiralling arrangement in some species. Sperm of Urastoma cyprinae do not resemble those of the
other prolecithophorans examined [46], There are two initially free axonemes that become
completely incorporated in the mature sperm body, peripheral microtubules that are crowded into
the centre of the shaft at one end, at least two mitochondria of regular appearance, no dense
bodies and no membranous system. It is possible that Urastoma may not belong to the
Prolecithophora, as suggested by NOURY-SRAIRI et al [46],
Proseriata
There are several reports and a few micrographs of some aspects of sperm [22, 25, 65, 69]
and three reports of some aspects of spermiogenesis from three of the seven families [65-67, 69].
Sperm have two free, sub-terminally inserted axonemes, peripheral microtubules, an elongate
nucleus, numerous dense bodies, and mitochondria arranged in a tightly packed longitudinal row
or rows (Fig. 4). During spermiogenesis, a well developed ICB lies between the basal bodies,
and rootlets (sometimes multipartite) extend from the basal bodies towards and alongside the
nucleus. In three species of Parotoplaninae (Otoplanidae), a distinctive striated distal appendage to
the ICB forms during cell elongation. SOPOTT-EHLERS [67] suggested the structure to be an
autapomorphy of Parotoplaninae. Changes in the ICB were also documented in a nematoplanid
[66] and a coelogynoporid [65, 69], involving considerable stretching and partial splitting in the
first, and transformation of some elements and partial splitting in the second.
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ADVANCES IN SPERMATOZOA!, PHYLOGENY AND TAXONOMY
43
Tricladida
Species from all three sub-taxa (Paludicola, Maricola and Terricola) have been examined by
EM [17-19, 28, 30, 31, 37, 40, 59, 62, 63] and some common characteristics identified [59]. All
have two tree, sub-terminally inserted flagella which arise together on one side of the shaft, a
distal projection beyond the flagellar insertion, and nucleus and a single fused mitochondrion
(Fig. 8) extending throughout most of the remaining length of the sperm (in contrast to many
other turbellarians, in which the nucleus is restricted to a shorter proportion of the sperm body).
The nucleus has two distinct components, one (the protein component [62]) less dense than the
other, coiled around each other in screw-like fashion (Figs 6, 8). In at least some species, nucleus
and elongate mitochondrion also coil around each other. Flagella of mature sperm contain many
small granules and the tips split into smaller units (Fig. 6) (Paludicola, Terricola). Mature sperm
have a short inner row of microtubules in addition to the full peripheral row (Paludicola,
Maricola) and lack the numerous dense bodies characteristic of many turbellarians (Paludicola,
Maricola, Terricola). During spermiogenesis, the nucleus has a dense apical layer, there are short
cross-striated rootlets from the basal bodies to the nucleus, and dense, half-moon shaped plates
around the basal bodies opposite the rootlets (Fig. 5) (Paludicola, Maricola, Terricola). In at least
two paludicolans and one maricolan the ICB splits into two halves attached to the basal bodies
during rotation [18, 59].
Rhabdocoela
Typhloplanida. There are LM reports of sperm with and without free flagella [e. g. 22], but
only detailed EM reports of three species, Bothromesostoma personation [13] and Phaenocora
anomalocoela [80], both Typhloplanidae, and Anthopharynx sacculipenis, Solenopharyngidae
[68], B. personation sperm have two free, subterminally inserted flagella emerging together on
the same side of the shaft, dense bodies, mitochondria and a complete ring of cortical
microtubules. During spermiogenesis, dense heels form at the ends of the basal bodies which then
rotate around the shaft, resulting in compression of one semicircle of microtubules into a tight
double row. P. anomalocoela sperm (Fig. 12) have two fully incorporated axonemes of unequal
length, numerous mitochondria and dense bodies. Basal bodies are close together and axonemes
also remain adjacent for their entire length, situated between the plasma membrane and the row of
microtubules. The nucleus extends almost the entire length of the sperm. During spermiogenesis,
dense heels form at the ends of basal bodies, and massive rootlets are attached to the sides of
basal bodies as they rotate around the shaft, compressing some of the peripheral microtubules into
a central double row. The manner in which the compressed row rejoins the outer microtubules
does not result in a spiral formation (cf. Temnocephalida below). A. sacculipenis sperm are
filiform, totally enclosed by cortical microtubules, possess two free flagella, one or two rows of
tightly packed mitochondria, and have regular rows of granules beneath the plasma membrane.
There are numerous dense bodies along the shaft and terraced elaborations of the nuclear
membrane. Similarly, two free flagella, regular rows of granules, dense bodies and a tightly
packed row of mitochondria are present in the sperm of Maehrenthalia sp. (Byrsophlebidae) [75].
Typhloplana virida has two free, 9+“l” flagella [42]
Kalyptorhynchia. By LM and EM, no sperm have been found with free flagella [22, 25,
39],
— Eukalyptorhynchia. EMgraphs of sperm of Gyratrix sp. (Polycystidae) and
Odontorhynchus sp. (Fig. 14) (Gnathorhynchidae) [52] and Polycystis naegelii (Polycystidae)
[39] show dense bodies, probably of two kinds, an elongate mitochondrion, peripheral
microtubules and two fully incorporated axonemes. In P. naegelii the nucleus contains a number
of dense bodies (long rods or short rounded structures?). Basal bodies are staggered and slightly
44
N. A. WATSON & K. ROHDE : "TURBELLAR1A " (PLATYHELMINTHES)
subterminal. L'HARDY [39] also studied spermiogenesis in Polycystis naegelii - initially free
flagella are incorporated in a distal to proximal manner.
— Schizorhynchia. Baltoplana magna sperm have a single incorporated axoneme [23, 25].
WATSON & L'HARDY [76] found two basal bodies in the ZD, with an ICB between them. Only
one develops into a normal flagellum - the other remains as a short bud. Both are carried distally
by the elongating shaft and one is incorporated into the shaft in a distal to proximal direction while
the spermatid is still pyriform. The other degenerates and mature sperm have only a single
axoneme, from one end to almost the other end. The shaft is surrounded by cortical microtubules,
there is an elongate mitochondrion, no dense bodies and the nucleus has an unusual arrangement
of long dense chromatin rods. Preliminary observations by SCHOCKAERT (personal
communication) suggest that several schizorhynch families may contain taxa with monoaxonemal
sperm.
Dalyelliida. This diverse group contains free living, symbiotic, and parasitic species, but
there are few reports of detailed EM investigations.
— Dalyelliidae. Two micrographs of sections of Microdalyellia sperm [27] and a full study
of spermiogenesis and sperm of Gieysztoria sp. [82] show two free axonemes, multiple
mitochondria, dense bodies (2 kinds in Gieysztoria ), and in Gieysztoria the nucleus almost
completely envelops a string of mitochondria along the shaft. During spermiogenesis in
Gieysztoria , an ICB lies between the basal bodies, dense heels develop at the bases of basal
bodies, and flagella rotate around the shaft to lie adjacent to one another. Rotation causes
compression of one semicircular row of peripheral microtubules, so that they lie in a double row
in the middle of the spermatid, in the region of the basal bodies. The manner in which the
compressed row rejoins the outer row does not result in a spiral formation (cf. Temnocephalida).
Figs 1-14. — Spermatozoa of "Turbellarian” Platyhelminthes. 1: Transverse section of sperm of Amphiscolops sp.
(Acoela). Note incorporated axonemes (arrows) without central element, two sizes of dense bodies (D), central array
of microtubules (arrowheads). Bar = 200 nm. 2: Transverse section of sperm of Macrostomum lubum
(Macrostomida). Note numerous dense bodies (D), mitochondria (M), bristles (B) and contralateral rows of
peripheral microtubules. Bar = 200 nm. 3: Longitudinal section of sperm of Macrostomum tubum
(Macrostomida). Note terminal fringe of filaments (F) and separate nuclei or nuclear fragments (N). Bar = 500 nm.
4: Longitudinal section of sperm of Monocelis sp.(Proseriata). Note nucleus (N), dense bodies (D), row of
mitochondria (M) and flagella (F) with double-helical central element. Bar = 200 nm. 5: Zone of differentiation
of Romankenkius libidinosus (Tricladida). Note intercentriolar body (1), rootlet (R), flagellum (F) and dense plates
(arrowheads). Bar = 200 nm. 6: Transverse section of sperm of R. libidinosus (Tricladida). Note nucleus (N) with
dark and lighter (p) components, some additional internal microtubules (arrowhead), free flagellum (F), split tips of
flagellum (double arrowheads) and dense granules between spokes of flagellum (arrow). Bar = 200 nm.
7: Transverse section through sperm of Kronborgia isopodicola (Fecampiida). Note nucleus (N), mitochondria
(M), and incorporated axonemes with hollow central elements (arrowheads). Bar= 200 nm. 8: Longitudinal
section of sperm of R. libidinosus (Tricladida). Note single long mitochondrion (M), nucleus with spiralling
lighter component (p), and flagellum (F) with dense granules (arrowhead). Bar= 200 nm. 9: Zone of
differentiation of Decadidymus gulosus (Temnocephalida). Note intercentriolar body (I) beginning to split, and
dense heels (arrowheads) at the ends of the basal bodies of flagella (F). Bar= 500 nm. 10: Spermatid of
Decadidymus gulosus (Temnocephalida) after rotation of flagella. Note inner compressed double row of
microtubules (arrowhead) and spur on the basal body (arrow) of a flagellum (F). Bar= 500 nm. 11: Cross
sections through sperm of Craspedella spenceri (Temnocephalida). Note spiral microtubule arrangement
(arrowhead) and filaments (F) resulting from splitting of the proximal end of the sperm. Bar = 200 nm.
12: Cross sections through sperm of Phaenocora anotnalocoela (Typhloplanida). Note nucleus (N), dense bodies
(D) and one or two axonemes (arrowheads) incorporated just beneath the plasma membrane. Bar= 500 nm.
13: Transverse section of sperm of Prorhynchus sp. (Lecithocpitheliata). Note nucleus (N) and flagella (F) lying
in grooves of the shaft. Bar = 500 nm. 14: Transverse section through sperm of Odoniorhynclius sp.
(Kalyptorhynchia). Note nucleus (N), dense body (D), single mitochondrion (M) and incorporated axonemes
(arrowheads). Bar = 500 nm.
Source :
ADVANCES IN SPERMATOZOA!, PHYLOGENY AND TAXONOMY
45
Source :
46
N. A. WATSON & K. ROHDE : "TURBELLARIA ” ( PLATYHELMINTHES )
— Provorticidae. Provortex balticus has elongate sperm, without flagella [22].
— Graffillidae. The sperm of the parasitic species Paravortex cardii, P. karlingi, P. tapetis
and Graffilla buccinicola are aflagellate [12, 22, 45] while free-living Pseudograffilla arenicola
has two free, sub-terminally inserted flagella [22], In the three Paravortex species, regular
longitudinal rows of small dense granules extend throughout the sperm, beneath the peripheral
microtubules. They resemble regular rows found in temnocephalans and do not react positively to
the Thiery test for glycogen [45], Similar rows of granules (also negative to the Thiery test) arc
present in the sperm of Bresslauilla relicta [75] which have two superficially attached 9+“l”
axonemes, two kinds of dense bodies (one occurring outside of the cortical microtubules) and a
single row of mitochondria. NOURY SRAIRI et al. also found small numbers of membrane-bound
dense bodies in P. cardii and P. tapetis [45].
— Luridae [70]. In Luriculus australiensis [58] microtubules in a helical configuration
surround the spermatid, and progressive elongation results in formation of an aflagellate, filiform
spermatozoon, containing regular rows of dense bodies, mitochondria and a ring of a small
number of longitudinal peripheral microtubules, interrupted where organelles are close to the
surface.
— Umagillidae. From LM, Syndesmis echinorum sperm are reported to be aflagellate [22],
Cleistogamia longicirrus and Seritia stichopi sperm (EM) [56] have two free flagella, numerous
dense bodies, multiple mitochondria and peripheral microtubules. Syndisyrinx punicea has similar
sperm (and split flagellar tips) [54], an ICB is present during spermiogenesis, and although dense
floccular material surrounds the basal bodies, no rootlets were seen [41],
Pterastericolidae. Sperm of 5 species of Pterastericola and spermiogenesis of one of
these (P. astropectinis) have been examined by EM [33, 83], A faint ICB is present but apparently
no rootlets. Basal bodies and free flagella are carried distally at the end of the elongating
spermatid, before fusing with the shaft for most or all of their length. There are a few dense
bodies, a small number of proximally positioned, elongate mitochondria and a row of peripheral
microtubules which is discontinuous where one or sometimes both axonemes lie close to the
surface. Regular centrioles composed of triplets were seen in mature sperm.
Temnocephalida. Sperm and spermiogenesis have been studied in one species from each of
Actinodactylellidae, Scutariellidae and Didymorchidae and at least 12 from Temnocephalidae.
Temnocephalidae. There are more or less comprehensive reports of sperm and
spermiogenesis in seven species of Temnocephala, three of Craspedella, and Decadidvmus
gulosus and Diceratocephala boschmai [36, 82, 84-87]: they are compared by WATSON & ROHDE
[82]. They have in common the formation of dense heels on the basal bodies (Fig 9) and
compression of a semicircle of microtubules by rotation of the basal bodies around the shaft as
described above for Gieysztoria. However, the two edges of the compressed row rejoin the
uncompressed row in an unequal manner, resulting in a region of the spermatid and mature sperm
in which the peripheral microtubules are arranged spirally (Fig. 1 1). In addition, proximal ends of
mature sperm have either a terminal flange of microtubules within the plasma membrane or are
sp it into a fringe of fine filaments containing microtubules (Fig. 1 1). It is likely that flange and
split shaft represent modifications of the one feature, since both structures occur in the one genus
Temnocephala ) [84] and there is a tendency for the flange in T. dendyi to break up into filaments
(personal obseiwation). Membrane-bound dense bodies and numerous smaller granules, in regular
rows beneath the microtubules, are present in the sperm of most of the species examined. A dense
body is usually located very close to the end microtubule of the inner spiral row.
— Actinodactylellidae. Spermiogenesis in Actinodactylella blanchardi [82] involves
formation ol an ICB, rootlets and a similar dense heel on the basal bodies, flagellar rotation and
compression of one semicircular row of microtubules and a spiral region of microtubules where
the compressed region rejoins the other microtubules. The proximal end of the mature sperm is
split into a fringe of filaments, containing microtubule(s).
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ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY
47
Scutariellidae. Sperm of Troglocaridicola sp. have a similar proximal fringe of filaments
and dense heel formation at the ends of basal bodies during spermiogenesis [29] and compression
of a semicircular row of microtubules by rotation of the basal bodies. However, expansion of this
row apparently does not result in a spiral arrangement. Mature sperm have regularly arranged
longitudinal rows of small granules beneath the peripheral microtubules, similar to those found in
Paravortex (Graffillidae) and Temnocephalidae, but no membrane-bound dense bodies. The
distal, anterior region comprises an electron dense, not membrane-bound corkscrew originating
from the apical process of the spermatid. This structure appears to be unique among the free-
living platyhelminths but resembles terminal structures found in many cestodes [3] and the
monogenean Calicotyle [71, 81],
Didymorchidae [32]. Only Didymorchis sp. (from Cherax destructor) has been
examined [82], Sperm and spermiogenesis are as in Actinodactylellcv, the nucleus in the distal
region also has a thin trailer which extends to the plasma membrane (also present in
Temnocephala minor [84]).
Fecampiidci. Kronborgia isopodicola is the only species in which sperm or spermiogenesis
has been examined [77, 78, 88]. Sperm have two fully incorporated axonemes of unequal length,
longitudinal, peripheral microtubules, no dense bodies and a small number of elongate
mitochondria (Fig. 7). Spermiogenesis [78] involves formation of two free axonemes,
incorporated into the spermatid in a proximal to distal manner. There is no ICB nor are there
flagellar rootlets, but many microtubules attached to the basal bodies probably effect rotation of
the basal bodies to lie parallel from their initial orientation at right angles to each other. Both ends
of the sperm taper markedly, the proximal (anterior) end containing a dense rod which becomes
horseshoe shaped in cross section and in which the ends of the longitudinal microtubules are
embedded. The distal (posterior) end beyond the nucleus consists of a long narrow filament
containing a few, and eventually no microtubules.
Phylogenetic implications
The validity of comparisons of sperm and spermiogenesis of different taxa depends on
quality of fixation and “completeness” of the investigations. Concerning fixation, clarity of most
cellular components differs in different studies even if specimens are processed similarly. For
example, dense bodies sometimes appear very dense and/or membrane-bound and sometimes
very indistinct. Reasons could be real differences in composition, fixation artefacts or changes
with development. Smaller, not membrane-bound granules are sometimes noted in the cytoplasm
of the sperm body (Table 1) arranged in very regular rows, best seen in tangential longitudinal
section. Their absence in other groups could be of phylogenetic significance or a fixation artefact.
Concerning “completeness” of investigation, many studies were carried out to answer
particular questions, such as whether sperm are biaxonemal and have the 9+“l” arrangement.
Such brief investigations allowed some important generalisations leading to the establishment of
the taxon Trepaxonemata, the distinction between Cotylea and Acotylea sperm, and recognition of
the complete incorporation of axonemes in kalyptorhynchs (indicating monophyly). Some studies
are less complete than others because few cells of a particular stage were available or because
serial sectioning was not used. Thus it is unclear whether certain structures such as rootlets, ICB,
spiral of microtubules etc. were overlooked or are indeed absent. When comparing the
morphology of the nucleus, for example, degree of maturation of the spermatid is crucial and
sperm should be examined from the seminal vesicle or sperm ducts as well as from the testis.
Considering all this, we must conclude that only a small number of species has been
examined in appropriate depth for detailed phylogenetic considerations. Suitable data can be used
in two ways for such considerations - to examine the monophyly of established taxa, and to look
for evidence of relationships between such taxa. As a first step, autapomorphic feature states need
to be identified (refer to Table 1).
48
N. A. WATSON & K. ROHDE : “TUKBELLARIA ” (PLATYHELMINTHES)
1. Flagella: presence, kind, number and location. The primitive metazoan sperm is
considered to be monoflagellate [20], while most platyhelminths possess two flagella or
incorporated axonemes. The biaxonemal condition could be plesiomorphic for the whole phylum
(lost in Catenulida and Nemertodermatida, highly modified in Macrostomida) or independently
acquired in Acoela and Trepaxonemata (or in Acoela and Rhabditophora if the bristles of
macrostomids are modified flagella). Absence of flagella, reduction to a single flagellum, and the
state of incorporated axoneme(s) are undoubtedly apomorphic states in some taxa, and the unique
central element is a synapomorphy for Trepaxonemata (unless the central element in some acoels
also has the same structure, but see earlier note regarding possible inisidentification). All Acoela,
Kalyptorhynchia and Neodermata have axonemes incorporated in the sperm body, all acotylean
polyclads have axonemes superficially attached to the sperm body, and all Prolecithophora (except
Urastoma) lack axonemes. Paired incorporated axonemes in Acoela are not associated with the
presence of an ICB as they are in Kalyptorhynchia and Neodermata, and are therefore likely to be
autapomorphic. Incorporated axonemes are also found sporadically in other taxa (eg.
Pterastericolidae, Phaenocora in Typhloplanidae, Bresslauilla relicta in Graffillidae), where they
represent a derived condition relative to other species in the taxon. The same applies to aflagellate
sperm. Thus, all Catenulida and Prolecithophora (except Urastoma) lack flagella, but so also do
some species of rhadocoels, e.g. the only species of Luridae and Provorticidae examined, four of
the six species of Graffillidae examined, and one species of Trigonostomidae. With so few taxa
examined in detail, we cannot imply relationships between higher taxa on the basis of presence,
absence, incorporation or not of flagella, especially since loss of structures is common in
evolution and these traits may be more closely linked to reproductive biology than to ancestry.
Table 1 shows that flagella arise on opposite sides of the shaft in most taxa but adjacent to each
other in Tricladida, Dalyelliidae, Temnocephalida and Typhloplanidae. In the last three the
condition is likely to be synapomorphic, since it is associated with a unique mode of rotation of
the flagella during spermiogenesis; it may be of independent origin in Tricladida because different
structures are present in the ZD.
In at least four species, all symbiotic, Syndisyrinx punicea, Kronborgia isopodicola,
Pterastericola asamushii and the monogenean Anoplodiscus cirrusspiralis, the central element of
axonemes appears hollow in cross section instead of solid as in other trepaxonematan taxa,
although it does have the double helical arrangement in longitudinal section. The phylogenetic
significance of this difference is unknown.
2. Mitochondrion/mitochondria. Within the phylum a wide spectrum of mitochondrial
formations is present ranging from numerous small mitochondria, through regular, tightly packed
rows of individual mitochondria, to varying degrees of fusion and formation of mitochondrial
rods and derivatives. The fused forms derive from many small mitochondria during
spermiogenesis. Acoela all retain scattered individual mitochondria, triclads and kalyptorhynchs
appear to have a single rod-like mitochondrion, prolecithophorans (except Urastoma) have a
complex membranous derivative, at least two families of Proseriata have regular rows of tightly
packed mitochondria, whereas within the Dalyelliida and Temnocephalida a range of states
occurs. Presence of small and of fused mitochondria can be demonstrated, but it is very difficult
to prove that only a single mitochondrial rod exists because of the considerable length of many
sperm. It is likely that fused mitochondria have arisen many times independently. However, if it
can be shown that all species in the taxon exhibit the derived state (such as in Prolecithophora
(except Urastoma which may not be a prolecithophoran), Neodermata, Tricladida) then it is likely
to be an autapomorphy for that taxon, especially if there are other sperm or spermiogenetic
synapomorphies for the group.
, • ol|ler characters of sperm and spermiogenesis, a number of apomorphic states can
be identified and used for phylogenetic deliberations. These are as follows:
-r • i 4. . ^T1?e, 9+‘T ?xoneme in Trepaxonemata (Polycladida, Lecithoepitheliata, Proseriata,
lncladida, Typhloplamda, Kalyptorhynchia, Dalyelliida, Temnocephalida and Neodermata).
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
49
Prolecithophora are aflagellate (except Urastoma), therefore indeterminate on this basis.
MacroMomids are aflagellate or have a pair of stiff bristles, perhaps modified flagella, but there is
no 9+“l” structure to suggest inclusion in this taxon, although secondary modification cannot be
excluded. Some acoel species have a solid central element but its homology with 9+“l” has not
been established;
b. The ICB is present in most species of Trepaxonemata possessing axonemes
(notably absent in Kronborgia and monopisthocotylean monogeneans [35]). It does, however,
appear to vary in composition and in the changes which it undergoes during spermiogenesis in
different taxa, and these variations may be autapomorphic for smaller taxa;
c. Helical arrangement of two distinctive components of the nuclei of triclads;
d. Lobed nucleus of prolecithophorans;
e. Spiral arrangement of microtubules in the region of sperm adjacent to the flagellar
insertion in a taxon consisting of Temnocephalida minus Scutariellidae;
f. A proximal terminal flange or fringe of microvilli-like processes on mature sperm of
Temnocephalida. A terminal fringe is also present in some Macrostomum sp. and two acoels, but
clear differences in other sperm characters suggest independent origin in these groups;
g. A suite of characteristics seen during spermiogenesis in Temnocephalida,
Dalyelliidae, Byrsophlebidae and Typhloplanidae (dense heel formation at the end of basal bodies,
rotation of flagella around the shaft resulting in compression of a semicircle of microtubules,
formation of a spur on the basal bodies in mature sperm). In Decadidymus gulosus
(Temnocephalidae) the ICB splits completely and one half accompanies each basal body during
rotation - it is likely that the same occurs in the other taxa. This combination of characteristics
suggests a common ancestor for these groups. It is not present in Pterastericolidae and
Umagillidae where it may have been secondarily lost or the absence could indicate that these
families are misplaced. Some dalyelliid families have species with aflagellate sperm, hence no
ICB etc., some have not been studied, and no other typhloplanid families have been examined. It
is therefore premature to draw phylogenetic conclusions, but it does appear that detailed studies of
events in the ZD of many more species could help to clarify relationships within Rhabdocoela
(minus Neodermata). The ICB also splits into two in some triclad species, but different accessory
structures are involved;
h. The bristles, multiple nuclei and unusual spermiogenesis of Macrostomum have not
been seen in any other platyhelminth taxa and also differ significantly from the primitive sperm
and spermiogenesis models of many Metazoa: they must be seen as apomorphies.
The foregoing discussion indicates that sperm and spermiogenetic characters support the
monophyly of each of the Acoela, Polycladida Acotylea, Prolecithophora (except Urastoma),
Tricladida, Temnocephalida minus Scutariellidae, Kalyptorhynchia, Neodermata. They also
provide significant evidence to suggest that some taxa may be misplaced, e.g. Urastoma may not
be a prolecithophoran and Kronborgia is not a rhabdocoel.
Regarding relationships between higher taxa, an area of great interest concerns the origin of
the Neodermata, without doubt a monophylum as shown by a range of characteristics including
the following: sperm with completely incorporated axonemes (or single axoneme or none by
secondary reduction), a single elongate mitochondrion (or none by reduction), no dense bodies,
and incorporation of axonemes in the proximal to distal direction [4, 35]. Suggested sister group
relationships to Neodermata have been proposed on the basis of the common occurrence of one or
more of these character states in some turbellarian groups (in conjunction with other characters not
related to sperm and spermiogenesis), e.g. with Fecampiidae [77] and with a clade comprising
Pterastericolidae, Fecampiidae and Acholadidae [34]. However, the first has not been supported
by DNA studies and at least the inclusion of Pterastericolidae in the second is not supported by the
later finding of dense bodies and distal to proximal fusion in spermiogenesis of Pterastericolidae
[83], There is also no evidence from sperm or spermiogenesis in support of a close relationship of
Temnocephalida and Neodermata as suggested by BROOKS [9], or of “Dalyellioida” and
50
N. A. WATSON & K. ROHDE : " TURBELLARIA " (PLATYHELMINTHES)
Neodermata suggested by EHLERS [15]. The sister group of the Neodermata remains
undetermined.
The question of the relationship between Catenulida, Acoelomorpha and Rhabditophora has
been addressed by several authors [e.g. 2, 15, 64 and references in these] but recent studies of
sperm and spermiogenesis only confirm the divisions, without shedding light on their
interrelationships. Spermatogenesis and spermatozoa in the Catenulida are unlike those observed
in any other platyhelminth group. If the monoflagellate condition of Nemertodermatida is
plesiomorphic (i.e. if the last common ancestor of the Acoelomorpha was monoflagellate with a
9+2 flagellum), then presence of two flagella in Acoela and the remainder of the platyhelminths is
likely to be of independent origin. However, if a second axoneme has been lost in
Nemertodermatida, the biflagellate condition need only have arisen once in the stem species of
Acoelomorpha and Rhabditophora. Some sperm with two axonemes have been found in
Nemertoderma sp. A [73] but spermiogenesis has not been studied.
The taxa commonly known as rhabdocoels (Typhloplanida, Kalyptorhynchia, Dalyelliida
and Temnocephalida) appear from DNA and protonephridial ultrastructure to constitute a
monophylum, but relationships between the orders and placement of some families within orders
are uncertain. JONDELIUS & THOLLESSON [34] used parsimony analysis of 21 LM and EM
characteristics to derive a working hypothesis for the Rhabdocoela, but the homology of many of
the characters utilised is questionable. Sperm and spermiogenesis studies may prove useful
because certain taxa within the group have very distinctive structures and processes.
In conclusion, sperm and spermiogenesis data have the potential to contribute significantly
to phylogenetic analysis within the turbellarians, but many more subordinate taxa must be
examined for a parsimony analysis.
ACKNOWLEDGEMENTS
Support for the authors’ own investigations reported in this review was provided by the Australian Research
Council and the University of New England, Armidale. Ulf Jondelius, Swedish Museum of Natural History, provided
specimens of Mecynostomum auritum.
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Source : MNHN , Paris
Spermatozoal Ultrastructure and Phylogeny in the
Parasitic Platyhelminthes
Jean- Lou JUSTINE
Laboratoire de Biologic Parasitaire, Protistologie, Helminthologie,
Museum National d’Histoire Naturelle, 61 rue Buffon, F-75231 Paris cedex 05, France
ABSTRACT
The ultrastructure of spermiogenesis and spermatozoon is briefly described and illustrated in fifteen species: nine
Digenea, Prosorchis ghanensis , Apatemon graciliformis , Spirorchis sp., Aphalloides coelomicola , Proctoeces maculatus ,
Aporocotyle spinosicanalis , Schistosoma sp., Haematoloechus sp. and Echinostoma togoensis , two Monogenea
Polyopisthocotylea, Heteraxinoides sp. and Diplozoon gracile , two Monogenea Monopisthocotylea, Cleitharcticus sp.
and Furnestinia echeneis, and two Euccstoda, Moniezia sp. and Echeneihothrium sp. Most of these species have their
spermatozoon described for the first time. The literature shows that sperm ultrastructure is now known in about 140 genera
(almost 200 species) of parasitic Platyhelminthes (= Aspidogastrea, Digenea, Monogenea, Amphilinidea, Gyrocotylidea,
and Eucestoda). The general structure (two 9+“!” axonemes, microtubules only dorsal and ventral in the principal region of
the spermatozoon) originates from a process of proximo-distal fusion and is found in the Aspidogastrea, Digenea,
Gyrocotylidea and Amphilinidea, with, however, a few variations in the first two groups. The Monogenea
Polyopisthocotylea are differentiated by the presence of additional lateral microtubules. The Monogenea
Monopisthocotylea are characterised by the loss of certain structures and a general trend towards a simpler spermatozoon.
The Eucestoda are characterized by the loss of the mitochondrion and show some other deviations. The genus
Schistosoma , belonging to the Digenea, has a spermatozoal structure completely different from the general structure found
in the two supposedly closely related families of blood-flukes, the Spirorchidae and Sanguinicolidae, and the
spermatozoon is considered “pr°genelic”- A precise identification of the outgroup for the parasitic Platyhelminthes is
needed for a more reliable cladistic analysis of spermatozoal characters.
RESUME
Ultrastructure des spermatozoides et phylogenie des Plathelminthes parasites
L’ ultrastructure du spermatozoide et de la spermiogenfcse est decrite brievement et illustr6e chez quinze especes: neuf
Digenea, Prosorchis ghanensis, Apatemon graciliformis, Spirorchis sp., Aphalloides coelomicola, Proctoeces maculatus,
Aporocotyle spinosicanalis, Schistosoma sp., Haematoloechus sp. et Echinostoma togoensis, deux Monogenea
Polyopisthocotylea, Heteraxinoides sp. et Diplozon gracile, deux Monogenea Monopisthocotylea, Cleitharcticus sp. et
Furnestinia echeneis, et deux Eucestoda, Moniezia sp. et Echeneihothrium sp. Le spermatozoide est decrit pour la premiere
fois dans la plupart de ces especes. Les donn£es bibliographiques montrent que fultrastructure du spermatozoide est
maintenant connue chez approximativement 140 genres (presque 200 esp&ces) de Plathelminthes parasites
(= Aspidogastrea, Digenea, Monogenea, Amphilinidea, Gyrocotylidea, et Eucestoda). La structure g£neralc (deux
axonemes 9+‘T\ microtubules seulement dorsaux et ventraux dans la region principale du spermatozoide) resulte d*un
processus de fusion proximo-distalc et est rencontree chez les Aspidogastrea, Digenea, Gyrocotylidea et Amphilinidea,
avec toutefois quelques variations dans les deux premiers groupes. Les Monogenea Polyopisthocotylea se differencient de
Justine, J.-L., 1995. — Spermatozoal ultrastructure and phylogeny in the parasitic Platyhelminthes. In: Jamieson,
B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem . Mus. natn. Hist,
not., 166 : 55-86. Paris ISBN : 2-85653-225-X.
56
J.-L. JUSTINE : PARASITIC PLATYHELMINTHES
la structure symplesiomorphe par la presence de microtubules lateraux additionnels. Les Monogenea Monopisthocotylea
sont differences par la pertc de nombreuses structures et une tendance generale vers un spermatozoi'de plus simple. Les
Eucestoda sont caract6ris£s par Tabscnce de la mitochondrie et montrent d’autres deviations. Lc genre Schistosoma ,
appartenant aux Digenea, a un spermatozoide a structure totalement differente de la structure generale qui est pourtant
retrouvee dans les families supposees proches (Spirorchidae et Sanguinicolidae), et son spermatozoi'de est consider
comme "progenetiquc”. L’outgroup dcs Platyhelminthes parasites doit etre identifie de manure precise pour une analyse
cladistique plus liable des caracteres du spermatozoi'de.
The phylum Platyhelminthes represents an exemplary case of the use of comparative
spermatology for the understanding of phylogeny, because it combines several characteristics: 1.
absence of fossils, necessitating that characters may be sought only in living taxa; 2. small size of
organisms, which have directed the interest of researchers toward electron microscopy from the
onset of this technique; 3. sperm morphologies which, although they follow a general ground
plan, are extremely variable; 4. in most parasitic Platyhelminthes, an enormous development of
the reproductive apparatus and a constant fecundity, making the work of the electron microscopist
easier.
Moreover, some “historical” factors have emphasised the importance of comparative
spermatology in the Platyhelminthes. The first is that several schools of research have been
created in this field and continue to be investigating actively. Professor EUZET has established, in
the seventies, the first of these schools in Montpellier, from which were successively issued the
theses of Ktari (1971)[135], Mokhtar-Maamouri (1976)[152], FOURNIER (1980)[60], and
JUSTINE (1980) [88], (1985)[95j. The work of SwiDERSKI [58, 209-222] was linked with this
school. A second generation of researchers has been formed by the students of EUZET: AZZOUZ-
DRAOUI ( 1985)[6] and Noury-SRAIRI ( 1988)[ 162]. Xavier MATTEI, commencing with a thesis
in the same University of Montpellier on Fish comparative spermatology, went on to establish an
active group in Dakar, Senegal, where JUSTINE worked on various Platyhelminthes [88-95, 105-
123] and later MARCHAND and BA worked on Cestodes [8-21], Two other independent schools,
which are not limited to spermatology but also deal with other ultrastructures, were developed in
Australia by Klaus ROHDE (with Nikki WATSON [174, 175, 178, 179, 187-189, 191-193, 233-
235, 237-239, 241, 242, 244] and others [140, 141]) and in Germany by Ulrich EHLERS [49-53]
(with SOPOTT-EHLERS [202], XYLANDER [250-252], and others). In the past years, several
independent teams have begun original works in this field, and the establishment of *01116
researchers in China [40, 65, 142, 143, 199, 255-257] and Korea [2, 82, 83, 201] promises that
research on spermatozoa will benefit from the wide diversity of the fauna in these regions.
The second historical factor is that spermatozoal ultrastructure was recognised relatively
precociously as a source of characters for the cladistic analysis of the phylum Platyhelminthes. As
early as 1984-1985, EHLERS [49-51] and later BROOKS [30] used spermatozoal apomorphies for
defining major groups of the phylum. The Trepaxonemata Ehlers, 1984 was defined on the basis
of an ultrastructural character of spermatozoa (the 9+“l” axoneme). This a striking example of the
importance of comparative spermatology for the cladistic analysis of the Platyhelminthes: such a
definition would have been impossible ten years before, but it remains unquestioned ten years
later. Several groups of Platyhelminthes such as the Temnocephalida [77, 98, 129], the
Prolecithophora [53], the Cercomeridea [98] or others [50, 51, 97, 98] have also been defined on
the basis of spermatozoal ultrastructural characters.
Table 1 is a list of genera studied for sperm ultrastructure. Comparison with a similar Table
published in 1991 [98] shows that the number of genera studied for sperm ultrastructure in the
parasitic Platyhelminthes has increased from 98 to 141 (representing almost 200 species) in four
years, thus giving an idea of the vitality of these studies in the past years.
This chapter includes new results from several species and a general presentation of sperm
ultrastructure in the various taxa of the parasitic Platyhelminthes. The term “parasitic
Platyhelminthes” refers in this study to the major taxa (Aspidogastrea, Digenea, Monogenea,
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
57
Gyrocotylidea, Amphilinidea and Eucestoda) which include only parasitic species. This study
excludes, however, the various parasitic species (see [182]) which belong to the “Turbellaria”.
The parasitic Platyhelminthes defined above are considered monophyletic in all modern analyses,
but are called the Cercomeridea [30] or the Neodermata [181] according to the position assigned
to the Udonellidea. Data concerning the “Turbellaria” and Eucestoda are detailed in others chapters
of this book by WATSON & ROHDE [232] and BA & Marchand [21], respectively. The results
of the cladistic analyses of the Platyhelminthes previously performed by the author [97-99, 102]
will not be repeated here.
MATERIAL AND METHODS
Observations are presented here on the following parasitic species, for which results have not previously been
published or have been published in a very incomplete form. Digenca: Prosorchis ghanensis Fischtal et Thomas, 1972,
Sclerodistomatidae, from the fish Acanthurus monroviae Steindachner, 1876, Dakar; Apatemon graciliformis Szidat.
1928, Strigeidae, from the bird Cairina moschata, Guadeloupe, French West Indies; Spirorchis sp., Spirorchidae. from the
turtle Pelusios adansoni , Lac de Guiers, Senegal; Aphalloides coelomicola Dollfus, Chabaud et Golvan, 1957,
Cryptogonimidae, from the fish Gobius micropus , Mediterranean. France; Proctoeces maculatus (Loos, 1901) Odhner,
1911, Fellodistomatidae, from the fish Crenilabrus cinereus , Mediterranean, France; Aporocotyle spinosicanalis
Williams. 1958, Sanguinicolidae, from the fish Merluccius merluccius L., 1758, Mediterranean. France; Echinostoma
togoensis Jourdane et Kulo, 1978, Echinostomatidae, from mice, strain from Togo; Haematoloechus sp.,
Haematoloechidae, from the amphibian Rana sp., Dakar, Senegal (spermiogenesis has already been described [111] in this
species but one micrograph is added here for comparison); Schistosoma sp., Schistosomatidae. from sheep or cattle,
Dakar, Senegal (this species has been referred to as S. bovis in publications concerning sperm [90, 108] but it has been
shown later that certain specimens could belong to S. curassoni [3]); Monogenea: Furnestinia echeneis (Wagencr, 1857),
Diplectanidae, from the fish Sparus aurata L.. Mediterranean, France; Cleitharcticus sp., Ancyrocephalidae, from the Fish
Acanthurus monroviae Steindachner, 1876, Dakar; Heteraxinoides sp., Heteraxinidae, from the fish Pomadasys incisus ,
Dakar (the spermatozoon has already been described in Heteraxinoides sp. from an other fish [123], but not in these
specimens); Diplozoon gracile, Diplozoidae, from the fish Gobio gobio, small river near Montpellier, France (sperm in
this species has been described [106, 107] but a micrograph is presented here for comparison); Ccstodes: Moniezia
expansa Rud., 1810, Anoplocephalidae, from sheep, Dakar; cestode, probably Echeneibothrium sp., Phyllobothriidae,
from the fish Raja miraletus L., 1758, Dakar.
Living specimens were placed in cold (4 °C) fixative consisting of 2% glutaraldehyde in a buffer solution of 0.1
M sodium cacodylate, 0.1 M sucrose, and 0.2 mM CaCI2. at pH 7.2 at 4°C for 1 h. After washing in the same buffer, worms
were postfixed for 1 h in 1% osmium tetroxide in the same buffer, dehydrated in ethanol and propylene oxide, and
embedded in Epon. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and observed with a Siemens
Elmiskop 101 or a Hitachi H600 electron microscope.
OBSERVATIONS
Conventions and orientation
Antero-posterior orientation , or “where is the head ?” Platyhelminthes spermatozoa are
filiform and it is impossible to recognize a “head” or a “tail”, in contrast to many other groups.
For the orientation of the spermatozoon, some authors have chosen to consider the nucleus as
anterior, by analogy to most animal spermatozoa. However, in the Platyhelminthes there is a
fundamental antagonism between the orientation of the axonemes and that of the nucleus. In most
animal spermatozoa, the anterior extremity of the axoneme, the centriole, pushes the nucleus
forward and thus the orientation of the nucleus (considered as anterior in the sperm) and that of
the axoneme are the same. In the parasitic Platyhelminthes, the nucleus is located at the non-
centriolar extremity of the axonemes. Some limited observations about movement show that the
axonemes are motile at the anterior part and that the nucleus is dragged at the posterior part [111].
Observations of fertilization [118, 125] show that the nucleus is the last part of the spermatozoon
to enter the oocyte. Therefore, it is more logical to use the antero-posterior orientation in a
58
J.-L. JUSTINE : PARASITIC PLATYHELMINTHES
Table 1. — List of the genera of parasitic Platyhelminthes in which sperm ultrastructurc has been studied.
Subclass Trematoda
Infraclass Aspidobothrea
Infraclass Monogenea
Cohort Polyopisthocotylea
Atriaster ( A triaster ) [101]
A triaster ( A trispin um ) [94, 97]
Axine [120]
Cemocotyle [123]
ADVANCES IN SPERM ATOZOAL PHY LOGENY AND TAXONOMY
59
Table 1. — continued.
functional way rather than follow an unjustified analogy with spermatozoa of other groups which
have a completely different ontogeny. The nucleus should be considered as posterior , and the
extremity of the spermatozoon which is thinner, more motile, and devoid of nucleus should be
considered anterior , because it contains the centrioles.
Dorso-ventral orientation. There are no functional arguments here. The usage follows the
purely arbitrary orientation chosen by SATO et al. (1967) [194]: mitochondrion ventral, nucleus
dorsal. Note that the antero-posterior and dorso-ventral orientations together allow recognition of
a right side and a left side for the spermatozoon, a notion important for unilateral organelles such
as undulating membranes [121].
Observations on spermiogenesis of some Digenea ( Figs 1-3)
Spermiogenesis in Prosorchis (Fig. 1). Spermiogenesis in Prosorchis exemplifies the usual
process found in most Digenea. The early spermatid, with round nucleus, has a short
protuberance (termed zone of differentiation) from which two flagella grow in opposite directions
(Fig. la). Later, the zone of differentiation elongates (Fig. lb). The two centrioles are each
associated with a striated root (Fig. lb), and an intercentriolar body is located between them (Fig.
la). The zone of differentiation has three processes elongating from its distal extremity: a median
cytoplasmic process and two free flagella. Transverse sections show that the two flagella have the
9+“l” structure diagnostic of the Trepaxonemata, and that the median cytoplasmic process has
dorsal and ventral microtubules. Attachment zones are visible and indicate the place where the
flagella will fuse with the median process (Fig. lc). The process of fusion is proximo-distal, i.e.
it begins near the common cytoplasmic mass and ends at the elongating tip of the spermatid. After
this fusion, the elongating spermatid (Fig. Id) shows the same structure as mature spermatozoa:
transverse sections show two axoneme, microtubules, and the mitochondrion. In addition, a
section of the nucleus is visible in the nuclear region (Fig. le).
60
J.-L. JUSTINE : PARASITIC PLATYHELMINTHES
Fig.
' — Spcrmiogenesis in ihe Digenea, exemplified in Prosorchis ghanensis. Note the 9+“l” structure of axonemes and
microtubules restricted to the dorsal and ventral side of spermatozoa, a: Early spermatid, with early zone of
dillerennanon from which two flagella grow in opposite direction. IB, interccntriolar body, b: Mature zone of
ditlerentiation. The nucleus passes through the zone of differentiation, which bears three elongating processes: the
median cytoplasmic process (MCP) and two flagella (one visible here). Each centriole has a striated root (R)
c: Transverse section in median cytoplasmic process (MCP) and associated flagella. Note attachment zones (Z)
where the flagella will later tuse with the MCP. d: Transverse section of elongating spermatids, after the fusion;
attachment zones (Z) still visible, e: Transverse section of elongating spermatid, showing nucleus and
mitochondrion, a, x 20 000; b, d, e, x 24 000; c, x 90 000.
For all figures: F, flagellum; M, mitochondrion; N, nucleus.
Source : MNHN, Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
61
Fig. 2. — Homogeneity of spermiogenesis and spermatozoon structure in the Digenea. a-c: Proctoeces maculatus.
d: Haematoloechus sp. e, f: Apatemon graciliformis. g, h: Aphalloides coelomicola. i: Echinosioma
togoensis. a, e: Median cytoplasmic process of spermatid, d, f, g, i: Transverse section of spermatozoa in
the mitochondrial region, b: Transverse section of spermatozoa in the principal region, containing the nucleus,
c, h: Transverse section in a region (probably anterior) showing ornamentation (arrows) on the membrane, a, c-
h. x 60 000; b. x 36 000; i. x 48 000.
Source :
62
J.-L. JUSTINE : PARASITIC PLA TYHF.LMINTHES
F|G' 3'nukerorr!,0ammiu?ChiS!0S0>e!. anf °the.r b'°°d nukeS- a'd; Schistosoma sp. (Family Schistosomal, dae, blood
nukes ol mammals), a: longitudinal section of sperm body, with mitochondria at the anterior extremity and
P trm nucleus, b: longitudinal section of flagella, c: glycogen demonstrated in flagella by Thiery’s method
?FnmrfivSVrSe SeTT 0ru?agfIUm' showin2 a structure which is not a trepaxonematan 9+‘T”. e: Spirorchis sp
y Sp!r°r,Ci"daf' „blood 1 ukcs 01 lurlles)- Transverse section at various levels, f, g: Aporocoivfe
l STahnSulnicolidac’ bl™d fl^e of fishes). Transverse section at the mitochondrial level'll)
nd nuclear level (g). The spermatozoon ol schistosomes is not threadlike and is similar to the zone of
erentiation lound in spermatids ol the other Digenea (“progenetic spermiogenesis”[100]) In contrast the
permatozoon o. the other blood flukes is threadlike and similar to that of other Digenea in having two 9V>r
trepaxonematan axonemes. b-d. modified from [108]. a, x 24 000; b, c, e, x 90 000^0. f, g. x 60 000.
Source .
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
63
Spermiogenesis in Proctoeces (Fig. 2 a-c). The median cytoplasmic process (Fig. 2 a) has a
similar as in Prosorchis, with its attachment zones clearly visible. Transverse section of
spermatozoa in the testis show the usual structure with two axonemes, microtubules,
mitochondrion and nucleus. As usual in the Digenea, the microtubules are only dorsal and ventral,
and the lateral faces, along the axonemes, are devoid of microtubules (Fig. 2b). Flowever, some
transverse section (Fig. 2c) show a continuous microtubule row at the periphery of the
spermatozoon. These sections also show external ornamentation associated with the membrane.
These sections correspond with the anterior extremity of the spermatozoon, which originates from
the zone of differentiation.
Spermatozoon of Haematolechus (Fig. 2d). This genus, which is easily collected from
frogs, has been widely used for the early studies of spermiogenesis in the Digenea (see Table 1)
and has become a standard. Sections of spermatozoa are displayed here to show the similarities of
the other species with this standard. Sections show the mitochondrion and a small circular profile
of two membranes, which probably represents the extreme anterior extremity of the nucleus.
Spermiogenesis in Apatemon (Fig. 2e,f). The median cytoplasmic process (Fig. 2e) shows
the usual structure, but the number of microtubules is relatively high. Transverse sections of
spermatid after the fusion still show the attachment zones (Fig. 2f).
Spermatozoa of Aphalloides (Fig. 2g, h) and Echinostoma (Fig. 2i). Spermatozoa show the
usual structure, including a zone with external ornamentation in Aphalloides (Fig. 2h).
Spermatozoa in Schistosomes (mammal blood-flukes) and other blood-flukes:
Sanguinicolidae or fish blood-flukes (Aporocotyle) and Spirorchidae of turtle blood-flukes
(Spirorchis) (Fig. 3). Schistosomes show an aberrant sperm structure [90, 100, 104, 131]. The
spermatozoon is not filiform (Fig. 3a), and there is one single axoneme, which has not the 9+“l”
trepaxonematan structure. Instead, the axoneme shows 9 doublets and a central poorly contrasted
structure, termed 9+0 or 9+“l” [108], and is devoid of dynein arms (Fig. 3 b-d). The phyletic
interpretation of this aberrant structure is difficult and requires a study of the families considered
as close to the schistosomes, i.e. the other blood-flukes, the spirorchid and sanguinicolids. In the
spirorchid Spirorchis, the spermatozoon (Fig. 3e) is filiform and shows the usual structure found
in the Digenea, with two 9+“l” axonemes and dorso-ventral microtubules. In the sanguinicolid
Aporocotyle, the spermatozoon (Fig. 3 f-g) is filiform and shows two 9+‘T’ axonemes, but the
peripheral microtubules are absent in most sections, although a few microtubules can be seen in
some rare sections.
Observations on spermatozoa of the Monogenea Polyopisthocotylea (Figs 4-6)
Spermatozoon o/Heteraxinoides (Fig. 4). Observations are briefly reported to exemplify
the homogeneous structure found in most polyopisthocotylean monogeneans. The spermatozoon
shows two 9+“l” axonemes, the nucleus and mitochondrion (Fig. 4). The fundamental difference
from the Digenea is that the microtubule row, in the principal region of the spermatozoon, makes
a complete circle around the sperm cell, i.e. it is not interrupted at the axoneme level.
Spermatozoon o/Diplozoon (Fig. 5). The spermatozoon is highly aberrant [105-107]: it is
elongate, aflagellate and contains several hundreds of parallel longitudinal microtubules (Fig. 5b).
A view of the animal is presented (Fig. 6a) for the purpose of the discussion.
Spermatozoon of Gotocotyla (Fig. 6). This species has the general pattern found in the
polyopisthocotylean monogeneans but shows an outstanding additional structure: a lateral
undulating membrane, which contains a large number of parallel microtubules. The ultrastructure
of the spermatozoon has been described previously [121], but Fig. 6 is an unpublished artist’s
view of the mature spermatozoon drawn from micrographs.
64
J.-L. JUSTINE : PARASITIC PLATYHELMINTHES
Fig. 4. — Spermatozoa in the Monogenea Polyopisthocotylea, exemplified by Heteraxinoides sp. a, b: Transverse
sections showing two 9+4T* axonemes. mitochondrion and nucleus. Note that the microtubules form a complete
row around the spermatozoon (synapomorphy for the Polyopisthocotylea [97. 98]). a, x 90 000; b. x 120 000.
Fig. 5. — The aberrant case of Diplozoon (Monogenea Polyopisthocotylea). a: The two member of a pair are
permanently fused. Scanning electron microscope photograph of Diplozoon nipponicum by Nathalie LEBRUN,
b: Transverse section of spermatozoa of Diplozoon gracile . showing numerous parallel longitudinal microtubules
(modified from (106]). This is the only known case of aflagellate spermatozoon in the parasitic Platyhelminthes,
and this aberrant pattern is linked with the exceptional biology of reproduction.
Source MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
65
Fig.
— An artist’s view of the spermatozoon of Gotocotyla acanthura (Monogenea Polyopisthocotylea), drawn from
micrographs published in [121). The spermatozoon has an undulating membrane along part of its length. The
undulating membrane is an autapomorphy for this species. However, the general sperm structure is similar to the
classic pattern found in the parasitic Platyhclminthes. Drawing by Nathalie LeBrun.
Source . MNHN. Paris
66
J.-L. JUSTINE : PARASITIC PLA TYHELMINTHES
Observations on spermio genesis of the Monogenea Monopisthocotylea (Figs 7, 8)
The Monopisthocotylea show a variety of sperm structures. Observations reported here
concern two species in which the spermatozoon has one single axoneme, and thus has the
maximum deviation from the Digenea or the Polyopisthocotylea.
Fig. 7. Spermiogenesis in Furnestinia echeneis, a Monogenea Monopisthocotylea with uniflagellate spermatozoon,
a: Transverse section of zones of differentiation embedded in the common cytoplasmic mass (modified from
[105]). b: Isogenic group with 64 spermatids; c: Mature spermatozoa have only one axoneme, and the nucleus is
located in a region devoid of any other organelles, d: centriolar adjunct, a, d, x 48 000; b, c, 30 000.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
67
Fig. 8. — Spermiogenesis in Cleitharcticus sp., a Monogenca Monopisthocotylea with uniflagellate spermatozoon,
a: Longitudinal section of zone of differentiation containing one single axoncme. Note absence of intercentriolar
body and striated roots (compare with Fig. la, b). b: Transverse section of zones of differentiation, showing
single centriole and migrating nucleus and mitochondrion, c, d: Axonemes in growing spermatids show an
incomplete tubule b (arrows) in the axonemc. e: Mature spermatozoa, however, have complete tubules b in their
axoneme. f: Region with external ornamentation located at one extremity of the spermatozoon, a, d, x 48 000;
b, x 30 000; c, e, f, x 60 000.
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J.-L. JUSTINE : PARASITIC P LATY HELM! NTH ES
Spermiogenesis in Furnestinia (Monopisthocotylea) (Fig. 7). During spermiogenesis, the
zone of differentiation is deeply embedded in the common cytoplasmic mass, and shows one
single axoneme, the migrating nucleus and mitochondrion (Fig. 7a). It is not possible to recognize
the proximo-distal fusion in this species, since only one element exists from the beginning to the
end of spermiogenesis. Isogenic groups of spermatids comprise 64 elements (Fig. 7b). Mature
spermatozoa in genital ducts show a relatively simple structure, with a region showing the
axoneme and mitochondrion, and an other region showing the nucleus with no accompanying
element (Fig. 7c). The anterior extremity contains a centriolar derivative (Fig. 7c). Cortical
microtubules are absent at all stages of spermiogenesis.
Fig. 9. Spermatozoa of Eucestoda. Note the absence of mitochondrion, a synapomorphy for the Eucestoda.
a, b: Ech eneibothriu m sp. Microtubules and axoncmes are at focus on the same section. Note presence of two
kinds ol microtubules, with thin wall and thick wall, c: Moniezia sp. Note that the axoneme and the peripheral
microtubules are not in perlect cross section on the same sections, because of the peripheral microtubules twisting,
a synapomorphy for the Cyclophyllidea [98]. a-c, x 60 000.
Spermiogenesis in Cleitharcticus (Monopisthocotylea) (Fig. 8). The zone of differentiation
(Fig. 8a) is deeply embedded in the cytoplasmic mass and shows one single centriole; the
intercentriolar body and striated roots found in the Digenea are absent. As for Furnestinia , it is not
possible to recognize the proximo-distal tusion in this species. In maturing spermatids, the
axoneme shows incomplete b tubules (Fig. 8c, d), but these are complete in the mature sperm
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
69
cells (Fig. 8e, f). Mature sperm cells show one single axoneme, the nucleus and mitochondrion,
and are devoid of cortical microtubules (Fig. 8f). Some rare sections (Fig. 8f) show external
ornamentation on the membrane and may, with analogy with the Digenea, be considered as
anterior, although this has not been fully demonstrated.
Observations on spermatozoa of the Eucestoda (Fig. 9)
A few observations are reported here to demonstrate the major characters of spermatozoa.
The two species shown have a single axoneme, but others have two axonemes.
Spermatozoa of Echeneibothrium (Fig. 9a. b). Transverse sections show one single
axoneme of the 9+“l” structure. The nucleus, not cut at the levels shown here, is present in other
sections. Sections never show a mitochondrion. An interesting feature is the co-existence of two
kinds of microtubules, either with thin wall and thick wall.
Spermatozoa o/Moniezia (Fig. 9c). The spermatozoon has a single axoneme and is devoid
of mitochondrion. Transverse sections of sperm never show both the peripheral microtubules and
the axoneme perfectly transverse because the microtubules are twisted around the sperm body.
DISCUSSION
Structure of the spermatozoon in the major groups of parasitic Platyhelminthes
The basic structure: Digenea. Amphilinidea, Gyrocotylidea. The basic structure is here
described for the Digenea but appears to be similar in the Amphilinidea and Gyrocotylidea. This
structure is a synapomorphy for the Cercomeridea [98]. However, within the Cercomeridea, other
structures have evolved from this basic pattern, and this pattern may be considered the
symplesiomorphic structure for the parasitic Platyhelminthes.
The spermatozoon is filiform, very long, and therefore can be described only from
transverse sections. It is composed of two main regions: the principal region, posterior,
originating from the fusion of the three processes attached to the zone of differentiation of the
spermatid; and the anterior region, originating from the zone of differentiation itself [98].
Transverse sections of the spermatozoon in the principal region (which contains the nucleus)
show two axonemes, the mitochondrion and nucleus, and peripheral longitudinal microtubules
limited to the ventral and dorsal faces. The microtubules are not twisted along the long axis of the
sperm and are parallel. The anterior region is much shorter than the principal region. It generally
shows a continuous microtubule row, not interrupted at the axoneme level, and is often marked
by external ornamentations on the membrane.
It should be emphasized that, although the structure found in the principal region is
remarkably homogeneous, a greater variety of structure is found in the anterior region. The
anterior region of Proctoeces (Fig. 2c) has the structure described above, but Aphalloides (Fig.
2h) has ornamentation in a region which lacks a complete row of microtubules. In
Haematoloechus , the two regions (anterior and principal) are separated by a “collerette” or collar
which is visible with the light microscope [111]. The variations of the anterior region and its
associated membrane ornamentation would probably be valuable for the understanding of
phylogeny, because the principal region is too homogeneous to be useful. However, transverse
sections of the anterior region often represent only a small proportion of the sections available,
and sub-optimal fixations do not allow observation of the membrane ornamentation.
Variation in the Digenea. Sperm structure in the Digenea is homogeneous and variations are
relatively rare.
In the didymozoids, deviations from the classic pattern have been found in Gonapodasmius
and Didymozoon. Gonapodasmius has a relatively short spermatozoon, and the intercentriolar
70
J.-L. JUSTINE : PARASITIC PLATYHELMINTHES
body is lacking during spermiogenesis, but the ultrastructure of the spermatozoon is “classical”
and the morphology is filiform. Didymozoon has a filiform spermatozoon, but the two axonemes
show a 9+0 pattern and peripheral microtubules are absent.
In the Schistosomes (genus Schistosoma) the deviation from the symplesiomorphic pattern
is extreme (Table 2).
Table 2. — Characteristics of spermiogenesis in the schistosomes and other Digenea (See Table 1 for references)
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
71
Sperm structure in the schistosomes and its deviation from the classic pattern is difficult to
interpret if only a comparison between mature spermatozoa is performed. However, a comparison
of the spermiogenetic processes shows that the mature sperm of schistosomes can be compared to
a mature zone of differentiation in other Digenea. Spermiogenesis in the schistosomes therefore
may be considered “progenetic” (i.e. the mature spermatozoon is a spermatid’s zone of
differentiation having precociously reached maturity) [100], In addition, this zone of
differentiation has one single axoneme instead of two.
It is interesting to note that the deviation in sperm pattern found in Schistosoma is not found
in the other blood-flukes, the spirorchids and the sanguinicolids. These families, however, are
considered to be closely related to the schistosomes in most phyletic schemes of the Digenea [31,
37, 44, 136, 200], but a different opinion was recently expressed [25]. The schistosomal sperm
pattern is therefore restricted to the family Schistosomatidae. Moreover, in the genus
Schistosomatium, a member of the family Schistosomatidae, light microscope observations have
shown that there are clusters and that the spermatozoon is filiform, with two free flagella at one
extremity [161]. Thus, the schistosome aberrant pattern appears to be restricted to the genus
Schistosoma but is similar in all species of the genus [104],
The link between phylogeny and sperm ultrastructure is thus not obvious for the
schistosomatids and related families (spirorchids, sanguinicolids). The influence of the biology of
fertilization is probably important, and is discussed below.
Sperm ultrastructure in the Aspidogastrea. The Aspidogastrea are considered the most
primitive group of Neodermata by ROHDE [183]. Indeed, their spermatozoa correspond to the
classic, symplesiomorphic pattern, with two 9+‘T’ axonemes and dorso-ventral microtubules.
However, some deviations from the plesiomorphic pattern do exist in the Aspidogastrea and are
listed in Table 3 for three species; information available on Aspidogaster is scarce and therefore
not included. The dense region could be considered a synapomorphy for the Aspidogastrea [235],
The undulating membrane of Lobatostoma [176, 193] and Multicotyle [239] is similar to that of
the monogenean Gotocotyla [121] and should be considered a case of convergence.
Table 3. — Deviation from the basic pattern of the Cercomeridea found in the Aspidogastrea.
Spermatozoon structure in the Monogeneci Polyopisthocotylea. The deviation from the
symplesiomorphic pattern in the Polyopisthocotylea is the acquisition of supplementary
microtubules on the lateral faces of the mature spermatozoon. This has been proposed as a
synapomorphy for this group [97]. The Polyopisthocotylea are relatively homogeneous in their
sperm structure, but some original features have been found in a few cases. These are generally
restricted to one species and thus should be considered only as autapomorphies [101], Diplozoon
72
J.-L. JUSTINE : PARASITIC PLA TYHELMINTHES
has an aberrant spermatozoon with more than 400 parallel longitudinal microtubules and no
axonemes. This represents the only case of aflagellate sperm in the parasitic Platyhelminthes, but
aflagellarity has also evolved several times in the “Turbellaria” ([163, 190, 202]; see also [232]).
The case of Diplozoon is dealt with below, in the paragraph on biology of fertilization.
Sperm structure in the Monogenea Monopisthocotylea. The Monopisthocotylea are
characterised by synapomorphies such as the loss of the dorso-ventral microtubules, loss of
striated roots, loss of intercentriolar body. A general evolutionary trend in this group is a tendency
to shorter and simpler spermatozoa. Simplification of sperm includes the loss of one axoneme and
the loss of peripheral microtubules, with the result that the sperm cell contains only three
components, the nucleus, mitochondrion and one axoneme. A general cladistic analysis of sperm
structure has been performed on the Monopisthocotylea [97] and updated [102], These analyses,
among other results, have allowed the erection of the group Monoaxonematidea Justine, 1991
(etymology: one single axoneme) for several families which have uniflagellate spermatozoa [97].
Since new information has been acquired recently on several Monogenea (see Table 1), this
cladistic analysis should be updated. This would, however, exceed the scope of this general
review.
The recent literature shows that DNA studies generally accord with spermatology
concerning the systematic relationships within the Monogenea. JUSTINE (1991) [97, 98] insisted
that synapomorphies can be defined for the Monopisthocotylea and for the Polyopisthocotylea,
but not for the Monogenea as a whole (= Monopisthocotylea+Polyopisthocotylea). Subsequently,
several DNA studies have also concluded that no argument could be given to support monophyly
of the Monogenea [27, 46, 183-185], The position of the Gyrodactylids within the
Monopisthocotylea or the Polyopisthocotylea is uncertain in studies based on morphology [137]
but they are clearly assigned to the Monopisthocotylea on spermatological evidence [97, 105]; this
has been recently confirmed in a study of 18S DNA [46].
The convergence found between the Monopisthocotylea and the Eucestoda has received little
comment. Both groups, and these groups alone, within the parasitic Platyhelminthes, have
filiform spermatozoa with one single axoneme. This emphasizes a striking feature of the
Platyhelminthes: the plesiomorphic sperm in the parasitic group is biflagellate, and these two
groups have evolved toward a simpler structure.
Sperm structure in the Eucestoda. The Eucestoda are characterized by a synapomorphy
accepted by all authors [21, 30, 50], the absence of mitochondrion. This clearly apomorphic
condition separates the Eucestoda from the two other groups of the Cestodaria, the Amphilinidea
and Gyrocotylidea. These two groups have apparently kept the basic structure of the
Cercomeridea. This character-state “absence of mitochondrion” requires some comments.
Characters defined by “absence” are generally considered, in systematics and particularly in
cladistics, as less reliable than the acquisition of new structures. However, in this case, one may
note that the absence of mitochondrion is probably correlated with the acquisition of other
enzymatic systems in the spermatozoon, which are not revealed by ultrastructural methods.
Another comment is that researchers interested in the inheritance of paternal mitochondrial DNA
should perform a comparative study between a digenean, in which the volume of the spermatozoal
mitochondrion is important, and a member of the Eucestoda.
Other synapomorphies of Eucestoda spermatozoa include the twisting of peripheral
microtubules, a synapomorphy for the Cyclophyllidea [98] exemplified in the present paper. BA
& MARCHAND have detailed propositions of other synapomorphies in their chapter in this volume
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
73
Number of spermatids in an isogenic group (32 or 64), and value of this character for
phylogenetic studies
Table 4 gives a list of some references concerning the number of spermatids in isogenic
groups, i.e. groups of cells originating from a single spermatogonium, and fused together until
the separation of the mature spermatozoa. XYLANDER [250] has given some phylogenetic value to
this character, and considered that a group of 64 was a synapomorphy for the
Amphilinidea+Gyrocotylidea+Eucestoda. However, some problems make this character difficult
to use. The number is not homogeneous in the Monogenea Monopisthocotylea nor in the
Aspidogastrea. Also, it is difficult to polarise this character since the outgroup for the parasitic
Platyhelminthes is not precisely known. This character cannot be used at the present time owing
to these uncertainties.
Table 4. — Number of spermatids in an isogenic group
Source
74
J.-L. JUSTINE : PARASITIC PLA TYHELM INTHES
Organelles absent in parasitic Platyhelminthes but present in free-living Platyhelminthes
("Turbellaria ”): dense bodies and 25 nm granules
As for other characters mentioned above, the apomorphic character of the parasitic
Platyhelminthes is the absence of structures which are present in the plesiomorphic taxa.
Membrane-bound dense bodies. These are absent in all parasitic Platyhelminthes examined.
Several categories of granules are present in the same spermatozoon in the Acoela [170]. In other
taxa of the “Turbellaria”, one or two categories are found, or dense bodies are absent (see Table 1
in [232]). The absence of membrane-bound granules has been considered a synapomorphy for the
Neodermata by EHLERS [50, 51].
Cortical non-membrane bound 25-nm granules. These have been found in the
Temnocephalidea [77, 180, 240, 243, 245, 246] and in some Dalyellioida [41, 163, 202],
Detailed bibliographical information can be found in the chapter by WATSON & ROHDE in this
volume [232], These granules have been discussed by several authors [77, 163, 202, 232], It is
pertinent to this chapter that they have never been found in any parasitic Platyhelminthes. In our
present state of knowledge, it does not appear valid to attribute to the presence of these granules
the value of a synapomorphy for a specific taxon of the “Turbellaria” because their presence is not
regular in all taxa of a given group. However, they could have an important physiological role,
which apparently does not exist in any parasitic Platyhelminthes.
Spermatozoal structure and the biology of fertilization
The question of the relationships between spermatozoal structure and the biology of
fertilization in Platyhelminthes has been discussed before [98] but new data make it more precise.
It is impossible to link the aberrant structure found in Schistosoma (Digenea) and in Diplozoon
(Monogenea) with the assumed respective phylogeny of these two taxa. For the schistosomes, the
present study shows that related families have the plesiomorphic structure. For Diplozoon , a
recent study [70] has shown that the Octomacridae, considered very close to the Diplozoidae,
have the symplesiomorphic structure. All spermatologists know that the biology of fertilization
may affect the morphology of spermatozoa: in a taxonomic group, the species which evolve
toward a different biology of fertilization may be expected to acquire new sperm structures.
Functional interpretation of these new structure is sometimes possible. In the case of the parasitic
Platyhelminthes, functional interpretation is not easy. Individuals of schistosomes (male and
female) live in permanent pairs, and they have acquired small and simple spermatozoa.
Individuals of Diplozoon also live in permanent pairs [138] (Fig. 5a), are fused, and the genital
ducts communicate, and they have acquired a long and very complicated spermatozoon. Similar
factors in Schistosoma and Diplozoon seem to have produced an evolution of the sperm structure
in two completely opposite directions. The answer to this puzzling problem could be found if we
consider that in both cases, the permanent pairing implies the absence of inter-individual sperm
competition. I he fact that the pair is made up of one male and one female in Schistosoma and of
two hermaphrodites in Diplozoon does not seem to have an influence. Thus, in both cases,
spermatozoa have not been subjected to selection in terms of performance such as speed or
fertilization efficiency, and evolution could diverge in different directions.
77 le problem of an outgroup for the cladistic analysis of the parasitic Platyhelminthes
The problem of the recognition of a correct outgroup is central for the polarisation of
characters. I will here concentrate on the interpretation of characters emerging from sperm studies
although it is clear that other structures may be highly useful. JUSTINE [98] has used, for defining
an outgroup, the general system of EHLERS [50, 51]. The taxon Typhloplanida, which has
spermatozoa with non-incorporated axonemes, was used as an outgroup for the
Doliopharyngiophora, which comprise the Temnocephalidea, Dalyelliida, Udonellidea and
Cercomeridea. It was noted [98] that the Dalyellioida have a very heterogeneous structure.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
75
WATSON & ROHDE have systematically tried to find the outgroup for the Neodermata in the
past year. Pterastericola, which has two incorporated axonemes was first proposed as an
outgroup for the Neodermata [85]. Later, the analysis of spermiogenesis demonstrated that the
fusion is not proximo-distal [242] and this hypothesis has been abandoned. DNA studies also
concluded that Pterastericolidae are not the sister-group for the Neodermata [185],
Kronborgia (family Fecampiidae) was suggested as the outgroup [236, 237], The
spermatozoon of Kronborgia has two incorporated axonemes and a continuous row of cortical
microtubules [237, 247, 248] and thus resembles that of the Monogenea Polyopisthocotylea.
Kronborgia has a spermiogenesis with proximal centrioles, as in the parasitic Platyhelminthes.
However, the proximo-distal fusion is not observed, there are no free flagella nor median
cytoplasmic process, and the two axoneme grow within the elongating spermatid. The presence of
a dense body makes its spermiogenesis close to most “Turbellaria”. It is not certain that
Kronborgia represents the taxon closest to the parasitic Platyhelminthes in term of ultrastructure of
spermiogenesis. Should Kronborgia be considered the outgroup of the parasitic Platyhelminthes,
this would require a re-examination of the polarity of certain characters, including the lateral
microtubules (synapomorphy for the Polyopisthocotylea) which are present in Kronborgia and
thus would become the plesiomorphic pattern. Recently, a study of 18s ribosomal RNA [186] has
indicated that Kronborgia was not the sister-group of the Neodermata and could even be widely
separate from them. However, this need confirmation from longer RNA sequences.
Udonella has a spermiogenesis with proximal centrioles, but there is no median cytoplasmic
process nor fusion [189, 133, 189, 251], This spermiogenesis is in fact similar to that of
Kronborgia. However, the mature spermatozoon of Udonella has no microtubules [105],
Spermiogenesis in Udonella and Kronborgia resembles that found in certain Monogenea such as
capsalids [114] or certain didymozoid digeneans [116] where a fusion is not observed because the
axonemes grow within the elongating zone of differentiation. In these two latter cases, this
characteristic should be interpreted as secondary reduction. The phyletic position of the
Udonellidea is a matter of controversy: either outgroup for the Cercomeridea [30], or close to the
Monopisthocotylea [183].
In a strictly spermatological point of view, we have, at present, no candidate for the role of
outgroup of the parasitic Platyhelminthes. The proximo-distal fusion, with typical zone of
differentiation, median cytoplasmic process and free flagella, is found only in the Aspidogastrea,
Digenea, Monogenea Polyopisthocotylea, Gyrocotylidea, Amphilinidea, and certain Eucestoda.
The analogies of the zone of differentiation in the Monogenea Monopisthocotylea [97, 98] and in
certain Eucestoda [21] with the basic structure of the parasitic Platyhelminthes suggest that,
although they lack a true proximo-distal fusion, these taxa may be considered closely related.
However, the proximo-distal fusion has not been found in any taxon more primitive than the
Cercomeridea.
ACKNOWLEDGEMENTS
Professor Louis Euzet, Dr Alain Lambert and the late Dr Claude Maillard, from the University of Montpellier,
collected the specimens of Aphalloides, Proctoeces, Aporocotyle and Furnestinia. The monogeneans were mostly
identified by Professor Euzet. Prosorchis was identified by Claude Maillard. Apatemon and Echinostoma were collected
by Professor Claude Combes and Dr Annie Fournier, from the University of Perpignan. Spirorchis was identified by
Professor Caude Combes. Dr Nathalie LeBrun communicated the scanning electron micrograph of Diplozoon of Fig. 5a,
and kindly made the drawing presented here in Fig. 6 and which was formerly used for the cover of the author’s thesis [94).
Dr Nikki WATSON and Professor Klaus Rohde have generously communicated their manuscripts which are in press.
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Source : MNHN, Paris
Spermiogenesis, Spermatozoa
and Phyletic Affinities in the Cestoda
Cheikh Tidiane BA * & Bernard MARCH AND **
* Laboratoire de Parasitologie,
Departemcnt de Biologie animale, Faculte des Sciences, Universite Cheikh Anta Diop, Dakar, Senegal.
** Laboratoire Arago,
Universite Pierre et Marie Curie (Paris 6), CNRS URA 1 17, F-66650 Banyuls-sur-mer, France.
ABSTRACT
Comparative ultrastructural studies ot spermiogenesis and/or spermatozoa of 43 species of cestodes lead us to conclude
that flagellar rotation and the proximodistal fusion of one or two flagella with the median cytoplasmic extension is a
plesiomorphic character in the Eucestoda and that the absence of flagellar rotation is a synapomorphy for the
Cyclophyllidea. We consider the presence ot the crested-like body at the anterior extremity of the spermatozoa of cestodes
as a synapomorphy for the Eucestoda. We also confirm the absence of mitochondria as a synapomorphy for the Eucestoda
Previous phylogenetic diagrams are critically reviewed.
RESUME
Spermiogenese, spermatozoides et affinites phyletiques chez les Cestoda.
L’etude ultrastructurale compare de la spermiogenese et/ou du spermatozoide de 43 espfcces de cestodes nous a permis de
considerer la rotation flagellaire et la fusion proximo-distale du ou des flagelles spermatiques avec une expansion
cytoplasmique mediane comme un caract^re plesiomorphe des Eucestodes et P absence de rotation flagellaire comme une
synapomorphie des Cyclophyllidea. Nous considdrons la presence de corps en crete a l’avant du spermatozoide comme une
synapomorphie des Eucestodes. Nous confirmons egalement 1’ absence de mitochondrie comme une synapomorphie des
Eucestodes. Les schemas phyletiques prec6dents sont revises de maniere critique.
Cestodes have been described in all vertebrates except agnathans [44]. They now comprise
nearly 4 000 species spread over 600 genera, 63 families and 13 orders. The different authors
interested in their phylogenesis on the basis of their morphological characters [14, 16, 19, 20,
22], frequently came to contradictory conclusions. The absence of mitochondria in spermatozoa
has been considered as a synapomorphy of the Cestodes [17, 18], and the spiral coil of the
cortical spermatic microtubules as a synapomorphy of the Cyclophyllidea [26]. In the present
work, we propose two new synapomorphies, one for the Eucestoda and one for the
Cyclophyllidea. Moreover, we critically debate the two last phylogenetic diagrams proposed by
EUZET et al. [20] and BROOKS et al. [16].
BA, C. T., & Marchand, B., 1995. — Spermiogenesis, spermatozoa and phyletic affinities in the Cestoda. In:
Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy Mem Mus
natn. Hist, nat., 166 : 87-95. Paris ISBN : 2-85653-225-X.
88
C. T. BA & B. MARCHAND : CESTODA ( PLATYHELM1NTHES )
MATERIAL AND METHODS
We have studied the ultrastructure of spermiogenesis and/or the spermatozoa of 14 species of cestodes (Table 1).
The specimens of the different species were gathered from the intestines of their respective hosts (birds or mammals),
naturally infested, then placed, alive and active, in a physiological saline solution (9 %c NaCl). Portions of strobile, 3 to
6 cm long, consisting of mature proglottids, were quickly taken, then stretched out with a brush soaked in cold (4°C) 2.5 %
glutaraldehyde in a 0.1 M sodium cacodylate buffer at pH 7.2. The genital apparatus was removed under a binocular
microscope, fixed for about 24 hours in glutaraldehyde at 4°C, rinsed lor one night in the sodium cacodylate buffer, post-
fixed for one hour with cold \% osmium tetroxide, dehydrated with ethanol and propylene oxide, then embedded in epon.
Ultrathin sections were cut on a Porter-Blum MT1 and Reichert-Jung Ultracut-E ultramicrotomes, then stained with uranyl
acetate and lead citrate. They were examined in Siemens Elmikop 101 and JEOL 100 CX II electron microscopes.
RESULTS
The figures 1 to 5 and Table 1 present our observations and those of other authors on
spermiogenesis and/or the spermatozoa of cestodes. Whatever the cestode may be,
spermiogenesis always begins by the formation of a differentiation zone (Figs la, 2a, 3a, 4a).
This is delimited at the proximal extremity by arched membranes, and bordered by cortical
microtubules. This contains one or two centrioles separated (Figs, la, 2a) or not (Figs 3a, 4a) by
an intercentriolar body and surmounted (Figs, la, 2a) or not (Figs 3a, 4a) by striated roots or a
centriolar-adjunct (Fig. 4a). Each centriole very rapidly gives rise to a flagellum that grows within
(Figs 4b-c) or external to (Figs lb, 2b, 3b) the differentiation zone. Subsequently, the flagellum
(or the flagella) undergoes (Figs lc, 2c) or not (Figs 3c, 4c) a rotation, becomes parallel to the
cytoplasmic extension, and fuses with it. After the migration of the nucleus into the differentiation
zone and the formation of one or many crested-like bodies, the ring of arched membranes narrows
until the spermatid detaches itself from the residual cytoplasm. The mature spermatozoon of the
cestodes lacks mitochondria, is filiform and is tapered at both extremities (Fig. 5). Its anterior
extremity exhibits an apical cone of electron dense material and one or many crested-like bodies.
The cytoplasm contains proteinaceous material and a nucleus coiled or not in a spiral around the
axoneme. The proteinaceous material may be arranged in four different forms: granulations, rods
making intracytoplasmic walls, a periaxonemal sheath, submicrotubular thickenings (Fig. 5).
DISCUSSION
During spermiogenesis of the Cercomeridea (Aspidobothrea, Digenea, Monogenea,
Gyrocotylidea, Amphilinidea and Eucestoda), the flagellum (or the flagella) of old spermatids
undergoes a rotation and becomes parallel to a median cytoplamic extension with which it fuses.
This proximodistal fusion as it is termed [26, 27] is not found in the Turbellaria [26, 56], In the
cestodes in particular, it is encountered in the Tetraphyllidea-Onchobothriidae [33, 37], the
Tetraphyllidea-Phyllobothriidae [36], the Tetrarhynchidea [51], the Proteocephalidea [50, 51], the
Pseudophyllidea [54] and the Caryophyllidea [51], The proximodistal fusion has recently been
proposed as a synapomorphy for all the Cercomeridea [26]. Nevertheless, in three
Cyclophyllidea, Thysaniezia ovilla [13], Hymenolepis nana [3] and Aporina delafondi [8], the
single flagellum grows directly into the spermatid body. In three other Cyclophyllidea,
Nematotaenia chantalae [38], Mathevotaenia herpestis [7] and Raillietina (Raillietina) tunetensis
[9], the flagellum grows outside the differentiation zone, parallel to the cytoplasmic extension and
then fuses with it before nuclear migration. Dealing with the existence or not of a flagellar rotation
during spermiogenesis, we can distinguish in the Cercomeridea two types of spermiogenesis. The
former which involves a flagellar rotation and a proximodistal fusion, has been supposed to exist
in all the Cercomeridea [26], The latter which is characterized by an absence of flagellar rotation,
has been reported in the six Cyclophyllidea which we have previously cited [3, 7, 8, 9, 13, 38].
In the present work, we consider the absence of flagellar rotation as an apomorphic character for
all the Cyclophyllidea.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
89
1- — Cestodes whose spermatozoa have been studied by electron microscopy. The references quoted contain data on
spermiogenesis (*), the presence of one (+) or more than one (++) crested-like bodies and the presence of two
axonemes (2a).
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C. T. BA & B. MARCHAND : CESTODA ( PLATYHELMINTHES )
One or many crested-like bodies have been described at the anterior extremity of the
spermatozoa of 26 species of cestodes: one Pseudophyllidean, one Proteocephalidean, three
Diphyllidea, seven Tetraphyllidea and 14 Cyclophyllidea (Table 1). Although their presence has
not been reported by some authors [24, 29, 42, 43], we have observed them in their illustrations.
In a Caryophyllidean, Glaridacris catostomi [53], a Haplobothrioidean, Haplobothrium
globuliforme [32] and a Tetrarhynchidean, Lacistorhynchus tenuis [48], these formations have
neither been described nor figured. In fact, it is not easy to detect them owing to their very small
size. Thus, in spite of these few “exceptions”, and although our knowledge is still limited to a
small number of species, we believe that the crested-like body should be considered as a
synapomorphy for all cestodes. Moreover, we consider the presence of a crested-like body in the
spermatozoa of one species of monopistocotylean Monogenea, Calceostoma sp. [28], as a simple
phenomenon of convergence.
The phylogenetic systematics of the cestodes are still poorly understood and are much
debated. EUZET et al. [20] thought that the presence of a single axoneme in the cestode
spermatozoon is an evolved character. Thus, they considered the Cyclophyllidea as derived from
the Proteocephalidea, then the Proteocephalidea and the Tetraphyllidea-Phyllobothriidae as issued
from the Tetraphyllidea-Onchobothriidae and lastly the Pseudophyllidea and the Tetrarhynchidea
as coming from the Haplobothrioidea. The Caryophyllidea and the Diphyllidea were of unknown
origin. On the other hand, FREEMAN [22] estimated the Tetraphyllidea-Phyllobothriidae as the
ancestors of the Cyclophyllidea, taking into consideration their post-embryonic development.
Schmidt [44], considered the Haplobothrioidea and the Tetrarhynchidea respectively as families
belonging to the Pseudophyllidea and the Trypanorhynchidea. BROOKS et al. [16], using 12
synapomorphic characters, subdivided the cestodes into five orders: Pseudophyllidea,
Nippotaeniidea, Proteocephalidea, Lecanicephalidea and Tetraphyllidea. Additionally, they
included the Caryophyllidea, the Cyclophyllidea and the Trypanorhynchidea, respectively, in the
Pseudophyllidea, the Proteocephalidea and the Tetraphyllidea. The phylogenetic systematic
diagram of BROOKS et al. (1991) [16] involves the coexistence of spermatozoa bearing one or
two axonemes in the same order (Table 1), thus showing a contradiction between spermatological
and morphological characters. Moreover, their propositions do not clarify the evolution of the
number of axonemes within the Cestodes. When considering the pattern of the posterior extremity
of the spermatic flagella, some important differences between the Proteocephalidea and the
Cyclophyllidea can be pointed out. In the Cyclophyllidea Thysaniezia ovilla [13], Stilesia
globipunctata [2], Hymenolepis nana [3], Moniezia expansa and Moniezia benedeni [4],
Retinometra serrata [5 ], Aporina delafondi [8] and Raillietina (R.) tunetensis [9], the central
element of the axoneme disappears before the simplification of doublets into singlets. On the other
hand, in the Proteocephalidean Sandonella sandoni [10], the posterior extremity of the
spermatozoon consists of the axonemal central element, surrounded by nine singlets that
correspond to the A microtubules which are in close contact with the plasmic membrane.
Figs 1-4. Attempted reconstruction of the different types of spermiogenesis in the Cestodes. Am, arched membranes; C,
centriole; Ca, centriolar-adjunct; Ce, cytoplasmic extension; Cm, cortical microtubules; F. flagellum; lb,
intercentriolar body; Sr, striated root. 1 a-d: First type of spermiogenesis with flagellar rotations (c) and
proximodistal fusions (d) as described in the Tetraphyllidea-Onchobothriidae [30, 33], the Proteocephalidea [48,
49J the Tetrarhynchidea [49] and the Pseudophyllidea [52]. 2 a-d: Second type of spermiogenesis with a flagellar
rotation (c) and a proximodistal fusion (d) as described in the Tetraphyllidea-Phyllobothriidae [32] and the
Caryophyllidea [49]. 3 a-d: Third type of spermiogenesis without flagellar rotation but with a proximodistal
fusion (d). The single flagellum grows outside but parallel to the cytoplasmic extension (c) then fuses with it (d) as
described in some species of the Cyclophyllidea: Nematotaenia chantalae [34], Mathevotaenia herpestis [7] and
Raillietina (R.) tunetensis [9]. 4 a-d: Fourth type of spermiogenesis with neither flagellar rotation nor
proximodistal fusion. The single flagellum grows inside the cytoplasmic extension (b-d) as described in some
other species of the Cyclophyllidea: Thysaniezia ovilla [13], Hymenolepis nana [3] and Aporina delafondi [8],
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
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C. T. BA & B. MARCHAND : CESTODA (PLATYHELM/NTHES)
5
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
93
Thus, it becomes obvious that the phylogenetic systematics of the Cestodes is a complex
subject and requires serious revision.
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Source . MNHN. Paris
Source : MNHN , Paris
Immunocytochemistry of Tubulin in
Spermatozoa of Platyhelminthes
Carlo IOMINI *
Olga RAIKOVA
& Jean-Lou
**, Nezha NOURY-SRAIRI ***
JUSTINE *
* Laboratoire de Biologic Parasitaire, Protistologie, Helminthologic,
Museum National d’Histoire Naturelle, 61 rue Buffon, F-7523I Paris cedex 05. France
** Laboratory of Evolutionary Morphology, Institute of Zoology, 199034 Saint Petersburg, Russia
*** Institut Agronomique et Veterinaire Hassan II. B.P. 6202, Rabat, Morocco
ABSTRACT
Indirect immunofluorescence and electron microscope post-embedding immunocytochemistry were used to detect tubulin
in spermatids and spermatozoa of various Platyhelminthes. General sperm morphology is exemplified by conventional
electron microscope observations on Gorgoderci sp. and Haematolpechus sp. The utility of tubulin indirect
immunofluorescence for the understanding of the filiform spermatozoa of Platyhelminthes is underlined by a comparison
between Echinostoma (Digenea) and Symsagittifera (Acoela). This technique might be useful for further comparative
research with phylogenetic implications. In the digenean Echinostoma sp., alpha- and beta-tubulin are detected in the
doublets of the 9+‘‘l” axonemes and in the cortical singlet microtubules, but alpha-acetylated-tubulin is present in the
axonemal doublets and absent in the cortical microtubules. The central core of the Platyhelminthes 9+*T’ axoneme is not
labelled by any of the three monoclonal anti-tubulin antibodies used (anti-alpha-, anti-beta- and anti-alpha-acetylated-
tubulin).
RESUME
Immunocytochimie de la tubuline dans les spermatozoides de Plathelminthes
L’ immunofluorescence indirecte et I’ immunocytochimie ultrastructurale post-inclusion ont etc utilisees pour detectcr la
tubuline dans les spermatides et les spermatozoides dc Plathelminthes varies. La morphologie generale des spermatozoides
est explicitee par des observations de microscopic electronique conventionnelle de Gorgodera sp. et Haematoloechus sp.
L’interet de V immunofluorescence indirecte de la tubuline pour la comprehension de la structure des spermatozoides
filiformes des Plathelminthes est souligne par une comparaison entre Echinostoma (Digenea) el Symsagittifera (Acoela).
Cette technique pourrait ctre utile pour une recherche comparce a but phylog£nique. Chez Echinostoma sp.. I ’alpha-
tubuline et la beta-tubuline sont detectees dans les doublets des axonemes 9+“!" et dans les singulets corticaux, mais la
tubuline acetylee est presente seulement dans les doublets des axon&mes et absente dans les singulets corticaux. L’element
central des axonemes 9+‘T” de Plathelminthes n’est marque par aucun des trois anticorps utilises (contre la tubuline alpha,
beta, el alpha-ac6tylee).
Iomini, C., Raikova, O., Noury-SraIri, N. & Justine, J.-L., 1995. — Immunocytochemistry of tubulin in
spermatozoa of Platyhelminthes. In: Jamieson, B. G. M., Ausio, J.. & Justine, J.-L. (eds), Advances in Spcrmatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 97-104. Paris ISBN : 2-85653-225-X.
98
C. IOMINI. O. RAIKOVA. N. NOURY-SRAlRl & J.-L. JUSTINE : TUBULIN (PLATYHELMINTHES)
Tubulin, the protein constituting the microtubules, is one of the major proteins in
spermatozoa which have axonemes and/or microtubules. Tubulin shows a high degree of
heterogeneity owing to two factors, genetic diversity [21] and posttranslational modifications:
acetylation [19], glutamylation [8], detyrosylation [33], phosphorylation [10, 22] and the recently
discovered polyglycylation [28]. Because tubulins are well conserved during evolution [1] and
posttranslational modifications widely distributed among various species, antibodies raised
against tubulin or posttranslational modifications of one species generally recognize the same
epitope in other species and can thus be used as tool to investigate tubulin properties in many
systems.
In this study, we demonstrate that different anti-tubulin antibodies can differentiate distinct
subcellular population of microtubules in spermatozoa of Platyhelminthes. In addition,
immunocytochemistry of tubulin gives valuable information about the general morphology of the
spermatozoon, and new insight into the composition of the 9+“l” axoneme of the
Platyhelminthes. These results might be of phylogenetic interest when further comparative data
are obtained.
MATERIAL AND METHODS
Material. Digencans were collected from hosts which can be kept easily in the laboratory. Gorgodera sp. and
Haematoloechus sp. were collected from commercial frogs (Rana sp.) originating from various localities of Eastern
Europe. Echinostoma liei and Echinostoma caproni were collected from experimentally infested laboratory mice. The
Acoela Symsagitlifera schultzei was extracted from sand collected in Roscoff, France.
Light microscope immunocytochemistry. Germ cells were obtained by squashing worms in a drop of PBS
(phosphate buffer saline. Sigma) on a pit slide previously washed with alcohol and acetone. Slides were allowed to dry for
1 h under a fan. then kept at 4°C and processed within 24 h. Cells were pcrmcabilizcd in acetone (10 min) and rinsed (PBS.
3x5 min). Non-specific antigens were blocked with 2% Bovine Serum Albumin (Sigma) in PBS (BSA-PBS) for 45-90 min
at room temperature. A monoclonal anti-tubulin antibody (anti-alpha-tubulin, clone DM 1A. Sigma, or anti-beta-tubulin,
clone TUB 2.1, Sigma or anti-acetylated-tubulin, clone 6-1 IB- 1, Sigma [26]) diluted at 1/200 to 1/600 in BSA-PBS was
applied for 40 min at room temperature. After rinsing (PBS 3x5 min), a FITC-conjugated antibody (Goat anti-mouse,
Nordic, 1/40 in PBS) was applied for 40 min at room temperature. For double labelling, supplementary steps were added as
follows: anti-tubulin polyclonal antibody (Sigma, ref. T-3526) diluted at 1/40 in BSA-PBS applied for 40 min at room
temperature, rinsed (PBS 3x5 min) and followed by a TRITC-conjugated antibody (Goat anti-rabbit, Nordic, 1/40 in PBS)
for 40 min at room temperature. The nuclear dye Hoechst 33258 (1 pg/ml in PBS, 10 min) or DAPI (1 }ig/ml, 10 min) was
sometimes used for labelling the nucleus. After a final rinse (PBS 3x5 min), mounting was done in Citifluor (Citifiuor
Ltd, London, UK) and slides were sealed with nail enamel. Controls were done by omitting the first antibody or by using a
non-relevant mouse antibody. Observations were made with a Nikon Optiphot epi fluorescence microscope equipped with a
mercury lamp and a single band Nikon filter for FITC channel (B-2A), TRITC channel (G-2A) or Hoechst channel (UV-1 A),
or a double-band (FITC/TRITC) Omega filter (XF 52).
Conventional electron microscopy. Living specimens cut into pieces were fixed for 1 h in 2% glutaraldehyde in a
buffer solution of 0.1 M sodium cacodylate at pH 7.2 at 4°C. After rinsing in the same buffer, the worms were postfixed
for I h in 1% osmium tetroxide in the same buffer, dehydrated in ethanol and propylene oxide, and embedded in Spurr’s
medium or in Epon. Ultrathin sections were contrasted with Daddow’s method [7] or with conventional lead citrate and
uranyl acetate, and observed with a Hitachi H600 electron microscope.
Post-embedding electron microscope immunocytochemistry. Living worms were fixed for 1 h in 2% glutaraldehyde
in a buffer solution of 0.1 M sodium cacodylate at pH 7.2 at 4°C. After rinsing in the same buffer (3 x 10 min), the worms
were embedded in LR White resin (medium grade). The resin was polymerized in tightly capped gelatin capsules for 10 h at
60°C. Ultrathin sections were placed on nickel or gold grids. Grids were rinsed (PBS, 3x5 min), and non-specific antigens
were blocked with goat immunoglobulins (Sigma) diluted at 1/30 in PBS for Ih. A monoclonal anti-tubulin antibody (anti¬
alpha-tubulin, clone DM 1A, Sigma, or anti-beta-tubulin, clone TUB 2.1, Sigma or anti-acetylated-tubulin, clone 6-1 IB-
1, Sigma [26]) diluted at 1/100 in PBS, was applied for 40 min at room temperature. After rinsing (PBS, 3x5 min), a gold-
conjugated antibody (Goat anti-mouse, 15 nm gold beads, 1/20 in PBS) was applied for 1 h at room temperature. After a
final rinse (PBS 3x5 min. then distilled water, 3x5 min), the grids were dried on Whatman paper, stained with Daddow’s
method [7] and observed with a Hitachi H600 electron microscope.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
99
RESULTS
Conventional electron microscopy (Fig. 1)
Results of conventional electron microscopy are briefly presented in this paper to show the
structure described below with electron microscope immunocytochemistry. In the digeneans,
spermiogenesis is homogeneous in most species [16, 17]. The spermatid shows a protrusion
called the zone of differentiation (Fig. la). At the distal extremity of this zone of differentiation,
three processes elongate: a median cytoplasmic process, and two flagella. The median cytoplasmic
process contains singlet microtubules aligned below the membrane (Fig. lb). Each flagellum
contains an axoncme with the 9+‘T” trepaxonematan structure (Fig. lc). The spermatozoon (Fig.
lc) is later produced by the fusion of the three processes, and contains two 9+“l” axonemes,
dorsal and ventral microtubules, an elongate mitochondrion and an elongate nucleus. The 9+“l”
axonemes (Fig. lb, c) do not contain the pair of central microtubules of the almost ubiquitous 9+2
axonemes but have, instead, a solid central core.
Fig. 1. — Conventional electron microscopy of digenean spermatozoa, a, b: Gorgodera sp. a: Zone of differentiation
(ZD) in spermatid, longitudinal section showing median cytoplasmic process (MCP) and flagella (one sectioned
here, F) which will fuse to produce the mature spermatozoon, b: Median cytoplasmic process with singlet
microtubules (T) and free flagella containing 9+“l” axonemes (A), transverse section, c: Haematoloechus sp.,
mature spermatozoon, transverse section. A, axonemes with 9+“l” trepaxonematan pattern; M, mitochondrion; N,
nucleus; T, dorsal and ventral microtubules, a, x 20 000; b, x 48 000; c. 125 000.
100 C. IOMINI. O. RAIKOVA, N. NOURY-SRAIRI & J.-L. JUSTINE : TUBUIJN (PLATYHELM1NTHES)
Light microscope immunocytochemistry ( Fig. 2)
Digenean spermatozoa, in all species studied, are long, filiform cells. Spermatozoa contain
two parallel axonemes. These are labelled by the anti-tubulin antibodies (Fig. 2a) and appear as
two closely parallel lines in some cases, but often as a single thick line. In some cases, the
process of drying and re-hydrating during processing partly alters the cell and, in certain regions
of the spermatozoon, two lines, sometimes widely separate, are visible. The use of various
antibodies and the technique of double labelling (Fig. 2b, c) reveal that tubulin epitopes are
different in these two microtubular structures. The two lines are labelled by the polyclonal anti¬
tubulin (Fig. 2b), the anti-alpha-tubulin (Fig. 2a) and the anti-beta-tubulin monoclonal antibodies,
but the monoclonal anti-acetylated-alpha-tubulin antibody labels only one line (Fig. 2c). This
shows that two distinct subpopulations of microtubules are present, but interpretation of the
nature of these two lines requires the use of electron microscope immunocytochemistry, described
below.
Acoel spermatozoa also contain two parallel axonemes, which do not show the 9+“l”
pattern but instead a 9+0 or 9+2 pattern [27], Immunocytochemistry (Fig. 2d) reveals that these
axonemes are widely separated and show longitudinal undulations. The axonemes are visible with
the three antibodies used.
Fig. 2. — Light microscope immunocytochemistry of
platyhelminth spermatozoa, a-c: Echinostoma
caproni (Digenea). a: View of spermatozoon
showing filiform pattern. Anti-alpha-tubulin
labelling, n, nucleus (evidenced by a nuclear dye,
not shown here). Two lines are visible in limited
regions of the cell (arrows), b, c: double
labelling with polyclonal anti-tubulin antibody
(b) and monoclonal anti-acetylated-alpha-tubulin
antibody (c) in a region where two lines are
visible. Acetylated-alpha-tubulin is detected in
one line only. Interpretation of these labelling
requires electron microscope
immunocytochemistry (see Fig. 3 and
Discussion), d: Symsagiitiferci schultzei
(Acoela), anti-alpha-tubulin antibody. The two
axonemes are widely separated and undulate, a, d,
x 600; b, c, x 1 200.
Electron microscope immunocytochemistry. (Fig. 3)
Axonemal microtubules and cortical singlet microtubules show striking differences in their
labelling. The anti-alpha tubulin and the anti-beta-tubulin antibodies label the cortical singlets
together with the axonemal microtubules (Fig. 3a, b, d, e). The anti-acetylated-alpha-tubulin
antibody (Fig. 3c, f, g) labels only the axonemal microtubules ; the cortical singlets are not
labelled.
None of the antibodies labels the central core of the 9+“l” axoneme (Fig. 3a-g).
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
101
Fig. 3. — Electron microscope immunocytochemistry of spermatids of Echinostoma liei (Digenea). a-c: Median
cytoplasmic process and flagella, transverse section (compare with figure lb), a: anti-alpha-tubulin, b: anti¬
beta-tubulin. c: anti-acetylated-alpha-tubulin. d-g: Spermatids, longitudinal section, d, e: anti-beta-tubulin;
f, g: anti-acetylated-alpha-tubulin. Axonemal microtubules are labelled in each case, but singlet microtubules
(T) in the median cytoplasmic process are labelled by the anti-alpha and anti-beta-tubulin antibodies but not by the
anti-acetylated-alpha-tubulin antibody. The central core (star) of the 9+*T’ axoneme is never labelled by the anti¬
tubulin antibodies, a-c, x 50 000; d-f, 70 000; g, x 60 000.
Source MNHN , Paris
102
C. 10MINI. O. RAIKOVA, N. NOURY-SRAIRI & J.-L. JUSTINE : TUBULIN (PIATYHELMINTHES)
DISCUSSION
Tubulin immunocytochemistry: an innovative technique for understanding the general
morphology of sperma tozoa
Spermatozoa of Platyhelminthes are generally very long and filiform [16, 17], without a
“head” and a “tail”, and thus are chiefly described by means of cross sections. Ordering the
various cross sections along a scheme of the sperm body from random sections is not trivial and
serial sectioning is very time consuming. Anti-tubulin immunocytochemistry, although a routine
technique in cell biology, has not previously been used for purely morphological studies. This
technique allows a view of the whole filiform spermatozoon and of the arrangement of the
microtubular organelles along its length. The two examples presented in Figure 2 show the
differences between a “turbellarian” spermatozoon, here an acoelan, with almost independent
axonemes which undulate along the cell, and a digenean, with very close axonemes. Anti-tubulin
immunocytochemistry is an innovative technique for the understanding of spermatozoa in two
very different cases: filiform spermatozoa which are almost “two-dimensional” such as those of
Platyhelminthes (this study), and complex three-dimensional spermatozoa such as those of
crustaceans [24, 25, 34].
Tubulin subpopulations in Platyhelminthes spermatozoa: axonemes versus cortical microtubules
The results of electron microscope immunocytochemistry allows the interpretation of the
double labelling observed in Echinostoma with indirect immunofluorescence (Fig. 2b, c) as
follows: one line, labelled by all the antibodies used, is made up of the two closely associated
axonemes; the other line, labelled by all antibodies except the anti-acetylated-alpha-antibody,
represents the singlet microtubules.
The location of tubulin in Platyhelminthes has received little attention [32], Results
presented here show that different tubulin epitopes are located in the axonemes and in the cortical
microtubules. The detection of alpha and beta-tubulin in the axonemes was expected, since alpha
and beta tubulin are constituents of the microtubules. It, however, reveals that anti-tubulin
antibodies developed against non-Platyhelminthes antigens can be used for this purpose.
The differential location of acetylated tubulin (present in axonemes, absent in cortical
microtubules) is a more interesting finding. Posttranslational varieties of tubulin have been
investigated in spermatozoa of only a few species: glutamylated in mammals [1 1, 18], tyrosinated
in sea urchin, man [13] and rat [15], polyglycylated in Drosophila [2], Acetylated tubulin has
been detected in male germ cells of sea urchin [26], insects [26, 35], fish [20], and various
mammals including man [11, 12, 15], but is apparently absent in the transient microtubules of
aflagellate nematode spermatids (MANS1R & Justine, this volume) [23]. In mammals, a
subcellular partition of acetylated tubulin has been found: it is present in axonemes, but not in the
singlet microtubules of the manchette [1 1, 12, 15]. The finding reported here show a similarity
between mammalian spermatids and platyhelminth spermatozoa, both having acetylated axonemes
and non-acetylated cortical microtubules.
The absence of tubulin in the central core of the 9+ “1 ” axoneme
The 9+“l” axonemal structure is a synapomorphy for the Trepaxonemata [9], Although the
9+“l” axoneme has been the subject of detailed studies with various methods of electron
microscopy [3-6, 14, 29-31], the chemical nature of the central core has not been investigated.
This study is, as far as we know, the first attempt to characterize the proteins present in the central
core. The central core of the 9+‘T” axoneme is not labelled by any of the three monoclonal anti¬
tubulin antibodies used (anti-alpha-, anti-beta- and anti-alpha-acetylated-tubulin), therefore
suggesting the absence of tubulin in the central core. The absence of tubulin in the central core, if
confirmed, would underline the uniqueness of the 9+“l” trepaxonematan structure in the Animal
Kingdom.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
103
ACKNOWLEDGEMENTS
Dr Annie Fournier (Perpignan) generously provided the specimens of Echinostoma. Dr Jean-Louis ALBARET (Paris)
helped in the identification of frog digeneans and in various techniques. Professor Claude Petter (Paris) generously
provided the frogs. Dr Franck Gentil (Ro scoff) kindly collected the sand. Dr Denise Escalier ( Kremlin- Bicetre) gave us the
original encouragement and advice for immunocytochemistry. Dr Laura Gea helped for electron microscope
immunocytochemistry. The author gratefully acknowledge the valuable comments and suggestions received from Dr Anne
Fleury (Orsay) and Professor Bjorn Afzeuus (Stockholm). Partially funded by an INTAS grant (n° 93-2176,
“Ultrastructure and immunocytochemistry of the cytoskeleton of spermatozoa, eggs and fertilization in selected
invertebrates species, for the understanding of Phytogeny") to O. R. and J.-L. J., a CNRS/CNR grant to N. N. S., a
B. Q. R. grant from the Museum national d’histoire naturelle to J.-L. J., and an international student grant from the
University of Milano (Italy) to C. I.
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Source MNHN, Paris
Comparative Spermatology of Gastrotricha
Marco FERRAGUTI* & Maria BALSAMO **
* Dipartimento di Biologia,
Universita di Milano, Via Celoria 26, I - 20133 Milano, Italy
** Dipartimento di Biologia Animale,
Universita di Modena, Via Universita, 1-41100 Modena, Italy
ABSTRACT
Sperm morphology of nine gastrotrich species belonging to the two orders Macrodasyida and Chaetonotida has been
examined. By comparison with the limited data in the literature it has been possible to determine a sperm model for
macrodasyids: long, corkscrew-shaped acrosome often showing tw'o different portions and containing a striated tube;
spring-shaped nucleus surrounding the mitochondria; tail with an axoneme surrounded by a striated cylinder. Within
Chaetonotida the spermatozoa are highly diverse: in the family Xenotrichulidae they are characterized by an uncondensed
nucleus, two extremely long paraacrosomal bodies, a single mitochondrion and accessory fibres in the tail. The other
chaetonotid families show- extremely atypical spermatozoa, formed by simple rods of chromatin. There are no evident
characters in common among the spermatozoa belonging to the two orders, nor similarities with other aschelminth sperm
models.
RESUME
Spermatologie comparee des Gastrotricha
La morphologic du spermatozoide a £te 6tudiee dans neuf espfcces de Gastrotriches appartenant aux deux ordres
Macrodasyida et Chaetonotida. Grace a une comparison avec les donnees limitees de la literature, il a etc possible de
definir un modcle spermatique pour les Macrodasyida: long, avec un acrosome en tire-bouchon qui presente souvent deux
portions et contient un tube strie, un noyau helicoidal entourant la mitochondrie, une queue avec un axoneme entoure par
un cylindre strie. Chez les Chaetonotida le spermatozoide est tres diversifie: dans la famille Xenotrichulidae, il est
caracterise par un noyau non condense, deux corps para-acrosomaux extremement longs, une mitochondrie unique et des
fibres accessoires dans la queue. Les autres families de Chaetonotida montrent des spermatozoides tres atypiques, formes
par de simples baguettes de chromatine. Il n’y a pas de caractfcres spermatologiques en commun unissant les deux ordres. ni
de similarites avec les modeles spermatiques des autres Aschelminthes.
Gastrotricha are small (80 (iin - 1 .5 mm) pseudocoelomate animals living in the sediments
of aquatic environments. The order Macrodasyida comprises about 230 species exclusive of
marine and brackish interstitial environments, whereas the order Chaetonotida, with about 320
species, is mainly freshwater dwelling, both in interstitial and epibenthic habitats. The two orders
have different reproductive modes: Macrodasyida are all amphimictic and hermaphrodite, whereas
Chaetonotida are thought to be mainly parthenogenetic with the exception of three hermaphrodite,
marine genera [3].
FERRAGUTI, M., & Balsamo, M., 1995. — Comparative spermatology of Gastrotricha. In: Jamieson. B. G. M.,
Ausio, J., & JUSTINE. J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. rutin Hist, ncit., 166:
105-117. Paris ISBN: 2-85653-225-X.
106
M. FERRAGUTI & M. BALSAMO : GASTROTRICHA
Table 1. — List of gastrotrich species included in the present review and relevant literature references.
Gastrotrich spermatozoa are only imperfectly known (Table 1). In fact only two sperm
models of Macrodasyida have been completely described [6, 12, 13], and among Chaetonotida
we know with some details only the extremely atypical, perhaps relictual spermatozoon of
Lepidodermella squamata [9]. Recently some data on the xenotrichulid spermatozoa have been
reported [7], Data on other species are scattered, often restricted to single micrographs, and in
some cases inconsistent even within single families. With this poor knowledge, delineation of a
generalized sperm model for Gastrotricha is extremely difficult.
In the order Macrodasyida the spermatozoa are filiform cells comprising a sequence of
acrosome, nucleus and tail, and devoid of any recognizable midpiece (Fig. 3). Both acrosome and
nucleus are corkscrew-shaped: the acrosome shows different regions with complex
differentiations [6, 10, 12, 13]; the nucleus is spring-shaped in the lepidodasyid Mesodasys
laticaudatus [6] and in the turbanellid Turbanella comuta [12, 13], the nuclear spring surrounding
the mitochondria in both species. In the lepidodasyd Cephcilodasys maximus , the nucleus is
Fig. 1. — Ultrastructural features of macrodasyid gastrotrich spermatozoa, a: Tetranchyroderma sp. 1: anterior part of an
acrosome with the striated tube, x 21 000; b: Diplodasys ankeli : base of the acrosome with the wider portion of
the striated tube. A further tubular structure possibly continuous with the acrosome coils around the nucleus
(arrowheads), x 40 000; c, d: Pseudostomellci etrusca : main (c) and basal (d) portion of the acrosome: the striated
tubule ends in a dense material (asterisk), x 40 000; e: Turbanella ambronensis : anterior (A) and basal (B) part of
the acrosome; the obliquely striated sheath surrounding the basal portion of the acrosome is visible in grazing
sections (arrows), x 45 000; f: Diplodasys ankeli: basal part of the nucleus (arrows) surrounding the
mitochondrion (M) and involved by the coiled tubular structure (arrowheads). Note the curious structure at the base
of the flagellum (compare with Fig. 2a). x 40 000; g: Pseudostomella etrusca nuclear region: in this species the
chromatin is reduced to a thin lamina (arrows) involving the cytoplasm with some mitochondria (M). x 40 000;
h: Turbanella ambronensis: the nucleus (arrow) is spring-shaped and surrounds a single, long mitochondrion (M).
x 60 000.
Source MNHN, Paris
fetf&ftj
ADVANCES IN SPERMATOZOALPHYLOGENY AND TAXONOMY
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Source : MNHN , Paris
108
M. FERRAGUTI & M. BALSAMO : GASTROTR1CHA
twisted in the apical portion and spring-shaped, containing the single mitochondrion only in the
basal portion [8], In contrast, in the macrodasyid Macrodasys sp. [10] the nucleus is straight and
surrounded by a mitochondrial helix involving also most of the acrosome. A similar situation was
reported also in Thaumastoderma sp. [11].
The tail has a 9+2 axoneme enclosed in a striated cylinder in all the macrodasyid species
studied so far with the sole exception of Turbanella cornuta [12, 13] and, perhaps, Acanthodasys
sp.[l 1] (however, the only micrograph published refers to spermatids).
For the order Chaetonotida we only have a single published micrograph of the
spermatozoon of the interesting genus Neodasys from the monogeneric family Neodasyidae [11]:
it shows a very simple and undifferentiated conical acrosome followed by a nucleus perhaps
surrounded by a mitochondrial helix. Of the three genera composing the family Xenotrichulidae,
Draculiciteria is parthenogenetic, whereas Xenotrichula and Heteroxenolrichula have very peculiar
spermatozoa [7] with a small and simple acrosome followed by an uncondensed rectilinear
nucleus, a single mitochondrion and a tail with nine peripheral accessory fibres. Furthermore, two
of the xenotrichulid species show extremely long paraacrosomal bodies of unknown function. All
the other chaetonotid families, comprising hundreds of species, are parthenogenetic. However,
hermaphroditic individuals of most genera have been discovered both in culture and in nature and
production of the spermatozoa during the long post-parthenogenetic phase of the life cycle has
been demonstrated in at least four genera of the family Chaetonotidae [3, 9]. Low numbers of
these spermatozoa are usually present, with four different morphologies. In Lepidodermella
squamata [9] and Chaetonotus maximus [3] the spermatozoa are only rods of condensed
chromatin surrounded by plasma membrane without organelles of any kind.
Thus, comparative spermatology of Gastrotricha is still in its infancy. For many years we
have undertaken a study of selected gastrotrich species to extend our knowledge of their
spermatozoa. However, the technical problems in working with such material (extremely small
size of these animals; difficult fixation; problems with embedding and orientation of specimens
within the resin; failure of attempts to rear macrodasyids in the laboratory) have impeded our
project. Knowing the importance of the spermatozoa as systematic characters in comparisons
within and among taxa, in this paper we will present some work in progress on previously
undescribed gastrotrich species. We will focus our attention on the family Thaumastodermatidae,
with Pseudostomella etrusca, three species of Tetranchyroderma, and Diplodasys ankelv, we will
describe a previously unknown spermatozoon from the Turbanellidae, Turbanella ambronensis ;
finally we will extend the description of the sperm of the three Xenotrichulidae [7]. We will
present some ultrastructural details of the species described and three-dimensional reconstructions
of four gastrotrich spermatozoa.
Fig. 2. — Ultrastructure of the spermatozoa of gastrotrichs. a-h: macrodasyids; i-1: chaetonotids. a, b: Diplodasys
ankeli: longitudinal (a) and cross (b) section of a tail to show the structure of the striated cylinder, x 60 000; c,
d: Pseudostomella etrusca: longitudinal sections of the crystalline structure situated between the nucleus (arrow)
and the tail. M, mitochondrion, x 60 000. e: Tetranchyroderma sp. 1: longitudinal, sagittal (top) and tangential
(bottom) section of the tail showing the structure of the striated cylinder, x 60 000; f: Tetranchyroderma sp. 2:
cross section of a tail showing the double striated cylinder, x 60 000; g, h: Turbanella ambronensis:
longitudinal (g) and cross (h) sections of a tail showing no striated cylinder. N, nucleus; B, basal portion of the
acrosome. x 60 000; i, j, k: Xenotrichula punctata: i: low power view of some spermatozoa: N. nucleus; A,
acrosome; P, paraacrosomal body; M. mitochondria, x 20 000; j: cross section of a tail. Note the prominent
accessory fibres, x 60 000; k: longitudinal section of an acrosome (bottom) and a paraacrosomal body (P).
x 60 000; I: Heteroxenolrichula squamosa: S.E.M. view of a whole spermatozoon. P. paraacrosomal body; A.
acrosome; N. nucleus; T. tail, x 2 800.
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M. FERRAGUT1 & M. BALSAMO : GASTROTRICHA
MATERIAL AND METHODS
Gastrotrichs were extracted from sandy sediments collected in some localities of the Tyrrhenian and Adriatic coasts
of Italy by decantation and narcotization with 7% MgCl2. Observations on living specimens were carried out by means of
a Leitz Dialux microscope equipped with Nomarski optics and phase contrast. Moving spermatozoa within the testes, and
after isolation, were recorded on videotape.
Transmission Electron Microscopy (TEM). Specimens were fixed in a 0.1 M phosphate buffered (pH 7.3) mixture
of paraformaldehyde, glutaraldchyde and picric acid with addition of sucrose (SPAF) [5], postfixed in 2% aqueous osmium
tetroxide, washed in 0.1 M cacodylate buffer, dehydrated in a graded acetone series, pre-stained en bloc with uranyl acetate
in 70% acetone and embedded in araldite. Sections were cut with an Ultrotome Nova LKB. triple stained following Daddow
[4], carbon coated and observed under a JEOL 100 XS electron microscope.
Scanning Electron Microscopy ( SEM ). The spermatozoa were isolated from living animals and fixed with the same
mixture used for TEM, dehydrated in a graded ethanol series, critical point dried with C02, coated with gold-palladium and
observed with a Philips XL40 or a Cambridge Stereoscan 250Mk2.
OBSERVATIONS
Macrodasyida
Family Thaumaslodennatidae . The spermatozoa of Pseudostomella etrusca (Fig. 4) show a
complex, corkscrew-shaped acrosome 5-7 pm long containing a matrix in which a tubular
striated structure is immersed (Fig. lc, d). The striated tube ends basally in an area of dense
material which forms the base of the acrosome (Fig. 1 d).The nucleus follows with the typical
shape of a hollow corkscrew (Fig. lg), about 20 pm long. The chromatin is visible as a 0. 1 pm
thick external sheath whereas the inner cavity contains some cytoplasm with numerous
mitochondria (Figs lg, 2c). Between the nucleus and the tail there is a crystalline structure, about
1.6 pm long and 0.5 pm in diameter (periodicity of about 13 nm) (Fig. 2c, d). The tail consists
of a 9+2 axoneme surrounded by a striated cylinder formed by a pile of rings about 13 nm thick
connected by thin threads (Fig. 2 d).
We have examined three species of the genus Tetranchyroderma : an unidentified species
from Tyrrhenian sea ( T . sp. 1); an undescribed species from the Maidive archipelago ( T '. sp. 2);
and T. papii from the Tyrrhenian sea. The acrosome of T. sp.l and T. papii show an apical
filament followed by a corkscrew region for a total length of approximately 8 pm ( T . papii). The
acrosome axis is a striated tube (Fig. 1 a) ending in a basal mass of dense material touching the
nucleus ( T . sp. 1). The ribbon-like nucleus coils around the single long mitochondrion in T.
sp. 1 . An additional tubular structure runs for two gyres around the nuclear apex in T. sp. 1
whereas it wraps around the whole nucleus in T. papii. The tubular structure continues the pitch
of the acrosome, and may also be structurally continuous with it. A crystalline structure similar to
the one already described in P. etrusca is visible in T. papii and in T. sp. 2, but is absent in T.
sp. 1 . The tail axis is a 9+2 axoneme surrounded by a striated cylinder formed by a pile of disks
15 nm thick connected by thin threads ( T . sp.l and T. sp 2) (Fig. 2e). In T. sp. 2 a second
external layer is present, similar in texture to the crystalline structure at the base of the tail. This
additional sheath is thicker (up to 120 nm) at the base of the flagellum (Fig. 2f), then flattens to a
size comparable to that of the striated cylinder. Images of “empty” tails, in which the striated
cylinder is present but there is no axoneme, suggest that the axoneme is shorter than the striated
cylinder.
Diplodasys ankeli has a long, thin, corkscrew-shaped acrosome (Fig. lb) in which two
regions can be recognized: the anterior one, 0.15 pm in diameter with a helical pitch of about
0.26 pm contains a twisted column with alternate dark and pale cross bands, whereas the basal
portion, about 1 pm long, contains a twisted striated tube. The following nucleus is a spring of
condensed chromatin surrounding the single mitochondrion (Fig. lb, f). A tubular structure
possibly continuous with the acrosome coils around the nucleus for its whole length (Fig. lb, f)-
The tail has a 9+2 axoneme enclosed by a striated cylinder formed by a pile of discs (Fig. 2 a, b).
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Fig. 3. — Schematic drawing of Mesodasys laticaudatus spermatozoon. A, anterior portion of the acrosome: the striated
column is visible in the detail (right); B, basal portion of the acrosome. A detail is visible at right; N, nucleus; M,
mitochondria; T, tail.
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M. FERRAGUTI & M. BALSAMO : GASTROTR1CHA
Family Turbanellidae. The acrosome of Turbanella ambronensis has two different regions
(Figs le, 5): the anterior one is thin (0.2 |im), corkscrew-shaped and contains a twisted axis
made of alternating dense and transparent bands; the basal portion is long and rectilinear and
shows an obliquely striated sheath surrounding the acrosome content (Fig. le). This is formed by
a pile of thick (60-80 nm) disks immersed in a transparent matrix. The nucleus is ribbon-like, D-
shaped in cross section (Fig. lh), and coiled to form a spring inside which a single, long
mitochondrion is located. The tail has a normal 9+2 axoneme but no striated cylinder (Fig. 2g, h).
Chaetonotida
Family Xenotrichulidae. We have studied the spermatozoa of three xenotrichulid species:
Heteroxenotrichula squamosa, Xenotrichula intermedia, and X. punctata. The spermatozoa of
these species have many characters in common, thus they will be described together. The
xenotrichulid spermatozoa are characterized by a sequence of acrosome, nucleus, mitochondrion
and tail (Fig. 6). The acrosome is a small structure (3.8 pm long with a diameter of 0.1 pm in
H. squamosa and 2.9 pm long in X. punctata) containing an acrosome vesicle and a large amount
of periacrosomal material (Fig. 2k, i, 1). Two projecting structures parallel to the acrosome are
inserted at the anterior extremity of the nuclear region in X. punctata and H. squamosa (Fig. 2i, 1)
where they are 20 pm long with a diameter of 0.2 pm . These structures are not surrounded by a
plasma membrane and consist of electron dense disks 0.1 pm thick connected by thin threads
(Fig. 2k); we have called them paraacrosomal bodies [7]. In the insertion area the plasma
membrane of the spermatozoon is uninterrupted [7], suggesting that the paraacrosomal bodies are
external to the cell. X. intermedia is an exception in having no acrosome nor paracrosomal bodies.
The nucleus is characterized by a scarcely condensed chromatin in the three species examined.
Only in X. punctata scattered dense globules of condensed chromatin were observed (Fig. 2i),
whereas in the other species examined the chromatin resembles that of a somatic cell. A single,
conventional mitochondrion of variable length follows the nucleus (Figs 2i, 6). A short tail is the
last portion of the xenotrichulid spermatozoon. In its principal tract the axoneme is surrounded by
nine large electron dense accessory fibres which are external to and in correspondence with each
axonemal doublet (Fig. 2j). They have a somewhat striated appearance in grazing longitudinal
sections whereas in cross section they appear pear-shaped with the thinner extremity directed
towards the cell membrane and the larger one towards the doublets. The last portion of the
X. intermedia spermatozoon shows a short tract of the axoneme devoid of accessory fibres.
DISCUSSION
Macrodasyida
The spermatozoa of members of four macrodasyid families have been examined up to now:
Lepidodasyidae, Macrodasyidae, Thaumastodermatidae, and Turbanellidae (Table 1). The
spermatozoa of Lepidodasyidae, Thaumastodermatidae (with the reported exception of
Thaumastoderma [11]) and Turbanellidae have a common general architecture: the acrosome is at
least in part corkscrew-shaped and contains various materials with different aspects; the nucleus is
always, at least in part, spring-shaped and contains some cytoplasm and one or more
mitochondria; a true basal body is lacking. Interspecific variations affect the acrosome which may
be divided in two portions, the anterior one containing a twisted axis made of dense and pale
disks, as in Mesodasys laticaudatus [6], D. ankeli and T. ambronensis. The basal portion of the
acrosome may be rectilinear (T. ambronensis, M. laticaudatus ) and contain a hollow striated tube,
as in D. ankeli and Cephalodasys maximus [8], A twisted hollow striated tube may run for the
whole length of the acrosome, as in P. etrusca and T. sp.l where a mass of dense material is also
present at the base of the acrosome. Cephalodasys maximus has an apical acrosome vesicle
protruding anteriorly [8]. If this report were confirmed, the real nature of the long structure
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
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Fig. 4. _ Schematic drawing of Pseudostomella etrusca spermatozoon. AC, acrosome: in the enlarged detail (right) the
inner structure of the acrosome is visible; N, nucleus; M, mitochondria; C, crystalline structure; T, tail.
Source : MNHN, Paris
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M. FERRAGUTI & M. BALSAMO : GASTROTRICHA
Fig. 5. — Schematic drawing of Turbanella ambronensis spermatozoon. A, anterior portion of the acrosome with the
striated column inside (detail at right); B, basal portion of the acrosome with the obliquely striated sheath; N,
nucleus; M, mitochondria; T, tail.
Source : MNHN . Pahs
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Fig. 6. — Schematic drawing of Heteroxenotrichula squamosa spermatozoon. AV, acrosome vesicle; PB, paraacrosomal
body; N, nucleus; M, mitochondrion; T, tail.
Source : MNHN, Paris
116
M. FERRAGUTI & M. BALSAMO : GASTROTRICHA
defined as an “acrosome” in this species would remain to be established. The acrosome of
Macrodasyidae briefly described by RUPPERT [10] may share some features with that of the other
Macrodasyida. In Macrodasys sp.. in fact, the acrosome has two portions: the basal one with a
“hollow central acrosomal filament" and the anterior one with the filament widening and twisting.
A marked difference lies in the mitochondrial spiral winding around the acrosome in Macrodasys.
The sperm nucleus of all the species, with the exception of Macrodasys sp. and, reportedly, of
Thaumastoderma sp.[l 1] is a spiral surrounding the cytoplasm. Variations concern the number
and morphology of mitochondria and the presence of the additional tubular structure coiled around
the nucleus. This additional tubular structure runs along the whole nucleus in some species but is
restricted to the nuclear apex in others and may be continuous with the acrosome. In
Cephalodasys maximus, part of the acrosome is reported to involve the first gyre of the nuclear
helix [8]. Mitochondria may retain their conventional aspect, as in P. etrusca , or be fused in a
single, twisted column-shaped, mitochondrion filling the whole space delimited by the nuclear
spiral, as in Turbanella (this study and [12, 13]) and Cephalodasys [8].
The tail shows a characteristic sheath called the striated cylinder [6], or the spiralled band
[8], enclosing the 9+2 axoneme in all families except the Turbanellidae. In Cephalodasys
maximus in addition to the striated cylinder there are nine dense strings [8] (probably accessory
fibres), one corresponding with each flagellar doublet. In this last species, a manchette of oblique
microtubules is reported beneath the flagellar plasma membrane.
Chaetonotida
The spermatozoa of Chaetonotida show a much greater interfamilial diversity with respect to
Macrodasyida. The sperm models of Neodasys [11], Xenotrichulidae ([7] and this chapter), and
Chaetonotidae[3, 9] studied so far are different from one another and bear no resemblance to
those of Macrodasyida. In particular the spermatozoa of Xenotrichulidae are unique not only
among Gastrotricha , but in the entire animal kingdom in the large paraacrosomal bodies (not
present, for unknown reasons, in one of the three species examined). The other outstanding
characters of the xenotrichulid spermatozoa are: the presence of a single, untransformed
mitochondrion placed between the nucleus and the tail and of the large accessory fibres of the
flagellum. The shape and position of the single mitochondrion is a curious convergence with leech
spermatozoa [14], whereas the accessory fibres are an independent invention of many
evolutionary lineages with internal fertilization [2].
Present knowledge of gastrotrich sperm morphology does not allow the delineation of a
general sperm model for Gastrotricha. Furthermore, if some characters are common among most
Macrodasyida (complex corkscrew-shaped acrosome, spring-shaped nucleus containing
mitochondria, striated cylinder in the tail), the three Chaetonotida models known so far present a
puzzling diversity.
Comparisons with the spermatozoa of other aschelminthes are also deceiving. The general
architecture of gastrotrich spermatozoa seems unique among pseudocoelomates. We may note that
if the tubular spiral structure involving the nucleus of some Thaumastodermatidae is proved to be
continuous with the acrosome, this could be an important resemblance with the spermatozoon of
the priapulid Tubiluchus corallicola [1],
REFERENCES
1 . Alberti, G. & Storch, V. 1983. — Fine structure of developing and mature spermatozoa in Tubiluchus (Priapulida,
Tubiluchidae). Zoomorphology , 103: 219-227.
2. BACCETTI, B., 1982. — The evolution of the sperm tail. In: W. B. AMOS & J. G. DUCKETT, Prokaryotic and
Eukaryotic Flagella. Cambridge, Cambridge University Press: 521-532.
3. Balsamo, M., 1992. — Hermaphroditism and parthenogenesis in lower Bilateria: Gnathostomulida and
Gastrotricha. In: R. Dallai, Sex Origin and Evolution. Modena, Mucchi: 309-327.
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
117
4. DADDOW, L., 1986. — An abbreviated method of the double lead stain technique. Journal of Submicroscopic
Cytology , 18: 221-224.
5. Ermak, T. H. & Eakin, R. M., 1976. — Fine structure of thecerebral pygidial ocelli in Chone ecaudata (Polychaeta:
Sabellidae). Journal of Ultrastructure Research , 54: 243-260.
6. FERRAGUTI, M. & Balsamo, M., 1994. — Sperm morphology andanatomy of the genital organs in Mesodasys
laticaudatus Remane, 1951 (Gastrotricha: Macrodasyida). Journal of Submicroscopic Cytology and Pathology,
26:21-28.
7. Ferraguti, M., Balsamo, M. & Fregni, E., 1995. — The spermatozoa of three species of Xenotrichulidae
(Gastrotricha, Chaetonotida): the two “diinne Ncbcngeisscln” of spermatozoa in Heteroxenotrichula squamosa
are peculiar paraacrosomal bodies. Zoomorphology, in press.
8. Fischer, U., 1994. — Ultrastructure of spermiogenesis and spermatozoa of Cephalodasys maximus (Gastrotricha,
Macrodasyida). Zoomorphology, 114: 213-225.
9. Hummon, M. R., 1984. — Reproduction and sexual development in a freshwater gastrotrich. 2. Kinetics and fine
structure of postparthenogenetic sperm formation. Cell and Tissue Research, 360: 619-628.
10. Ruppert, E. E., 1978. — The reproductive system of gastrotrichs. II. Insemination in Macrodasys : a unique mode of
sperm transfer in Metazoa. Zoomorphologie, 89: 207-228.
1 1. Ruppert, E. E., 1991. — Gastrotricha. In: F. W. HARRISON & E. E. Ruppert, Microscopic Anatomy of Invertebrates.
Vol. 4. Aschelminthes. New York, Wiley-Liss: 41-109.
12. Teuchert, G., 1975. — Differenzierung von Spermien bei den marinen Gastrotrich Turbanella cornuta Remane
(Ordnung Macrodasyoidea). Verb. Anat. Ges., 69: 743-748.
13. Teuchert, G., 1976. — Elektronenmikroskopische Untersuchung iiber die Spermatogenese und
Spermatohistogenese von Turbanella cornuta Remane (Gastrotricha). Journal of Ultrastructure Research, 56:
1-14.
14. Wissocq, J. C., & Malecha, J., 1975. — Etude des spermatozoVdes d’hirudin£es h l’aide de la technique de coloration
negative. Journal of Ultrastructure Research, 52: 340-361.
Source : MNHN. Paris
Source : MNHN, Paris
Centrioles with Ten Singlets in Spermatozoa of the
Parasitic Nematode Heligmosomoides polygyrus
Ai'cha MANSIR & Jean-Lou JUSTINE
Laboratoire de Biologie Parasitaire, Protistologie, Helminthologie,
Museum National d'Histoire Naturelle, 61 rue Buffon, F-75231 Paris cedex 05, France
ABSTRACT
The spermatozoon of Heligmosomoides polygyrus is elongate and aflagellate. The nucleus, in a posterior position, is
arrowhead-shaped and has an anterior fossa. Two centrioles, which are the only microtubular organelles of the mature
spermatozoon, are mutually perpendicular in the nuclear fossa. These two centrioles are made up of 10 singlets, closely
arranged, forming an ellipse 140 x 160 nm. This is the first description of a centriole with 10 singlets.
Immunocytochemistry demonstrates that spermatids have a transitory system of microtubules converging toward the
centrioles. Spermatozoal centrioles may provide additional character for the understanding of nematode phylogeny.
RESUME
Centrioles a dix singulets dans les spermatozoi'des du nematode parasite Heligmosomoides
p oly gy rus
Le spermatozoi'de de Heligmosomoides polygyrus est allonge et aflagell£. Le noyau, place post£rieurement, est en forme
de pointe de fleche et pr£sente une fossette & sa partie anterieure. Deux centrioles, qui sonl les seuls organites composes de
microtubules dans le spermatozoi'de mur, sont disposes perpendiculairement dans la fossette nucleaire. Ces deux centrioles
sont composes de 10 singulets juxtaposes, formant une ellipse de 140 x 160 nm. Ceci est la premiere description d'un
centriole ^ 10 singulets. L’immunocytochimie montre que les spermatides possedent un systfcme transitoire de
microtubules convergeant vers les centrioles. Les centrioles des spermatozoi'des peuvent fournir de nouveaux caractferes
pour la comprehension de la phylog6nie des Nematodes.
The spermatozoa of the Nematoda are all aflagellate. Axonemes are always absent but
microtubules are sometimes present during spermiogenesis. In mature spermatozoa, microtubules
have been described only in a few species [6]. Table 1 is a list of species in which the
spermatozoon has been described by electron microscopy.
In Heligmosomoides polygyrus , a parasitic nematode often used in laboratory experiments,
we found an outstanding centriolar structure, which is described here. The spermatozoon has
been described previously in this species [81, 84], but centriolar structure was not addressed.
In addition, the present paper gives some information on the fate of the microtubular system
of spermatids, observed by mean of immunocytochemistry of tubulin.
Mansir, A., & Justine, J.-L., 1995. — Centrioles with ten singlets in spermatozoa of the parasitic nematode
Heligmosomoides polygyrus. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hisi. nat., 166 : 119-128. Paris ISBN : 2-85653-225-X.
120
A. MANSIR & J.-L. JUSTINE : HELIGMOSOMOIDES POLYGYRUS (NEMATODA)
MATERIALS AND METHODS
Material. Samples of Heligmosomoides polygyrus (Dujardin, 1845) were obtained from laboratory white mice,
strain CDL This species is sometimes named Nematospiroides dubius but this is a junior synonym [20, 21]. The strain
used here belongs to the subspecies Heligmosomoides polygyrus bakeri Durette-Dcsset, Kinsella & Forrester, 1972 [19],
originally collected from Peromyscus maniculatus in North America and transferred to laboratory mice, strain CDL in
which it has been maintained for more than twenty years [46]. Adults were collected two to four weeks after oral infestation
with 300 larvae. Males were isolated under a binocular microscope and kept for a few hours in salt water (NaCl 9 %c).
Electron microscopy. Living specimens were placed in cold (4 °C) fixative, the head being immediately cut to
allow the fixative to penetrate into the body. Specimens were fixed for 1 h in 2% glutaraldehyde in a buffer solution of
0.1 M sodium cacodylate at pH 7.2 at 4°C. After rinsing in the same buffer, the worms were postfixed for 1 h in 1%
osmium tetroxide in the same buffer, dehydrated in ethanol and propylene oxide, and embedded in Spurr’s medium [69].
Ultrathin sections were contrasted with Daddow’s method [15], and observed with a Hitachi H600 electron microscope.
Immunocytochemistry. Germ cells were obtained by dissecting each male in a drop of PBS (phosphate buffer
saline. Sigma) on a pit slide previously washed with alcohol and acetone. The genital system was gently pressed with thin
needles to release germ cells. Slides were kept in a humid chamber for 1-2 hours to allow cells to sink and adhere to the
slide. The PBS was then removed and replaced by a drop of 3.7% formaldehyde in PBS for fixation. After 15 min the pit
was rinsed with PBS (3x5 min). Cells were then permeabilized in 0.1% Triton X-100 in PBS and rinsed (PBS, 3x5 min).
Non-specific antigens were blocked with 2% Bovine Serum Albumin (Sigma) in PBS (BSA-PBS) for 45-90 min at room
temperature. Without intermediary washing, a monoclonal anti-tubulin antibody (anti-alpha-tubulin, clone DM 1A,
Sigma, or anti-beta-tubulin, clone TUB 2.1, Sigma or anti-acetylated-tubulin, clone 6-11B-1, Sigma) diluted at 1/200 in
BSA-PBS was applied for 40 min at room temperature. After rinsing (PBS 3x5 min) the FITC-conjugated antibody (Goat
anti-mouse, Nordic, 1/40 in PBS) was applied for 40 min at room temperature. After a final wash (PBS 3x5 min),
mounting was done in Citifluor (Citifluor Ltd, London, UK) and slides were sealed with nail enamel. Controls were done by
omitting the first antibody or by using a non-relevant mouse antibody. Observations were made with a Nikon Optiphot
cpi fluorescence microscope equipped with mercury lamp and a single band Nikon filter for FITC channel (B-2A).
RESULTS
Electron microscopy of mature spermatozoa (Fig. 1)
The spermatozoon of Heligmosomoides polygyrus , observed in the testis, is an elongate
and a flagellate cell (Fig. la), about 17 pm in length and 3 pm in width. It comprises three
region: 1. The anterior region, opposite the nucleus, is devoid of organelles and contains a
fibrillar cytoplasm. It is known that this region produces pseudopods in activated spermatozoa
and is functionally anterior [81, 84]. 2. The median region contains round mitochondria and
membranous organelles, which are similar to those described in other nematode spermatozoa. 3.
The posterior region contains an arrowhead-shaped nucleus, with highly condensed chromatin.
The nuclear envelope is absent, as in most nematodes. The anterior fossa of the nucleus contains
two centrioles, which are the only microtubular elements of the mature spermatozoon.
The two centrioles have different orientations (Fig. lb, c). One is aligned or slightly oblique
along the longitudinal axis of the spermatozoon, and will be here termed longitudinal. The other
centriole is perpendicular to the other and will be termed perpendicular.
The perpendicular centriole is made up of 10 singlets (Fig. Ib-e). Each singlet is in contact
with its two neighbours. The singlets are not arranged in a perfect circle, but in an ellipse (140 x
160 nm). Ten dense peripheral elements are visible at the periphery of the centriole: relatively
Fig. 1. — Electron microscopy of Heligmosomoides polygyrus spermatozoon, showing centrioles with 10 singlets,
a: Longitudinal section of spermatozoon, showing anterior region (A), median region (M) and posterior region
with nucleus (N). b, c: Sections showing the two centrioles in the nuclear fossa, b: Longitudinal, relatively
thick, section of spermatozoon. The perpendicular centriole (right) shows 10 peripheral spokes (arrows);
c: Transverse section, d, e: Perpendicular centriole in longitudinal section of the spermatozoon. Note the 10
peripheral dots, f-h: Longitudinal centriole. f: Longitudinal section of nucleus, showing centriole in nuclear
fossa, g, h: Transverse section of spermatozoon, showing longitudinal centriole with 10 singlets and peripheral
dots. In e and h, note elliptic shape of centriole and singlets closely associated, a, x 12 000; b, d, x 45 000; c,
x 30 000; e, x 100 000; f, x 15 000; g, x 34 000; h, x 150 000.
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A. MANSIR & J.-L. JUSTINE : HELIGMOSOMOIDES POLYGYRUS (NEMATODA)
Table 1. — Spermatozoa of nematodes described by electron microscopy. Classification according to Inglis (1983) [32].
Many orders of the Nematoda have not been studied for spermatozoal ultrastructure and thus are not cited here.
thick sections (Fig. lb) show a triangular spoke 30 nm long extending from the peripheral limit of
two singlets, and thin sections (Fig. Id, e) show a dot located at a distance of 30 nm, and located
at the level of this limit. The centre of the centriole shows no dense structure.
The longitudinal centriole (Fig. lb, c, f-h) is also made up of 10 singlets and has the 10
peripheral elements. Lengths ranging from 200 nm (Fig. If) to 330 nm (Fig. lb) have been
found, possibly reflecting a change of length during the maturation of the spermatozoon.
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Immunocytochemistry of tubulin in spermatids and spermatozoa (Fig. 2)
The immunolocalization of tubulin in mature spermatozoa gives absolutely no reaction, and
thus is not illustrated here. However, spermatids show the presence of tubulin. Early elongate
spermatids (Fig. 2a) show a heavy labelling of tubulin around the nucleus and in the elongating
part of the cytoplasm. The most intense labelling is found in a triangular region located inside the
anterior fossa of the nucleus. More advanced spermatids (Fig. 2b) show labelling only in the
fossa and in a longitudinal central core in the cytoplasm. Finally, only a few parallel longitudinal
microtubules are visible, labelling of which is most intense in the fossa (Fig. 2c). Mature
spermatids (Fig. 2d) show no labelling.
Fig. 2. — Immunocytochemistry of alpha-tubulin in spermatids of Heligmosomoides polygyrus, a-d: Successive stages
of spermiogenesis, showing the decreased amount of tubulin in spermatids. The nuclear fossa is strongly labelled,
thus indicating that the centriolar region located here may act as a microtubule organizing centre. Mature
spermatozoa are not labelled and thus are not pictured here, a-d, x 2 500.
This labelling is consistent with the view that the centriolar region, located in the nuclear
fossa, plays the role of a microtubule organizing centre (MTOC). However, centrioles in mature
spermatids and spermatozoa are no longer labelled by the antibodies. This could be due to a
penetration problem, since the centrioles are deeply located in the tossa in the mature spermatozoa
and are embedded in an electron-dense material. An alteration of tubulin antigenicity in mature
spermatozoa cannot, however, be excluded.
The labelling illustrated in Fig. 2 was performed with an anti-alpha tubulin antibody. 1 he
anti beta-tubulin antibody gives a similar pattern. The anti-acetylated tubulin antibody gave no
labelling at any stage.
DISCUSSION
The spermatozoon of Heligmosomoides poly gyrus has been previously described by
Wright & SOMMERVILLE [81, 84], Our study confirms their observation on gross morphology
and on the presence of microtubules in spermatids, which are no longer present in mature
spermatozoa. However, the centrioles were not described in their study.
Centrioles have been described in nematode spermatozoa in a limited number of species
(Table 2).
124
A. MANSIR & J.-L. JUSTINE : HELIGMOSOMOIDES POLYGYRUS (NEMATODA)
Table 2. — Ccntrioles in nematode spermatozoa.
Species Centriole structure Reference
Heterakis gal lino rum
Dipetalonema viteae
Caenorhabditis elegans
TrichineUa spiralis
Sphaerolaimus hirsutus
Ascaris megalocephala
Nippostrongyl us brasil iensis
Gastromermis sp. (spermatid)
Heligmosomoides poly gyrus
Table 2 demonstrates that singlets are the usual structure found in nematode spermatozoal
centrioles. The description of doublets in Gastromermis refers only to spermatids [53] and the
structure is not described in mature spermatozoa. It is not unlikely that the doublets are simplified
into singlets in mature spermatozoa in this species; such a process is know in many
Platyhelminthes (see [36]).
The number of singlets in nematode sperm centrioles is generally 9. The centriole of
Nippostrongylus has been described as having 18 singlets by Jamuar [33], but examination of
the photographs suggests that it is made of 9 singlets separated by regular spaces, as later
correctly described by WRIGHT & SOMMERVILLE [83]. The case of Heligmosomoides poly gyrus
described here is the first case with 10 singlets, and therefore 10-fold symmetry.
The 9-fold symmetry is almost ubiquitous in axonemes. A few exceptions have been noted:
axonemes made up of 3 [54], 6 [62], 8 [55], 12 [5, 75], 13 [17, 86], 14 [5, 18], 16 doublets [18]
have been described in spermatozoa of certain animals. Axonemes with a 9+‘T” structure (nine
doublets) but with centrioles made up of 18 singlets have been recently described in a flatworm
[36]. The only known case of a ten-fold symmetry in a spermatozoal axoneme has been very
recently described by DALLAI et al. [16] in the spermatozoa of a dipteran, which has a 10+0
structure. DALLAI et al. (1995) [16] have remarked that “in order to accommodate a tenth doublet,
the axoneme either must increase its cross-sectional diameter or the doublets have to be more
closely packed. The former alternative seems to be realized as the axoneme is seen to have an
elliptic shape...”. In Heligmosomoides polygyrus centrioles, both conditions (closer microtubules
and elliptic shape) are found.
It is known that the ciliary' apparatus is generally very reduced in nematodes [8]: flagella are
absent from spermatozoa, motile cilia are unknown, and an axonemal structure is found only in
sensory cells. Moreover, sensory cilia of nematodes often show a deviation from the 9+2 pattern.
Cilia with 10+0 structure (10 peripheral doublets) have been described in larval Haemonchus
contortus [60],
In spermatozoa which have a flagellum, the presence of a centriole is correlated with the
existence of the flagellum. In the Nematoda, flagella are never present and the role of the
centrioles may be questioned. Centrioles seem to act as MTOC during spermiogenesis, but might,
on first consideration, appear redundant in mature sperm. Their presence in mature sperm may
indicate a role during fertilization. However, the participation of the paternal centriole during
fertilization is not ascertained in all animal species [61]. In the nematodes, a comparative study of
the role of centrioles in fertilization in species with centrioles with 9 singlets and in
Heligmosomoides poly gyrus with 10 singlets would be interesting.
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
125
Spermatozoa of nematodes have been tentatively used for the understanding of phylogeny
by BACCETTI et al. [6]. The position of the centrioles may provide additional characters. For
instance, if we consider only spermatozoa with elongate shape, the position of centrioles is
different in Gastromermis, where they are at the distal (posterior) tip of the nucleus [53], whereas
they are at the anterior extremity of the nucleus in the Trichostrongyloidea [33, 44, 82-84], The
symmetry of the centrioles may provide further useful character.
ACKNOWLEDGEMENTS
Dr Dominique Kerboeuf (INRA. Nouzilly) provided us with larvae of Heligmosomoides polygyrus. Dr Marie-
Claude Durette-Desset helped in the resolution of systematic problems.
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Source . MNHN. Paris
Ultrastructure of Sperm and Sperm-Egg Interaction in
Aculifera: Implications for Molluscan Phylogeny
John BUCKLAND-NICKS
St Francis Xavier University,
P.O. Box 5000, Antigonish, Nova Scotia, B2G 2W5, Canada
ABSTRACT
All Aculifera, other than neomenioids, fertilize externally with ectaquasperm but these vary greatly in structure between
Polyplacophora and Aplacophora, reflecting different modes of fertilization. These structural differences provide us with
an additional set of relatively conservative characters with which to examine the phylogeny oi Aculifera in relation to
other Mollusca and to Bilateria as a whole. Primitive chitons, perhaps similar to Leptochiton asellus , probably had
ectaquasperm with a large acrosome that penetrated a smooth thick-hulled egg by the release ot digestive enzymes and the
extrusion of a perforatorium (as is typical of other molluscs). In the majority of extant chitons, such as Cryptochiton
stelleri , the acrosome has been reduced to a tiny vesicle that digests a pore in the egg envelopes, through which a needle¬
like portion of the nucleus (replacing the perforatorium), penetrates to fuse with an egg microvillus. The egg hulls ol these
chitons are elaborated into complex spines or cupules, which may be open or closed. In closed cupule species sperm
penetrate between cupules, whereas in open cupule species sperm are attracted inside the cupules to fertilize. Ectaquasperm
of Chaetodermomorpha are highly unusual with many derived features indicating a long divergence oi the group. Sperm
exposed to egg water exhibited an elongation of the apical tube, which suggests a novel polymerization process since a
typical subacrosomal granule is absent. Introsperm of Neomeniomorpha are similar to those ot internally fertilizing
prosobranch snails as well as some polychaete annelids, but are closest in morphology to the parasperm of Protodrilus
sp. a primitive annelid. This provides additional support for the theory that molluscs and annelids are sister tax a and also
suggests that internal, not external, fertilization, may be plesiomorphic to both groups. This would require secondary
evolution of ectaquasperm, an idea that has also been suggested for teleost fishes to explain the occurrence oi introsperm
in basal groups. Furthermore, all extant Platyhelminthes have introsperm but only Nemertoderma sp., considered a basal
member of the group, has a plausible acrosome, filiform nucleus and midpiece, and typical 9+2 flagellum. Platyhelminthes
are widely regarded as basal to the Bilateria which suggests, in contradiction to the widely held belief that early bi ater.ans
fertilized externally, that the common bilaterian ancestor of Platyhelminthes and Eubilatena fertilized internally with
introsperm. This indicates that Bilateria are a monophyletic group, their basal members being united by the shared
synapomorphies of similar introsperm and internal fertilization, which are absent in all extant Radiata.
RESUME
Ultrastructure des spermatozoides et des interactions spermatozoides-oeuf chez les Aculifera:
implications pour la phylogenie des Mollusques
Tous les Aculifera autres que les Neomenioidac ont une tecondation exteme avec ectaquaspermatozoidcs dont la structure
varie beaucoup entre les Polyplacophora et les Aplacophora, refletant des modes d.fferents de fecundation Ces differences
structures nous fournissent un ensemble supplemental de earaches relativement conserves avec lesquels ^on peut
examiner la phylogenie des Aculifera en relation avec les autres Mollusques et 1 ensemble des Bilateria. Les chitons
primitifs, peut-etre similaires a Leptochiton asellus , avaient probablement un ectaquaspermatozo.de avec un grand
BUCKLAND-Nicks J , 1995. — Ultrastructure of sperm and sperm-egg interaction in Aculifera: implications lor
molluscan phylogeny. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 166: 129-153. Paris ISBN : 2-85653-225-X.
130
J. BUCKLAND-NICKS : AC U LI F ERA (MOLLUSC A)
acrosome qui penetrait un oeuf h coque lisse et 6paisse grace a la liberation d’enzymes digestives et 1’extrusion d’un
perforatorium (situation typiquc des autres Mollusques). Dans la majorite des chitons, tels que Cryptochiton stelleri,
Tacrosome a etc reduit h une petite vesicule qui digere un pore dans les enveloppes de l'oeuf, h travers lequel une portion en
aiguille du noyau (qui remplace le perforatorium) penetre pour fusionner avec une microvillosite de l'oeuf. La coque de
l'oeuf de ces chitons est compliquee par des opines ou des cupules, qui peuvent etre ouvertes ou fermees. Dans les esp&ces &
cupules fermees les spermatozoides pendtrent entre les cupules, alors que dans les especes & cupules ouvertes les
spermatozoides sont attires & l’interieur des cupules pour la fecondation. Les ectaquaspermatozoides des
Chaetodermomorpha sont tfes differents, avec de nombreux caracferes d6riv£s indiquant une longue divergence du groupe.
Les spermatozoides exposes aux secretions des oeufs montrent une elongation du tube apical, qui sugg£re une
polymerisation d’un nouveau type puisque le granule subacrosomien est absent. Les introspermatozoides des
Neomeniomorpha sont similaires & ceux des Mollusques Prosobranches & fecondation interne et & certaines Ann£lides
Polychetes, mais ont une morphologie tres proche du paraspermatozoide de Protodrilus sp., une Annelide primitive. Ceci
fournit des arguments additionnels a la theorie selon laquelle les Mollusques et les Annelides sont des groupes-freres et
suggfcre aussi que la fecondation interne, et non externe, pourrait etre pfesiomorphe dans ces deux groupes. Ceci
demanderait une Evolution secondaire de l'ectaquaspcrmatozoide, une idee qui a deja ete suggdfee chez les Poissons
Tefeosfeens pour expliquer la presence d’ introspermatozoides dans les groupes basaux. De plus, tous les Plathelminthes
ont des introspermatozoides mais seul Nemertoderma sp., considere comme un membre basal du groupe, a un acrosome
plausible, un noyau et une pfece intermediate filiforme, et un flagelle typique 9+2. Les Plathelminthes sont g^neralcment
consid^res comme dtant a la base des Bilateria ce qui suggere, en contradiction avec la croyance largement repandue que les
premiers Bilateria avaient une fecondation externe, que l’ancetre commun des Plathelminthes et des Eubilateria avail une
fecondation interne avec introspermatozoide. Ceci indique que les Bilateria sont un groupe monophyletique, leurs membres
basaux <§tant unis par des synapomorphies d'un introspermatozoide similaire et de la fecondation interne, caracferes qui
sont absents chez tous les Radiata.
Aculifera consist ol two classes, Polyplacophora (chitons) and Aplacophora (chaetoderms
and neomenioids) [67, 69]. Among Metazoa, the most primitive groups, Porifera and Radiata,
fertilize externally with sperm that have the typical shape, round head, four or five spherical
mitochondria and a centriolar satellite complex that harnesses the flagellum at the base of the
sperm. To avoid prior connotations of “primitive” and “modified” being associated with sperm
shape, JAMIESON and Rouse [47] devised a new system of nomenclature based on the mode of
fertilization, in keeping with FRANZEN's original hypothesis that sperm structure correlates with
biology of fertilization [33, 34], Two key terms emerged, “ectaquasperm” are characteristic of
external fertilizers, whereas “introsperm” characterize internal fertilizers. This system provides for
intermediate conditions such as “entaquasperm” [60, 61] and “ect-terrasperm” [80], as well as
“plesiosperm” for the archetype [45],
Chitons and chaetoderms have external fertilization with ectaquasperm, although sperm
structure and mode of fertilization vary greatly between the groups indicating a long divergence.
Neomenioids fertilize internally with introsperm [32], which have a similar morphology to sperm
of some primitive annelids [53], gastropods, such as Neritimorpha [15] and Caenogastropoda
[13, 40], as well as other invertebrates [2, 3, 35], The eggs of Aplacophora are relatively
unspecialized in relation to surface features but in chitons the hull is usually elaborated into a
series of complex spines or cupules, which may be closed or open to the external environment
[21 22, 29], In hermaphroditic and brooding species the cupules tend to be reduced, sometimes
to flattened plates [20, 29], Self-fertilization is predominant in hermaphroditic species but sperm
morphology is unchanged presumably because sperm encounter eggs in a sea water medium in
the pallial grooves [20],
Aplacophorans are easily separated from the chitons on the basis of synapomorphies for
distichous radula, reduced foot, gonads that open into the pericardium, and small posterior mantle
cavity [67, 69]. Furthermore, neomenioids are quite distinct from chaetoderms on the basis of
apomorphic characters for sperm structure, other aspects of reproductive biology, as well as
general morphology [66, 67, 69], The main problem arises in trying to distinguish between the
different families of chitons.
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131
Chiton phylogeny has been difficult to resolve because potentially useful characters such as
radula morphology, which have an extensive fossil record, are virtually unchanged in the chitons
[28, 31, 781. Other characters such as girdle morphology and hull spine tip structure, are highly
variable and may have arisen independently in several lineages, or may be polymorphic in a single
taxon [28,71], Furthermore, previous classifications have been constructed as a series of
progressive grades or ascending levels of organization rather than in a way to test phylogenetic
affinities [28, 79],
Several authors advocate the use of a classification system for generic and higher taxa based
on shell valve morphology alone, because these characters can be corroborated with the fossil
record [for review, see 78]. However, as EERNISSE [28] pointed out, any phylogenetic analysis
would be strengthened by inclusion of independent morphological characters to avoid coincidental
resemblance of form (i.e. of shell valves). EERNISSE first revealed the potential advantages of
using gill placement and morphology, as well as egg hull morphology, as independent character
sets to test the validity of previous phylogenies based on shell valve morphology alone [28],
SIRENKO followed up on this and studied gill placement and egg hull morphology in 1 17 species
of chitons many of them for the first time [71]. This analysis produced not only a re-arrangement
of existing families but the addition of several new ones, including two new super-families,
Tonicelloidea and Schizochitonoidea. Lepidochitoninae were included as a subfamily within
Tonicellidae, in the superfamily Tonicelloidea. Furthermore, all chitons were grouped in three
suborders, Lepidopleurina, Chitonina and Acanthochitonina.
This chapter examines sperm structure and sperm-egg interactions in Aculifera. It has been
restricted largely to those species for which complete sperm and egg hull data are available. Thus,
families for which sperm and egg data were not verifiable by the author have been omitted from
the cladistic analysis. These include Ochmazochitonidae, Loricidae, Schizochitonidae,
Juvenochitonidae, Schizoplacidae, and Cryptoplacidae (see [71]). In a few instances key species
have been included from other studies to ensure a more complete analysis of a family. This
analysis of new sperm and egg characters, when combined with existing morphological
characters, has enabled a reassessment of phylogenetic relationships among Aculifera and
between them and other taxa.
RESULTS
Sperm structure in Polyplacophora
Chiton sperm can be classified into one of four main types, depending on the presence of a
typical acrosome, type of chromatin condensation, number and position of mitochondria, and
presence of an axonemal fibrous complex. The appropriate families for each sperm type are listed
on the first line, and follow the recent reclassification of chitons by SIRENKO [71], with the
exception of Lepidochitonidae [see phylogeny].
Sperm type 1 : (Acanthochitonidae, Mopaliidae, Tonicellidae, Lepidochitona). This sperm
type is probably the most derived as well as the best characterized [16, 17, 19-22, 42, 64, 65]. A
detailed description will be given based on this information and following the four arbitrary stages
of spermiogenesis described elsewhere [19] and illustrated here in Figure 37. The other sperm
types will then be compared to it.
At the onset of spermiogenesis tetrads of Stage A spermatids are interconnected by
cytoplasmic bridges (Fig. 4). Chromatin in the spherical nucleus is patchy and granular in
appearance. Two centrioles are positioned adjacent to the plasma membrane with the distal one
being attached to it via the centriolar satellite complex and annulus (Fig. 1). The proximal centriole
is perpendicular to the distal centriole and anterior to it. There are six or more
132
J. BUCKLAND-NICKS : ACUL/FERA (MOLLUSCA)
Table 1. — List of species used in the present study, classified according to Sirenko [71).
Species studied Family Reference
Leptochiton asellus (Linnaeus, 1767)
Callochiton castaneus (Wood, 1875)
Chaetopleura apiculaia (Say, 1834)
Accmthopleura granulata (Gmelin, 1791)
Callistoplax crassicostatus Pilsbry, 1893
Stenoplax conspicua (Pilsbry, 1892)
Lepidozona retiporosci (Carpenter, 1 864)
Chiton tuberculatus (Linnaeus, 1758)
Onithochiton quercinus (Gould. 1846)
Ischnochiton albus (Linnaeus, 1767)
Lepidochitona dentiens (Gould, 1 846)
Lepidochitona fernaldi Eernisse, 1986
Tonicella lineata (Wood, 1815)
Tonicella marmorea (Fabricius, 1780)
Nuttalina flexa (Carpenter, 1 864)
Mopalia muscosa (Gould, 1846)
Mopalia lignosa (Gould, 1 846)
Mopalia ciliata (Sowerby)
Katharina tunicata (Wood, 1815)
Acanthochitona viridis (Pease, 1872)
Acanthochitona pygmaea (Pilsbry, 1893)
Cryptochiton stelleri (Middendorff, 1857)
Chaetoderma argenteum Heath, 1911
Chaetoderma canadense Nierstrasz, 1 902
Epimenia australis (Thiele, 1897)
Figs 1-9. — Transmission electron micrographs of spermiogcnesis in Polyplacophora. 1: Stage A type 1 spermatid of
Tonicella marmorea showing Golgi body (G) and associated lysosome (L), multi vesicular body (MB) and vesicles
(arrowhead). Distal centriole (DC) is attached to plasma membrane (PM) by the annulus. Scale bar = 0.2 pm.
2: Longitudinal section (L.S.) of stage B type 2 spermatid of Lepidozona retiporosa showing condensation of
chromatin in 20 nm line fibres. The proximal centriole (PC) is attached by fibrogranular secretions to the
thickened region of nuclear envelope (arrowhead). Scale bar = 1 pm. 3: L.S. stage C type 2 spermatid of L.
retiporosa , showing formation of 70 nm coarse fibres. Scale bar = 0.5 pm. 4: L.S. stage C type 1 spermatid of
Cryptochiton stelleri showing tight spiralling of fine chromatin fibres. Coarse fibre stage is absent. Note:
cytoplasmic bridge joining spermatids (arrowhead); lacuna (L) in nucleus and cross section of flagellum at level of
annulus (small arrow). Scale bar = 0.2 pm. 5: L.S. stage C spermatid of Tonicella lineata with fine chromatin
fibres twisted anteriolaterally forming extension of nucleus in anterior filament (arrowhead). Two microtubules of
manchette surrounding nucleus are visible in superficial section (arrows). Scale bar = 0.5 pm. 6: Stage D
spermatid of C. stelleri showing aggregation of nucleopores (P) where nucleus extends into anterior filament. Scale
bar= 0.3 pm. 7: lip of anterior filament of stage D type 1 spermatid of Mopalia muscosa showing acrosome (A)
separated from nuclear extension (N) by basal (= subacrosomal) plate (arrowhead). Scale bar = 0.3 pm. 8: L.S.
stage D type 1 spermatid of M. muscosa in longitudinal section showing basic form of sperm and position of
fibrous complex (FC). Scale bar = 0.2 pm. Inset: Close-up of fibrous complex (arrowhead). Scale
bar = 0.2 pm. 9: L.S. stage D type 2 spermatid of Chaetopleura apiculata in longitudinal section showing basic
lorm of sperm with large round posterior mitochondria, surrounded by glycogen granules (Gl) on opposite side to
basal body (arrowhead). Scale bar= 0.5 pm. Inset: Tip of anterior filament of L. retiporosa showing acrosome (A)
atop nuclear extension (N). Scale bar= 0.1 pm.
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134
J. BUCKLAND-NICKS : ACULIFERA (MOLLUSCA)
mitochondria at this stage but as spermiogenesis proceeds they migrate posteriorly and fuse to
form between 4 and 7 in the mature sperm. A prominent Golgi body secretes several small
proacrosomal vesicles and is usually associated with a lysosome and multivesicular body (Fig. 1).
Stage B in all chitons is characterized by the development of the centriolar fossa at the base of the
nucleus accompanied by a thickening of the outer nuclear membrane (Fig. 2). The centrioles
migrate into this region and the proximal centriole becomes attached by fine fibrous strands to the
thickened nuclear membrane (similar to Fig. 2). The flagellum elongates posteriorly. Chromatin
condensation progresses forming fine fibres about 20 nm in diameter, which later in this stage
become twisted anteriolaterally towards the pointed end of the nucleus (Fig. 5). A microtubular
manchette emanates from the centrioles, twists around the nucleus and extends into the pointed tip
of the spermatid (Fig. 5). In the Stage C spermatid two granules begin to develop in the acrosome
by the fusion of proacrosomal vesicles. The nuclear filament becomes more elongate (Fig. 5) and
the nucleopores aggregate at its base (Fig. 6). The rest of the nucleus appears to be devoid of
nucleopores at this stage. Glycogen granules are formed in the midpiece, near the Golgi body and
mitochondria. The basal body is slightly offset and mitochondria begin to group above it on the
same side, and below it on the opposite side. In Stage D the spermatids mature and the
microtubular manchette disappears. The acrosome, containing two granules, is separated from the
nuclear filament by a basal (= subacrosomal) plate (Fig. 7). The nucleus and nuclear filament
become homogeneously dense with occasional lacunae in the main body (Fig 8). The nucleus is
bullet-shaped and subtends an offset basal body comprising proximal and distal centrioles
interconnected by fibrogranular secretions. Mitochondria are found laterally above the centrioles
and basally opposite them. The centriolar satellite complex and annulus are housed in the collar,
which is an evagination of plasma membrane that extends posterior to the distal centriole and
around the axoneme. A fibrous complex forms adjacent to the axoneme near the annulus, on the
same side as the basal body (Fig. 8, inset). The elongate flagellum terminates in a narrow
endpiece, which contains only the central microtubules and is similar in length to the nuclear
filament. The Golgi body, rough endoplasmic reticulum, lysosome, multivesicular body, as well
as excess cytoplasm are eliminated via the cytoplasmic bridge into a central cytoplasmic droplet,
which is sloughed off thus freeing the four mature spermatozoa of the cohort.
Sperm Type 2: (Chaetopleuridae, Chitonidae, Callistoplacidae, Ischnochitonidae). Type 2
chiton sperm has been described previously for 12 species [4, 19, 42, 63-65], and is represented
here by Lepidozona retiporosa. Type 2 sperm develop similarly to Type 1 with the following
exceptions: firstly, following the fine fibre phase (fibres are 20 nm in diameter) of chromatin
condensation (Fig. 2), there is a coarse fibre phase (fibres are 70 nm in diameter) (Fig. 3), before
the nucleus becomes homogeneously dense (Fig. 9). Secondly, the midpiece has a distinctive
Figs 10-20. — Sperm-egg interactions in Polyplacophora. 10: Mature open cupule egg of Mopalia ciliaia. Scale bar =
50 pm. 11: Open cupules of polyspermic egg of M. muscosa. Scale bar = 20 pm. 12: Polyspermic egg of
M. muscosa with cupules removed revealing preponderance of sperm inside cupule profiles. A few sperm penetrate
area between cupules. Scale bar = 10 pm. 13: Mature closed cupule egg of Lepidochiiona dentiens. Scale bar =
50 pm. 14: Mature spinous egg of Stenoplax conspicua. Scale bar = 50 pm. 15: Polyspermic eggs of S.
conspicua with sperm between spines. Bifurcations of spines now are closed, but were spread apart when spawned.
Scale bar = 10 pm. 16: Polyspermic egg of M. muscosa: sperm penetrating hull inside cupule. Scale bar= 2 pm.
17: Light micrograph of 1 pm section of sperm of T. lineata penetrating hull and vitelline layer to inject nuclear
material into egg (arrowhead). Scale bar= 2 pm. (From [16]). 18: Portion of maturing egg of Acanthopleura
granulata, showing polymorphic spines. Short bifurcating spines occurring between medium to long single tipped
spines with caps. Scale bar = 10 pm. 19: Polyspermic egg of S. conspicua in plan view showing sperm
penetrating hull at junctions of cupule bases (arrowheads). Scale bar = 5 pm. 20: T.E.M. of fertilizing sperm
penetrating hull of S. conspicua. Characteristic dense outer layer of hull, has been dissolved around penetrating
sperm. Scale bar = 2 pm.
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136
J. BUCKLAND-NICKS : ACULIFERA (MOLLUSCA)
appearance because of the lack of lateral mitochondria and the position of basal mitochondria (Fig.
9). Frequently two mitochondria are adjacent to the nucleus with a third one below them. This
extends the midpiece and collar posteriorly. Thirdly, the basal body is more offset than in Type 1.
This and the position of mitochondria gives the Type 2 sperm its characteristic shape (Fig. 9).
Fourthly, the fibrous complex is absent or poorly developed, but at least in some species the
plasma membrane is thickened at the same site as the fibrous complex of Type 1 sperm. The
acrosome is more uniform in appearance, lacking distinct apical and basal granules (Fig. 9, inset).
Sperm type 3: (Callochitonidae). The third type of sperm has been described only in
Callochitonidae [42, 64], represented here by Callochiton castaneus. Sperm of Hanleyidae, a
fairly primitive family, have not yet been examined.
Type 3 sperm is characterized by the plesiomorphic condition of a central basal body and
flagellum, surrounded by five mitochondria at the base of the nucleus. The mitochondria are
slightly derived because two of them are elongate whereas three are spherical [42]. There is no
fibrous body or unilateral reinforcement of the plasma membrane near the annulus. In other
respects the sperm is similar to Type 2 with a similar pattern of chromatin condensation.
Sperm type 4\ (Lepidopleuridae). This sperm type, considered plesiomorphic to chitons, is
known only from the species, Leptochiton asellus [42], The sperm is unique among chitons in
having a fairly typical molluscan acrosome. A detailed diagram summarizing spermiogenesis in
L. asellus is given in Figure 36 (based on [42] and unpublished observations).
In Stage A spermatids a large proacrosomal vesicle (1 |im in diameter) is evident posterior
to the nucleus and connected to the plasma membrane by flocculent material, as occurs in
aplacophorans and other molluscs. During Stages B and C the nucleus becomes oval and
chromatin condensation proceeds from granular to short fine fibres 20 nm in diameter, then to
short coarse fibres, 70 nm in diameter. The fibres appear much shorter than in other chiton
groups, reflecting variation in the pattern of condensation. The mitochondria fuse to form five
spheres surrounding a central basal body and unspecialized flagellum. In Stage C the
proacrosomal vesicle, independent of the Golgi body, differentiates into the elongate acrosome
cone and subacrosomal granule, which becomes separated from the nucleus by the subacrosomal
plate. The subacrosomal granule apparently lacks filaments, as in some gastropods. In Stage D,
the nucleus becomes homogeneously dense and bullet-shaped with no anterior filament. The
flagellum terminates in a short filamentous endpiece containing only the central microtubules.
Sperm-egg interaction in Polyplacophora
Chiton eggs are surrounded by a vitelline layer as well as a hull, both of which appear to be
secreted by the egg [58, 59], With few exceptions the hulls of chiton eggs are characterized by
elaborate projections which vary from spinous to cupular [16, 17, 20-22, 28, 29, 31, 43, 56, 58,
70, 71], By contrast the hull of Leptochiton asellus (Lepidopleuridae) is smooth [42]. In brooding
forms, which include several hermaphroditic species, the cupules are often reduced but retain the
basic morphology of the group [20, 28, 29]. There are three main types of sperm-egg interaction
which correlate with egg hull structure as follows: Type 1 . Cupular projections; Type 2. Spinous
projections; Type 3. Smooth hull. Variation in the size, shape and structure of hull projections has
profound influences on; the site and mechanism of fertilization [16, 17, 20-22], the drag on the
egg and therefore its sinking rate [21], the aggregation of eggs in strings and clusters and
attachment to substrates [28, 29, review: 56] and therefore the target size for sperm, as well as
the limitation of numbers of eggs that can be brooded [20, 28, 29].
Egg type 1: Cupular projections. (Lepidochitonidae, Tonicellidae, Mopaliidae,
Acanthochitomdae). In cupular species the projections may be open or closed, which directly
influences where the sperm penetrate the egg. In species with open cupules, such as in Mopalia
ciliata, (Fig. 10), sperm preferentially swim inside the cupules, become segregated into one of
seven channels and penetrate the hull where it overlies the egg membrane (Figs 12, 16). At this
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137
point the hull is thinnest. The hull can be penetrated between cupules but this is rare and has only
been observed in polyspermic eggs, created artificially by using a sperm concentrate (Fig. 12).
No micropores have been observed in the hull of these species. The acrosome appears to contain
two granules, which in Tonicella lineata are exhausted sequentially as the sperm penetrates the
two egg envelopes, fuses with an egg microvillus and injects the nuclear material into the egg [16,
17] (Fig. 17).
During ontogeny cupules are covered by follicle cells [58, 70], which are thought to be
involved in their morphogenesis but not their secretion [58, 59] distinguishing this envelope from
the chorion of other animal groups. An immature open cupule egg can easily be mistaken for a
closed cupule one. A good example is Tonicella lineata , which only becomes obviously open
cupule in form when it sheds follicle cells close to spawning [16, 17]. Thus it is very important to
make definitive observations only on naturally spawned eggs. Chitons collected during the
breeding season [review: 56] may be kept in culture dishes in running sea water, until spawning
occurs. Usually this happens one or two days after collection. Males usually spawn first and
appear to stimulate females [56]. In species with closed hull cupules, such as Lepidochitona
dentiens (Fig. 13), sperm must penetrate the egg envelopes between projections. In L. dentiens ,
micropores are present between cupules in the mature egg that provide sperm with direct access to
the vitelline layer [20, 21], Preliminary evidence suggests that sperm acrosomes may be further
simplified in this and similar species by having a single granule [21]. In the brooder L.fernaldi,
the structure of the egg is the same as L. dentiens except that cupules are reduced to flattened
plates [20].
Egg type 2: Spinous Projections. (Hanleyidae, Callochitonidae, Schizochitonidae and
Loricidae [71]; Chaetopleuridae, Chitonidae, Ischnochitonidae, Callistoplacidae). In chitons with
spinous projections, such as Stenoplax conspicua (Fig. 14), the sperm penetrates the hull between
spines, frequently at the junction of their bases (Figs 15, 19). Spines assume a wide variety of
forms from complex, as in Chaetopleura apiculata, to simple as in Hanleyella asiatica [71]. Simple
spines have a wide variety of tip formations, including one to seven-fold symmetry. Spinous tips
may be pointed and bifurcate, as in S. conspicua (Fig. 15) or petalloid, as in Chiton tuberculatus.
However, there appears to be considerable polymorphism within genera [71], making it difficult
to use tip form as a character in phylogenetic analysis. The egg hull of 5. conspicua (and
possibly other spinous hulled species), has an outer dense layer in addition to the typical hull.
This thin dense layer is digested by the entrance of the sperm, presumably because certain
acrosomal enzymes are released (Fig. 20). The area of the hull disrupted by sperm entrance is
much greater than the width of the anterior filament and cannot be accounted for just by gaps
between spine bases, visible in some micrographs (Fig. 19).
Egg type 3: Smooth Hull. (Lepidopleuridae). In Leptochiton asellus , the only chiton species
known with a smooth-hulled egg, fertilization is presumed to occur anywhere on the egg surface.
However, sperm-egg interaction has not been observed in any lepidopleurids. The presence of a
typical molluscan acrosome is coincident with an egg hull that is ten times thicker than in other
chitons and probably reflects penetration by a perforatorium as is typical of other molluscs and
invertebrates [42]. For these and other morphological reasons (see phylogeny section),
Lepidopleuridae are considered basal to Polyplacophora.
Sperm structure and sperm-egg interaction in Aplacophora
Class Aplacophora may be divided into two subclasses, the Neomeniomorpha and the
Chaetodermomorpha [67, 69]. Neomenioids are considered basal to Aculifera and to Mollusca as
a whole, based on recent morphological [67] as well as molecular analyses [62], whereas
chaetoderms are considered to be derived, having diverged early from the molluscan lineage [69],
Neomenioids and chaetoderms have radically different modes of reproduction.
Neomenioids are monoecious and fertilize internally, with introsperm being stored in seminal
138
J. BUCKLAND-NICKS : ACUL1FERA ( MOLLUSCA )
receptacles [68, 69]. Chaetoderms are dioecious and fertilize externally, by releasing
ectaquasperm into the ambient sea water [18, 69]. These sperm types are quite different from
those of chitons.
Sperm of Neomeniomorphcr. Epimenia australis. Spermiogenesis in E. australis is
summarized diagrammatically in Fig. 38. Spermiogenesis of the introsperm of Epimenia is very
similar to that of the parasperm of the primitive annelid, Protodrilus, as well as introsperm of
some prosobranchs [13, 15, 40], Stage A is similar to what has been described for chitons. In
stage B the sperm still has the plesiosperm form (Fig. 21). During this stage the centrioles migrate
to the centriolar fossa dragging the plasma membrane with them and causing it to invaginate
where it is attached to the annulus. This creates a temporary flagellar canal. The mitochondria
move posteriorly and begin to fuse at the base of the nucleus, initially into four or more spheres.
At this time a Golgi body is posterior to the nucleus and mitochondria (Fig. 21). In stage C,
chromatin condensation progresses from granular to fine fibres 20 nm in diameter, which become
twisted as the nucleus elongates (Fig. 22). During this stage the Golgi body secretes numerous
vesicles which contribute to a large mass of dense granular material at the base of the nucleus
around the axoneme. The annulus breaks away from the distal centriole and begins migrating
posteriorly towards the junction between midpiece and glycogen-piece. The dense granular mass
becomes reorganized around the proximal and distal centrioles where it contributes to the
formation of the basal body as well as to the adjacent peribasal body. Similar granular secretions
are organized into a spiral ridge around the axoneme down to the annulus (Fig. 38C).
The Golgi body plays an active role in forming the spiral ridge much like that observed
previously in Nerita picea [15]. Subsequently, the Golgi body migrates again to the apex of the
sperm adjacent to the acrosome cone, which to this point has been developing in the absence of
the Golgi body (Fig. 38C). An extracellular striated cone, coated with fine fibrous strands, begins
Figs 21-28. — Spermiogenesis in Neomeniomorpha: Epimenia australis. 21: L.S. stage B spermatid showing basic
plesiosperm form. Apical acrosome (A), Golgi body (G) and spherical mitochondria (M) at base of nucleus
containing granular chromatin (N). Scale bar = 1 pm. 22: L.S. stage C spermatid showing twisting and spiralling
of chromatin fibres in nucleus (N). Acrosome (A) is at tip of spiralled nucleus (out of section). Golgi body (G) is
once again associated with the acrosome (or else there 2). Large fused mitochondria (M) are beginning to elongate
around axoneme, which has been reinforced by spiral ridge (arrowhead). Scale bar = 1 pm. 23: Stage D spermatid.
L.S. of acrosome (A), subtending sub-acrosomal granule (SG) and separated from dense nucleus by sub-acrosomal
plate (arrowhead). Note: striated extracellular cone (EC) on thickened membrane at tip of acrosome. Scale bar =
0.2 pm. 24: L.S. stage D spermatid showing basal body (BB) in posterior indentation of nucleus, which is
blocked by peri-basal body (PB). Note also: mitochondria (M) and sections of spiral ridge (R). Scale bar =
0.2 pm. 25: Stage D spermatid. L.S. of junctions between mid-piece and glycogen piece showing annulus
adjuncts (AnA), mitochondrion (M), spiral ridge (R) and annulus (arrowhead). Scale bar = 0.2 pm. 26: Stage D
spermatid. L.S. glycogen piece showing glycogen granules (arrowheads). Scale bar= 0.3 pm. 27: Cross Section
(C.S.) stage D spermatids at level of acrosome (A), nucleus (N) and mitochondrion (M). Note single break in
mitochondrion (arrowhead), which forms longitudinal slit. Scale bar = 0.2 pm. 28: L.S. end-piece of stage D
spermatids, showing density (arrowhead) at termination of central microtubules. Scale bar = 0.2 pm.
FIGS 29-35. — Sperm and eggs of Chaetodermomorpha. 29: Stage C spermatid of Chaetoderma canadense with granular
chromatin in nucleus (N), Golgi body (G) is in process of forming apical horn and apical tube in anterior of
spermatid. Scale bar = 0.5 pm. 30: Stage C spermatid of C. canadense showing acrosome (A) at tip of apical tube
(AT), that caps apical horn (AH). Apical tube is projected over large Golgi-derived vesicle (V). One of five
mitochondria (M) lies at base of nucleus. Scale bar = 0.5 pm. 31: C.S. mid-piece of C. argenteum showing five
mitochondria encircling centrioles. Scale bar = 0.5 pm. 32: Light micrograph of mature egg of C. argenteum
with apparent hull (arrow). Scale bar = 30 pm. 33: Enlarged area of C. argenteum egg showing periodic bumps
with short spinous or tubular projections (arrows). Scale bar = 20 pm. 34: L.S. of acrosome (A) and apical tube
(AT) of C. canadense. Note attachment of acrosome to plasma membrane (arrowhead). Scale bar = 0.2 pm.
35: S.E.M. of C. argenteum sperm exposed to egg water for ten minutes. Apical tube (AT) of upper sperm is
longer than lower sperm. Elongation can be at least 1.5x original length. Note also apical horn (AH), acrosome
(A) and mitochondria (M). Scale bar= 2 |im.
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139
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140
J. BUCKLAND-NICKS : ACUUFERA ( MOLLUSCA )
to form near the tip of the acrosome, between the sperm and the somatic (Sertoli) cell. Its function
is unknown.
The basal body becomes housed in a posterior indentation of the nucleus, which is closed
off by the peribasal body (Fig. 24). The annulus breaks away from its connections to the distal
centriole and leads the posterior migration of the mitochondria. The acrosome cone is fully
developed and subtends a subacrosomal granule, separated from the dense nucleus by a
subacrosomal plate. The two mitochondria begin to elongate and later fuse to form a single
Nebenkern enclosing the axoneme but for one longitudinal slit down its length (Fig. 27). In stage
D the mitochondrion extends from the base of the nucleus to the tip of the annulus adjuncts,
which are two hollow, semicylindrical structures that form above the annulus on either side of the
axoneme (Fig. 25). The annulus occupies an invagination of the plasma membrane at the junction
between midpiece and glycogen piece (a site typical of many metazoan introsperm) (Fig. 25). The
glycogen piece is filled with granules of glycogen (Fig. 26), which tend to be grouped over the
nine axonemal doublet microtubules. This aggregation is similar but less ordered than in
gastropods. The axoneme tapers towards the endpiece, where it is reduced to just the central
microtubules that terminate in a characteristic density (Fig. 28).
Sperm-egg interaction in Neomeniomorpha : Sperm-egg interaction in Epimenia has not been
observed. The outer egg envelope appears to be unspecialized, which suggests that fertilization
may occur anywhere on its surface. Fertilized eggs are brooded prior to release as juveniles [68].
Sperm of Chaetodermomorpha: Chaetoderma canadense, and C. argenteum. See
diagrammatic summary of spermiogenesis in Fig. 39. The ectaquasperm of C. argenteum [18]
and C. canadense [22] are highly unusual. Spermiogenesis begins in a similar way to
Leptochiton but after forming the proacrosomal vesicle the Golgi body migrates to the apex of the
nucleus (Fig. 29) where it becomes involved in producing three key structures: the apical horn,
the apical (dense) tube and a large vesicle below the tip of the nucleus (Fig. 30). The apical tube
grows out over the large vesicle to contact the proacrosome on the plasma membrane (Fig. 30).
Glycogen granules are deposited around the apical horn as well as the mitochondria. At this time
there are two flagella, proximal and distal, produced by the respective centrioles. The distal
flagellum encircles the nucleus and exits posteriorly. The proximal flagellum grows anteriorly
through the cytoplasm and exits at the tip of the nucleus, alongside the developing apical tube,
perhaps acting as a guide [18]. Mitochondria fuse to form five spheres surrounding the centrioles
at the base of the nucleus. At the completion of spermiogenesis the proximal flagellum separates at
the centriole and is eliminated anteriorly with the residual cytoplasm (including Golgi body, large
vesicle, lysosome, multivesicular body, endoplasmic reticulum, etc.) [18], Conversely, in most
Metazoa the residual cytoplasm is eliminated from the midpiece region. The distal flagellum
unwinds and exits posteriorly in the usual position for molluscs. The chromatin condenses from
granular, through patchy, to homogeneously dense with a few lacunae in the mature spermatid
[18]. The distal centriole is harnessed to the plasma membrane by the annulus via the centriolar
satellite complex, which typifies ectaquasperm [74] and is plesiomorphic for introsperm [2, 73,
77]. The axoneme has a typical 9+2 configuration and terminates in just the two central
microtubules as in other Aculifera.
The mature sperm is easily mistaken for a chiton sperm because the apical tube resembles
the anterior filament (Fig. 35) [32], However, the apical extension is directed anteriolaterally and
is ol completely diflerent construction [18], It contains no nuclear material and comprises two
unique structures, the apical horn and apical tube (Figs 30, 35), the functions of which remain
unknown. There is, however, little doubt that the acrosome lies at the tip of the apical tube, as this
vesicle forms from the Golgi body in much the same way as proacrosomes of some other
molluscs, including Leptochiton.
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Figs 36-39. Diagrammatic representation of four arbitrary stages (A-D) of spermiogenesis in Aculifera following
descriptions given in text.
Fig. 36. — Spermiogenesis in Leptochiton asellus. A: Pro-acrosome (arrowhead) formed from Golgi vesicles, is migrating
to apex of nucleus along plasma membrane. Annulus (An) connects distal centriole to plasma membrane. Nucleus
contains granular chromatin and in the cytoplasm arc several small mitochondria and rough E. R. B: Pro-acrosome
(A) is atop nucleus, chromatin is forming short fine fibres in nucleus. Mitochondria are at base of nucleus with
Golgi body. C: Acrosome (A) is elongating in absence of Golgi body. Chromatin fibres are thicker and fused,
enlarged mitochondria surround centrioles. D: Fully developed acrosome subtends a subacrosomal granule and is
separated from dense nucleus by sub-acrosomal plate. Five uniform spherical mitochondria surround centrioles.
Annulus (An) attaches distal centriole to plasma membrane via satellite complex [24, 42].
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J. BUCKLAND-NICKS : ACUL1FERA (MOLLUSC A)
Fig. 37. — Spermiogenesis in Cryptochiton stelleri. A: Similar to L. aseilus but no pro-acrosome forms. Individual pro-
acrosomal vesicles arc visible near Golgi body. B: Chromatin condensing in fine fibres is twisting anterio-
laterally in nucleus. ProacroSotnal vesicles are visible at apex of spermatid (arrowhead). Mitochondria have
accumulated at base of nucleus with Golgi body. C: Anterior filament is elongating with acrosome forming at its
tip. Nuclcopores (Nu) have aggregated at base of anterior filament. Tip of tail contains only central microtubules
(arrowheads). I): Bullet shaped sperm has long ncedlc-like filament with acrosome (A) at its tip. Acrosome is
separated from dense nuclear extension by a basal (- subacrosomal) plate (P). Mitochondria are positioned
laterally and below nucleus, adjacent to centriolcs. Annulus (An) attaches distal centriole to plasma membrane.
Fibrous complex (FC). characterizing Type 1 sperm, is visible below annulus.
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ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
143
Fig. 38. — Spcrmiogencsis in Epimenia australis. A: Plesiosperm stage like that in Figs 36 & 37. Pro-acrosomal granule
(arrowhead) is attached to plasma membrane by fibro-granular material. B: Pro-acrosome (A) is visible atop
nucleus. Chromatin is granular but more tightly packed than in stage A. Mitochondria are fusing and aggregating at
base of nucleus. Basal body, comprising fused proximal and distal centrioles, has migrated into centriolar fossa.
Large active Golgi body is positioned next to temporary flagellar canal (arrowheads). C: Pro-acrosome (arrowhead)
is attached to plasma membrane atop nucleus with Golgi body nearby. Chromatin has condensed into elongate fine
fibres which are twisted inside nucleus. Annulus has broken away from distal centriole and is migrating posteriorly
everting flagellar canal as it does so. Dense granular material surrounds apex of axoneme below basal body and a
spiral ridge is evident posterior to this. Two large mitochondria are elongating posteriorly. D: Late spermatid with
introsperm form. Extracellular striated cone (EC) is atop acrosome (A), which subtends acrosomal granule (SG) and
is separated from dense nucleus by sub-acrosomal plate (SP). Basal body (BB) is housed in basal indentation of
nucleus, blocked posteriorly by peri-basal body (arrow). Mitochondria (M) form an almost complete sheath around
axoneme, reinforced by spiral ridge (R). Annulus (An) is in characteristic junction of mid-piece and glycogen piece
and extends anteriorly as annulus adjuncts (AnA). Note also: Glycogen (G) and terminal density (D).
Source . MNHN, Paris
144
J. BUCKLAND-NICKS : AC U LI F ERA (MOLLUSCA)
Fig. 39. — Spermiogenesis in Chaetoderma argenteum. (redrawn from [ 1 8J). A: Earliest pari of stage A, showing distal
centriole attached to plasma membrane by annulus (An) via centriolar satellite complex (CS) with nearby Golgi
body (G) producing pro-acrosomal granule (AG). B: Proximal (PF) and distal flagella (DF) are visible.
Mitochondria are grouped at base of nucleus; Golgi body (G) and proacrosome (A) have migrated anteriorly.
Numerous secretory vesicles are fusing to form one larger vesicle (V). C: Proximal flagellum (PF) is aligned with
apical tube (AT). The acrosome (A) at end of apical tube is connected to large vesicle (V) by fibro-granular material.
Golgi body (G) is visible in an apical indentation of nucleus, adjacent to developing apical horn (AH) and apical
tube. Distal flagellum exits anteriorly but will eventually encircle spermatid. D: Late spermatid showing
homogeneously condensed nucleus with few lacunae. Spherical mitochondria (M) surround centrioles. Glycogen
granules are visible near apical horn but are also sequestered in mid-piece. Cross sections 1-5: (1) acrosome; (2)
apical tube; (3) apical horn; (4) flagellum; (5) end-piece.
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145
Sperm-egg interaction in Chaetodermomorpha : The egg of Chaetoderma, regrettably, has
not been observed with the scanning electron microscope. The only two ripe eggs observed with
light microscopy, appeared to have a distinct hull with tiny bumps and small spinous projections
(Figs 32, 33). However, at the moment there is no way to be certain that this resembles the hull of
chitons, or is formed in a similar way. The mechanics of fertilization may be similar to chitons in
that the acrosomal vesicle may digest a pore through the egg envelopes, followed by the apical
tube penetrating down to the egg membrane. In both cases the perforatorium that characterizes
other molluscs, and Metazoa in general, has been replaced by an anterior projection. In chitons
this is a finite extension of the nucleus but in Chaetoderma the equivalent projection, the apical
tube, is extensible. In response to egg water the apical tube elongated up to 1.5 times its original
length, suggesting a novel polymerization process (Fig. 35). T.E.M. sections have not yet
revealed what is changing in the anterior structures during elongation, but one suspects the
polymerization of actin, which is almost universal among the eukaryotes. Elucidation of the
details of sperm-egg interaction in Chaetoderma will have to await that elusive but opportune
moment.
Phylogenetic analysis of Aculifera
Phylogenetic analysis (Tables 2-4) in MacClade, as well as “branch and bound” analysis in
PAUP of 25 morphological characters (including new sperm/egg characters), produced a single
minimum length tree (tree length = 67). The tree showed support for classification of three
suborders of chitons, in two orders: Lepidopleurina in Lepidopleurida; Chitonina and
Acanthochitonina in Chitonida. The Chitonina formed a clade based on synapomorphies for
spinous hull projections that are narrow-based, posterior extension of midpiece altering sperm
shape, as well as production of long coarse fibres in chromatin condensation. Of the families
included in this study, Chitonina included: Callochitonidae, Chaetopleuridae, Acanthopleuridae,
Chitonidae, Callistoplacidae, and Ischnochitonidae. Acanthochitonina are separable as a more
recent clade by synapomorphies for mitochondrial arrangement in sperm, a fibrous complex in the
sperm flagellum, and overall sperm shape, as well as adanal gills and broadly based hull cupules
that are not spinous. Acanthochitonina included the families; Lepidochitonidae, Tonicellidae,
Mopaliidae, and Acanthochitonidae. The Neomeniomorpha were used for outgroup comparison.
The related Chaetodermomorpha clearly aligned with the neomenioids, in spite of different sperm
types, based on synapomorphies for spiculate cuticle, distichous radula and other characters
discussed below.
DISCUSSION
Phylogenetic relationships among Aculifera
Phylogeny of Aculifera currently is of great interest to malacologists because, although this
group has generally been given a primitive status, recent morphological and molecular analyses
place neomenioid aplacophorans basal to all extant Mollusca [67, 69].
Aplacophora (Neomeniomorpha and Chaetodermomorpha) are readily distinguished from
Polyplacophora by the following synapomorphies: vermiform body with spiculate cuticle,
distichous radula, reduced foot, gonads that open into the pericardium, and a cloaca-like posterior
mantle cavity [66, 67, 69]. Also Chaetodermomorpha may be distinguished from
Neomeniomorpha based on apomorphies for ectaquasperm structure, lack of seminal receptacles,
presence of gills, as well as other characters [reviews: 66, 67, 69].
146
J. BUCKLAND-NICKS : ACULIFERA ( MOLLUSCA )
Table 2. — Data matrix and reconstructed states for internal nodes (Note: * = outgroup)
Node
1111111111222222
1234567890123456789012345
Lep i dop 1 eur i dae
Chaetodermomorpha
Callochitonidae
Chae topi eur idae
Ac an t hop 1 eur i dae
Cal 1 is toplacidae
Chi ton idae
Ischnochitonidae
Lepidochitonidae
Tonicell idae
Mopaliidae
Acanthochi tonida
Neomen iomorpha *
1
2
3
4
5
6
7
8
9
10
11
001101142 00100212 110711??
12130214100470201000000??
2312111411021121311111133
2322111111031121412111122
2322111111032111412211354
2322111111732111412111222
2322111111732111412211254
2322111111 7321114 12 111 143
3340111211123231512121111
2340111212024231512121111
2340111212024231513121111
2340111211723231511331111
0000000000000000000000000
0010001410000020000000000
0010011410010021211011111
2310111411021121311111111
2312111411021121311111122
2322111111031121412111122
2322111111032111412111122
2322111111032111412111222
2322111111032111412211254
2340111211023231511121111
2340111211023231512121111
2340111212024231512121111
Lepidopleuridae
Callochitonidae
Chaetopleuridae
Acanthopleuridae
Chitonidae
Callistoplacidae
Ischnochitonidae
Lepidochitonidae
Tonicellidae
Mopaliidae
Acanthochitonidae
Chaetodermomorpha
Neomeniomorpha
Fig. 40. Single most parsimonious tree resulting from branch and bound analysis in Paup of the data matrix in Table 2.
Branch and bound search settings were, initial upper bound: unknown (compute via stepwise); addition sequence:
turthest; no multistate taxa; unrooted trees rooted using outgroup method. Character state optimization: accelerated
transformation (ACCTRAN); tree length = 67; consistency index = 0.970; homoplasy index = 0.030; retention
index = 0.967; HI excluding uninformative characters = 0.033; rescaled consistency index = 0.939. The ingroup
( ,menor nodes (2'H) are labelled. The apomorphy hypotheses supporting each interior node are shown
in Table 3.
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
147
Table 3. — Apomorphy hypotheses for internal nodes of cladogram of Aculiferan phylogeny (Fig. 40). Unless otherwise
stated character changes are from 0->l.
Node l->2:
Node 2 — >3:
Node 3— >4:
Node 4— >5:
Node 5— >6:
Node 6->7:
Node 7— >8:
Node 3 — >9:
Node 9— >10:
NodelO— >1 1 :
6,12,1 6,17(0— >2), 1 8,19,21 ,22,23,24,25
1 (0— >2), 2(0— >3), 5, 10, 12(1 — >2), 1 3,14,17(2— >3), 20
4(0— >2), 24(1 -»2), 25(1— >2)
3(1 — >2),8(4 — > 1 ), 1 2(2 — >3), 1 7(3 — >4), 19(1 — >2)
13(1— >2), 15(2— > 1 )
23(1— >2)
20(1 — >2), 24(2— >5), 25(2— >4)
3(1 — >4),8(4— >2),13(1 — >3),14(1 — >2),15(2— >3), 17(3— >5),21( 1— >2)
19(1— >2)
10(1 — >2), 1 3(3 — >4)
Table 4. — Characters used in cladistic analysis of Aculifera with character state codes. See Table 2 for data matrix.
Sperm and Egg Data
(1) . Acrosome: 0: cone with subacrosomal plate (SA.P.); 1: simple vesicle with SA.P.; 2: complex vesicle with SA.P; 3:
simple vesicle with SA.P.
(2) . SubAcrosomal Granule: 0: loose granular; 1: rod granular; 2: apical horn & tube; 3: absent
(3) . Mitochondrial Associations: 0: mitochondria (M) fused unspiralled; 1: M in ring around central proximal and distal
centrioles (PC/DC); 2: offset PC/DC with basal M; 3: offset PC/DC associated with basal not lateral M; 4: offset
PC/DC associated with basal & lateral M.
(4) . Chromatin Condensation: 0: develop fine long fibres; 1: develop fine then coarse short Fibres; 2: develop fine then
coarse long fibres; 3: “granular” chromatin.
(5) . Golgi Body Production of Acrosome: 0: proacrosomal granule migrates on plasma membrane; 1: proacrosomal
vesicles migrate, fuse; 2: proacrosomal granule migrates with Golgi body.
(6) . Glycogen Reserves: 0: stored in tail; 1: stored in midpiece; 2: stored in anterior & midpiece.
(7) . Flagellar Canal: 0: present but temporary, annulus breaks from D.C.; 1: absent.
(8) . Flagellum Reinforced: 0: thickening around axoneme; 1: unilateral thickening of plasma membrane; 2: unilateral
fibrous body; 3: absent.
(9) . Flagellum Termination: 0: 2 central microtubules elongate+density; 1: 2 centrals, short; 2: 9+2 configuration.
(10) . Fertilization Site: 0: anywhere on egg; 1: between hull projections; 2: inside hull projections.
(11) . Hull Pores: 0: absent; 1: present
(12) . Sperm Head Shape: 0: filamentous; 1: oval ; 2: oval w. nuclear Filament; 3: oval w. offset nuclear filament; 4: oval
w. apical tube.
(13) . Hull Type: 0: smooth; 1: complex spines; 2: simple spines w. modiFied tips; 3: closed cupules; 4: open cupules.
(14) . Cupule Base Size: 0: absent; 1: 5-30 pm; 2: 50-90 pm [71)
(15) . Mitochondria: 0: 1-2; 1: 2-4; 2: 4-6; 3: 6-8.
Shell Valve [28,78] and Gill Placement Data [71]
(16) . External Covering: 0: spiculate cuticle; 1: 8 shell valves; 2: smooth.
(17) . Gills: 0: absent; 1: 2 ctenidia posterior; 2: Type 1 at/anal; 3: Type 2 tfdanal; 4: Type 3 adanal, 5: Type 4 a/?anal.
(18) . Articulementum: 0: absent; 1: present.
(19) . Chiton Valve Morphology: 0: absent; 1: modern valve (V) shape; 2: multifissurated terminal Vs 3: post. V. w.
post, median sinus; 4: girdle & post.V. Fissured.
(20) . Insertion Plates: 0: absent; 1: slitted; 2: pectinated; 3: reduced slits.
(21) . Girdle Ornamentation; 0: absent; 1: girdle elements similar; 2: potential bristles; 3: girdle fissured; 4: sutural
tufts.
(22) . Radula Morphology: 0: distichous; I: docoglossan.
(23) . PHOTORECEPTORS: 0: absent; 1: macro & micro aesthetes; 2: intrapigmental aesthetes; 3: ocelli.
(24) . Gills From Nephropore: 0: absent; 1: 1-2 gills; 2: 3-4; 3: 5-6; 4: 7-8; 5: >9
(25) . Gills From Gonopore: 0: absent; 1: 1-3 gills; 2: 4-6; 3: 7-9; 4: >10.
148
J. BUCKLAND-N1CKS : ACULIFERA (MOLLUSC A)
Egg hull projections and associated follicle cells are synapomorphic to chitons, uniting the
entire group. Furthermore, all chitons more recent than lepidopleurids are united by having an
elongate sperm nuclear filament with reduced acrosome, cupular hull projections, slitted insertion
plates and modern gill placement. This evidence supports the theory of THIELE [75] that chitons
are a monophyletic group derived from a lepidopleurid-like ancestor, and argues strongly against
a polyphyletic origin as proposed by ASHBY [5]. Historically, Polyplacophora have been difficult
to resolve because potentially useful characters such as radula morphology, which have an
extensive fossil record, are virtually unchanged in the chitons [72], Other characters, such as
girdle morphology or hull spine tip structure, are highly variable and may have arisen
independently, or may be polymorphic for a single taxon [28, 71]. EERNISSE [28] first suggested
using placement of gills, sperm morphology and egg hull data as a way to develop independent
character sets to test the validity of previous phylogenies based on shell valve morphology alone
[reviews: 72, 78],
The addition of a new character set based on sperm morphology and mode of fertilization,
presented here for the first time, has enabled a preliminary reassessment of chiton phylogeny. The
present analysis combined sperm and egg hull data with gill placement and morphology [71], as
well as shell valve morphology [28, 78], The single minimum length tree showed strong support
for retaining three suborders of chitons, Lepidopleurina, Chitonina, and Acanthochitonina [71],
Acanthochitonina are more derived than Chitonina based on the sperm fibrous complex, adanal
gills and broadly based hull projections. Lepidochitonids clearly are included in Acanthochitonina
by possessing each of these synapomorphic characters. Previous classifications based largely on
shell valve morphology have given lepidochitonids a rather primitive status placing them between
lepidopleurids and other chitonids [reviews: 72, 78]. The present analysis reinstates
lepidochitonids in their own family, Lepidochitonidae, as they clearly fall outside the Tonicellidae
(based on apomorphies for hull pores, simplified acrosome, and cone-like hulls). This disagrees
with a recent classification that placed lepidochitonids within Tonicellidae, but supports the
inclusion of both Lepidochitonidae and Mopaliidae within the superfamily Tonicelloidea [71].
The Chitonina have been more difficult to resolve. However, the present analysis provides
support for maintaining the suborder Chitonina, as suggested by SlRENKO [71], rather than
Ischnochitonina [72, 78], The superfamily grouping Ischnochitonoidea Dali, 1889 [71], in this
analysis included the families Chaetopleuridae, Acanthopleuridae, Chitonidae, Callistoplacidae,
and Ischnochitonidae, but excluded the Callochitonidae. Relaxing parsimony by one tree length
(= 68) creates an even more paraphyletic assemblage and underscores the need for more
synapomorphic characters, particularly for this suborder. More detailed analyses of oogenesis and
spermiogenesis (such as acrosome substructure and hull deposition) may provide additional
characters that will enable better resolution of this complex taxon.
Phylogenetic relationship between Aculifera and other Bilateria: quest for the origin of internal
fertilization
FRANZEN's hypothesis that the biology of fertilization correlates with sperm morphology
works well in most situations [33, 34], Furthermore, the idea that the “primitive sperm” [33] or
plesiosperm’ [45], (which is found throughout the Radiata), represents the primitive condition in
basal Metazoa, is not questioned by most biologists. This is partly because sperm of more recent
groups usually pass through a stage in spermiogenesis that resembles the plesiosperm type, but
also because recent molecular and morphological analyses indicate a monophyletic origin of
Metazoa from a diploblastic ancestor [30, 50]. However, the idea that ectaquasperm generally
represent the primitive condition wherever they occur in more recent groups [8, 9, 33, 34, 44], is
actively debated [22, 23, 38, 46, 47, 54, 60]. For example, JAMIESON [46, page 2] discussed
this topic in his recent book on fish evolution: “The term plesiosperm recognizes that, whether or
not aquasperm have evolved in some sections of the Metazoa from more complex sperm, the
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149
“primitive” sperm facies may be genuinely plesiomorphic in many metazoan groups. This is not to
exclude from further investigation the possibility (a) that the earliest metazoans may have been
free-spawning with sperm of an even simpler form than the plesiosperm or (b) that they were
internally fertilizing with more or less complex sperm”.
It was once said that “it is unlikely that primitive spermatozoa from different animal phyla
should have evolved independently on several occasions obtaining the same morphology by
convergence. ”[9]. How much more unlikely is it that primitive members of at least twenty animal
phyla have evolved a basically similar, infinitely more complex introsperm by convergence? It
would seem more logical to assume secondary evolution of the simpler aquasperm from the more
complex introsperm. Recent molecular [62] and morphological [67, 69] analyses of neomenioids
place them basal to all extant Mollusca. It was surprising to find how typical is the introsperm of
neomenioids [22, 23], It is similar in basic structure and arrangement of organelles to introsperm
of primitive members of most bilaterian phyla, including some nemertines [2], bryozoans and
entoprocts [35], arthropods [3], annelids [53] as well as some deuterostomes [review: 46], In
particular, neomenioid introsperm closely resemble parasperm of protodrilids [53], a primitive
annelid group, formerly included in the now abandoned Archiannelida. The above evidence
provides support for current theories that recognize Eutrochozoa (animals with trochophore
larvae) as a distinct clade [30, 37, 50], which includes nemertines [76] and perhaps some other
invertebrate taxa. Besides the striking similarity to protodrilid parasperm, introsperm of
neomenioids have much in common with introsperm of Neritimorpha [15] and “primitive”
Caenogastropoda [13, 40], suggesting that current ideas on gastropod evolution [39] also may
require revision. Perhaps the Patellogastropoda, presently considered basal to Gastropoda [39],
diverged early from a neritimorph-like ancestor?
All Platyhelminthes have introsperm but until the key paper on the primitive turbellarian
Nemertoderma [77] demonstrated the typical introsperm pattern with a plausible acrosome,
rodlike nucleus, elongate midpiece, and single typical flagellum, it had been easy for scientists to
dismiss the sperm of Platyhelminthes as highly derived, which most of them are [41]. Since then
it has become obvious that introsperm are basal at least to the Platyhelminthomorpha
(Platyhelminthes and Gnathostomulida) [6, 73], Moreover, the above evidence strongly suggests
that introsperm are basal to all Bilateria [22, 23]. If true, this would unite Eubilateria and
Platyhelminthomorpha as a monophyletic clade.
The basal Platyhelminth groups (Nemertodermatida, Catenulida, Acoela and Macrostomida)
are minute organisms, attaining only a few millimeters in length and fractions of a millimeter in
width [6]. AX suggests that this is because they all possess the plesiomorphic condition of a
simple mouth pore, which limits nutrient uptake and hence body size [6], More importantly it
leaves only minute spaces between the endoderm and ectoderm with little room for expansion of
gonads. The corresponding small size of basal Gnathostomulida suggests that the bilaterian stem
was a microscopic organism [6]. Based on trace fossil evidence the earliest Bilateria are
considered to have emerged as minute wormlike organisms in the Proterozoic, between 600 M
and 1000 Myrs ago [25, 26]. BOADEN [11, 12] agreed with this and formulated the “Thyobios”
hypothesis which suggested that tiny anaerobic Bilateria evolved in shallow water, anoxic
sediments during the earliest emergence of the Metazoa. He suggested that as a consequence of
their small size they would have fertilized internally [12]. This correlates with geological evidence
of low atmospheric oxygen concentrations until the middle Vendian (550 Myrs) [27, 49]. A late
Proterozoic increase in atmospheric oxygen coincides with the emergence of macroscopic
Metazoa, first evident in the Ediacaran fauna [25, 27, 49]. However, the trace fossil evidence
indicates that Bilateria were well established before this time, and key ichnogenera are traceable
through all the early biozones [26].
Furthermore, the suggestion that free spawning always reflects primitive status [33, 44]
does not withstand close scrutiny [38, 54], A study of extant marine invertebrates has shown that
reproductive biology (including fertilization biology) depends on a series of covariable
150
J. BUCKLAND-NICKS : ACULIFERA (MOLLUSCA)
reproductive traits [54]. When body length is less than 1mm metazoans fertilize internally with
introsperm; only larger bodied forms successfully free-spawn ectaquasperm. Much of the reason
for this probably relates to fertilization success. Recent studies have shown that among free-
spawners, selection favours larger sperm numbers, larger egg numbers, as well as larger egg size
[51, 52]. Other key factors contributing to success include distance between mates, and spawning
synchronicity [7, 10, 51, 57]. Tiny triploblastic organisms would not be able to expand the
gonads to produce viable numbers of sperm and eggs for free-spawning and selection would
favour some form of internal fertilization [54], or hermaphroditism [36].
If it is true that introsperm were basal to Bilateria, then ectaquasperm must have been
secondarily developed many times. How could this have occurred? As mentioned, sperm
biologists are well aware that all sperm pass through a stage in their ontogeny that resembles the
plesiosperm [33, review:47]. Perhaps the re-expression of the plesiosperm form has occurred by
truncation of spermiogenesis, a type of progenesis? This has been advanced to explain the
presence of plesiomorphic structures in the sperm of schistosomes [48], and probably explains
secondary development of ectaquasperm in teleosts [47], as well as sabellid polychaetes [60]. If
progenetic spermiogenesis has occurred frequently among animal phyla, we might expect a
relatively simple mechanism to control it, perhaps a molecular switch. In vertebrates completion
of spermiogenesis is dependent on Sertoli cells [1]. Recently, a regulatory protein was isolated
from the round spermatid stage of rats (= plesiosperm stage), which was capable of turning on
the production of Sertoli cell total protein and transferrin secretion [55]. If these proteins are
found to play key roles in the completion of spermiogenesis, the regulatory protein produced by
the spermatids could be the kind of molecular switch we are looking for. In the absence of such a
regulatory protein, spermiogenesis could be truncated at the plesiosperm stage.
If secondary expression of ectaquasperm has occurred frequently, as suggested, there must
be a selective pressure favouring this type of sperm in external fertilization. Evidence indicates
that introsperm of some marine invertebrates are capable of swimming effectively in sea water
[14,60]. This suggests that the selective advantage to progenetic spermiogenesis may relate more
to lower energetic costs and shorter generation times in producing the less complex ectaquasperm,
rather than to more efficient locomotion in a sea water medium.
ACKNOWLEDGEMENTS
I would like to thank the following: Doug EERNISSE for collection and identification of some chiton species, as well
as for ideas on spermiocladistics; Doug Bright for collection, manipulation and fixation of sperm and eggs of
Chaetoderma argenteum; David Garbary for introducing me to Paup and MacClade and helping with analysis of
cladograms; Amelie Scheltema for supplying fixed specimens of Epimenia australis and for identifying Chaetoderma
canadense\ Leslie Hart and Shelley Hannigan for technical assistance. Finally I would like to thank Professor A. O. D.
Willows, Director Friday Harbor Laboratories, for periodic use of research facilities. This study was supported by an
NSERC of Canada research grant to J. B.-N.
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Source MNHN , Paris
Source : MNHN . Paris
Comparative Spermatozoal Ultrastructure and its
Taxonomic and Phylogenetic Significance
in The Bivalve Order Veneroida
John M. HEALY
Zoology Department, University of Queensland
Brisbane, Q 4072, Australia
ABSTRACT
A comparative study of sperm ultrastructure in the Veneroida, an ecologically and economically important order of
bivalve molluscs, has revealed significant differences between taxa. Most veneroid spermatozoa are of the aquasperm type
(general features: conical acrosome, nucleus usually short, midpiece consisting of a ring of round mitochondria
surrounding the centrioles, single simple flagellum). Five principle sperm morphologies can be recognized in the
Veneroida, these correlating to varying degrees of precision with groups of superfamilies. Group A (Lucinoidea,
Cardioidea (including Tridacnidae), Veneroidea, Mactroidea, Chamoidea, Solenoidea, Tellinoidea (one species of
Donacidae)): basal ring of acrosome without visible substructure and not developed longitudinally; nucleus curved if rod¬
shaped. Group B (Galeommatoidea ( Mysella , Scintilla , Divariscintilla )): sperm similar to Group A and especially C, but
with strongly tilted acrosomal complex and basal ring developed transversely. Group C (Tellinoidea (Donacidae,
Tellinidae), Arcticoidea, Dreissenoidea, Galeommatoidea ( Lasaea )): basal ring developed longitudinally, sometimes
showing substructure; overlap between mitochondria and nucleus only in Tellinidae. Group D (Tellinoidea
(Scrobiculariidae), Corbiculoidea): acrosome and nucleus elongate; pronounced overlap of mitochondria with nucleus.
Group E (Carditoidea + Crassatelloidea assemblage): acrosome and nucleus elongate; midpiece exhibiting usually 8
mitochondria; proximal centriole modified into well developed rootlet. The widespread occurrence of the Group A
spermatozoon within the Veneroida including the basal superfamily Lucinoidea, suggests that this sperm type was typical
of early veneroids. In contrast, the sperm morphologies encountered in Groups D and E are unknown elsewhere within the
Bivalvia, and undoubtedly represent modifications from a less complex sperm type (e.g. Group A sperm).
RESUME
Ultrastructure comparee des spermatozoides et sa signification taxonomique et phylogenetique
dans l’ordre des Veneroida (Bivalves)
L’etude compare de l’ultrastructure des spermatozoides chez les Veneroida, un groupe de Mollusques important du point
de vue 6cologique et economique, a montr6 des differences significatives entre les taxons. La plupart des spermatozoides
des Veneroida sont du type aquaspermatozoi'de, dont les caracteristiques generates sont un acrosome conique, un noyau
generalement court, une ptece intermediate consistant en un anneau de mitochondries rondes entourant les centrioles et un
flagelle simple et unique. Cinq morphologies principles de spermatozoides peuvent etre reconnues chez les Veneroida, et
peuvent etre corretees avec des degres varies de precision avec les groupes ou les superfamilies. Groupe A (Lucinoidea,
Cardioidea (y compris les Tridacnidae), Veneroidea, Mactroidea, Chamoidea, Solenoidea, Tellinoidea (une espece de
Donacidae)): anneau basal de facrosome sans substructure visible et non d6veloppe longitudinalement; noyau courbe si en
Healy, J. M., 1995. — Comparative spermatozoal ultrastructure and its taxonomic and phylogenetic significance
in the bivalve ’order Veneroida. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 155-166. Paris ISBN : 2-85653-225-X.
156
J. M HEALY : VENEROIDA (MOLLUSC A)
forme de baguette. Groupe B (Galeommatoidea (My sella. Scintilla, Divariscintilla )): spermatozoide similaire au groupe A
et specialement au groupe C, mais avec un complexe acrosomien fortement incline et un anneau basal developpe
transversalement. Groupe C (Tellinoidea (Donacidae, Tellinidae), Arcticoidca, Dreissenoidea, Galeommatoidea ( Lasaea )):
anneau basal developpe longitudinalement, montrant parfois une substructure; chevauchcment entre les mitochondries et
le noyau seulement chez les Tellinidae. Groupe D (Tellinoidea (Scrobiculariidae), Corbiculoidea): acrosome et noyau
allonges; chevauchement prononcc des mitochondries et du noyau. Groupe E (Carditoidea + assemblage des
Crassatelloidea): acrosome et noyau allonges; pidce interm&liaire montrant g6n6ralement huit mitochondries; centriole
proximal modifie en une racine bien developpee. La presence tr£s repandue du spermatozoide du groupe A chez les
Veneroida, y compris dans la superfamille primitive Lucinoidea, suggere que ce type de spermatozoide etait typique des
premiers Veneroida. Au contraire, les morphologies de spermatozoides rcncontrees dans les groupes D et F sont inconnues
dans d’autres groupes des Bivalvia, et representent de maniere certaine des modifications a partir d’un type de
spermatozoide moins complexe (par exemple le spermatozoide du groupe A).
The Veneroida constitute one of the most important extant orders of bivalve molluscs.
Included within the group are several marine families of economic and ecological significance
such as the cockles and giant clams (Cardiidae, Tridacnidae), venus shells (Veneridae and allies),
tellins (Tellinidae and allies) and the estuarine/ freshwater family Corbiculidae. Because of this,
studies of veneroid reproductive biology are of considerable importance in understanding the
success of the order. In addition, comparative work on sperm fine structure in this and other
molluscan groups continues to generate characters of taxonomic and phylogenetic significance,
most notably in the internally fertilizing Gastropoda which have complex, often polymorphic,
spermatozoa [12, 13, 17, 25, 26]. Although sperm morphology has been examined for several
bivalve species, it is only in recent years that a comparative approach has been applied within this
class at the uitrastructural level. In 1971 GHARAGOZLOU-VAN GlNNEKEN & POCHON-MASSON
[10] were the first authors to demonstrate significant differences between species and genera
within the Veneridae (Veneroidea, Veneroida, see Table 1). Similar studies have been carried out
by Hodgson and co-workers [19-21] on various Veneroida (congeneric species of Solenidae and
Donacidae, see Table 1) and Mytiloidea. Such information is not only useful in verifying the
validity of species, but also offers an opportunity to test their relationships to other congeners -
the latter exercise being dependent on the availability of other sperm data. On a broader scale
sperm uitrastructural studies by POPHAM [41] and HEALY [14, 17] have also been directed
towards investigating the taxonomic and evolutionary relationships between bivalve subclasses
and orders.
Above the species and genus levels, sperm ultrastructure appears to be a promising source
ol taxonomic and phylogenetic information in the Veneroida based on my own observations
(presented here, see Table 1) as well as data from published accounts (Table 2). The present study
examines comparative sperm morphology among veneroid bivalves and concludes with a
discussion of the possible taxonomic and phylogenetic implications of all available data for the
group. Although some authors exclude the Lucinoidea from the Veneroida (placed in a separate
order by MORRIS [33], in a separate subclass by POJETA [40]), I have adopted the more
traditional approach [1, 5, 32, 49] and retained it within the order.
MATERIAL AND METHODS
, PreSe,n‘ ^fVey °f ve"eroid sPerm ultrastructure is based on the author’s observations (using species collected
from the Queensland coast: see Table 1) combined with information already available in the literature (sec Table 2) For
n Tm Pnh^SKniVeltl8ffed here'n- f“ °' lesIicular tissue was camed out using ice cold (0-4°) glutaraldehyde (3.5% in
,or inP! , ua e buffer containing 10% w/v sucrose) followed by placing tissue pieces into 1% osmium tetroxide (at 0-
idiust'ed?' ei^nn^h Ph°sPhaIe buffer>- followed by three rinses in phosphate buffer (at 0-4°C, buffer sucrose
a,nd C,p d g ln Spurr's epoxy resin- For Codakia punctata (Lucinoidea) and Tellina
S S ,v ,n, T f0rmahn ’est,s tlssues only werc processed for TEM. Semithin and ultrathin sections were
fr,nf 8 , UM IV UI,rotome s,amcd according to the procedure of Daddow [7] and examined using an Hitachi H-300
transmission electron microscope.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
157
Table 1. — Veneroid taxa examined in the present study and dimensions of acrosome, nucleus and midpiece (dimensions
averaged, n = 5). ND = not determined. Dimensions expressed in pm. L = maximum length; D = maximum
diameter. Maximum diameter measurements for acrosomal vesicle and nucleus taken through base of these sperm
components.
158
J. M. HEALY : VENEROIDA (MOLLUSCA)
Table 2. — Vcneroid taxa previously investigated for sperm ultrastructure
Superfamily Cardioidea
Family Cardiidae
Donax serra [20]
Donax trunculus [47]
Family Scrobiculariidae
Scrobicularia plana [48]
Superfamily Dreissenoidea
Family Dreissenidae
Dreissena polymorpha [9]
Superfamily Galeommatoidea
Family Galeommatidae
Lasaea subviridis [35,38]
My sella tumid a [35, 36]
Pseudopythina rugifera [37]
Divariscintilla yoyo [8]
Divariscintilla troglodytes [8]
Scintilla sp. [8]
Superfamily Corbiculoidea [13)
Family Corbiculidae ( Corbicula )
Corbicula sandai [11]
Corbicula fluminea [28]
Superfamily Carditoidea
Family Carditidae
Cardita muricata [18]
Superfamily Crassatelloidea
Family Crassatellidae
Eucrassatella cumingii [18]
Eucrassatella kingicola [18]
Talabrica aurora [18]
RESULTS AND DISCUSSION
Aquasperm features ofveneroid spermatozoa
Most veneroid spermatozoa, like those of the majority of other bivalve taxa, could be
classed as unmodified or relatively unmodified aquasperm, the principal features of which are as
follows: (1) a well developed, conical acrosomal vesicle; (2) a short or relatively short nucleus
(with electron-lucent lacunae); (3) a short midpiece (containing round mitochondria, usually four
or five in number, surrounding a pair of orthogonally arranged, triplet substructure centrioles); (4)
a radial array of satellite fibres anchoring the distal centriole (basal body) to the plasma membrane;
(5) a simple flagellum (9+2 axoneme sheathed only by the plasma membrane). Although most
differences in sperm morphology between veneroid taxa involve acrosomal and/or nuclear
Fig. 1. — A-E: The five major sperm morphologies occurring within the Veneroida. a, acrosomal complex; av, acrosomal
vesicle; c, centrioles (proximal and distal); f, flagellum; m, mitochondria (of midpiece); n, nucleus; sm.
subacrosomal material. Scale bars = 0.25 pm, except where indicated. Sources of data: present study except
Chamoidea [2]; Solenoidea [4]; Galeommatoidea [5, 6]; Tellinoidea - Scrobiculariidae [9].
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
159
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160
J. M. HEALY : VENEROIDA (MOLLUSCA)
features, some useful variations in midpiece architecture are also apparent such as differing
mitochondrial number and presence/absence of an unmodified proximal centriole. The five main
sperm morphologies occurring in the Veneroida, and their chief characteristics, are as presented
below (taxa studied here indicated by *, other sources referenced by number).
Group A: Lucinoidea [*], Cardioidea [*, 46], Veneroidea [* 10, 20, 34, 39, 43], Mactroidea [*,
22, 29], Tellinoidea (Donacidae - Donax trunculus only, [47]), Chamoidea [22], Solenoidea [4,
21 ] (Figs 1A, 2A-E)
General features: (1) acrosomal vesicle short, conical, deeply invaginated, with thick,
highly electron-dense basal ring (longitudinal profile of ring round to pyriform); subacrosomal
material either diffuse or with axial rod differentiated; (2) nucleus short or rod-shaped (usually
curved if rod-shaped) depending on family or genus, typically with no or only a poorly developed
apical depression (apex often convex); (3) midpiece with two unmodified (triplet substructure)
centrioles surrounded by four or most commonly five rounded mitochondria; (4) satellite fibre
complex anchoring distal centriole to plasma membrane; (5) single flagellum with 9+2 axoneme.
The widespread occurrence of this sperm morphology within the Veneroida, including the
Lucinoidea (considered by many authors as the oldest extant veneroid superfamily), suggest that it
was probably characteristic of the earliest members of the order. Variation in the shape and
dimensions of the acrosomal vesicle and nucleus is considerable in the Veneroidea.
Group B: Galeommatoidea (Lasaea, Scintilla, Divariscintilla) [8, 35]. (Fig. IB)
Features as for Groups A and especially C, but differing in having the acrosomal complex
arranged at a considerable angle to the longitudinal axis of the spermatozoon. Basal ring crescentic
(as in Group C) but developed transversely.
This type of sperm morphology was probably derived from the more widespread Group C
type, through re-alignment of the acrosomal vesicle (and its crescentic basal ring). A detailed
study of Scintilla and Divariscintilla [8] shows that the tilted positioning of the acrosomal vesicle
seen in mature spermatozoa takes place in the final phase of spermiogenesis.
Group C Tellinoidea (Donacidae [20,*], Tellinidae [*]), Arcticoidea [*], Dreissenoidea [9],
Galeommatoidea (Mysella [35]). (Figs 1C, 2F-I)
Features resembling those of Groups A and especially B. Group C acrosomes differs from
those of Group B in not being tilted and in having the basal ring developed longitudinally rather
than transversely. Substructure sometimes visible within the acrosomal vesicle contents. Marked
overlap of midpiece and nucleus in Tellina (Tellinidae, Tellinoidea) similar to Group D.
Group D: Tellinoidea (Scrobiculariidae [48]), Corbiculoidea [11, 28]. (Fig. ID)
Acrosomal vesicle slender, almost totally invaginated with basal ring not clearly defined.
Subacrosomal material diffuse, without axial rod. Nucleus elongate and slender. Midpiece
Fig. 2. — A-E: Electron micrographs illustrating major variations between Veneroida examined. Group A. A: Sperm
head and midpiece of Codakia punctata (Lucinidae, Lucinoidea). B: Acrosome of Glauconome sp. (Glauconomidae,
Veneroidea). Arrowhead in this and other figures indicates basal ring component of acrosomal vesicle contents.
C: Acrosome of Spisula trigonella (Mactridae, Mactroidea). D: Acrosome of Fragum unedo (Cardiidae,
Cardioidea). E: Sperm head and midpiece of Circe cf plicatina (Veneridae, Veneroidea). Note curved nucleus.
F: Sperm head, midpiece and proximal portion of flagellum of Donax deltoides (Donacidae, Tellinoidea).
G: Acrosome of Trapezium sublaevigatum (Trapeziidae, Arcticoidea). H: Acrosome of Tellina rostrata
(Tellinidae, Tellinoidea). I: Nuclear base and midpiece of T. rostrata . Note extensive overlap of mitochondria and
nucleus, av, acrosomal vesicle; dc, distal centriole; f, flagellum; m, mitochondria (of midpiece); n, nucleus; pc.
proximal centriole; sm, subacrosomal material. Scale bars: A, E, F = 0.5 pm; B-D, G-I = 0.25 pm.
Source : MNHN, Paris
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
161
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162
J. M. HEALY : VENF.R01DA ( MOLLUSCA )
mitochondria oblong and overlapping significantly with base of nucleus (similar to Tellina from
Group C).
This represents one of the most modified sperm morphologies encountered in the
Veneroida. The type is of special interest because microtubules surround the spermatid nucleus
during condensation (at least in Scrobicularia', details unavailable for Corbicula). Spermatid
perinuclear microtubules occur in no other bivalves, but are widely reported in gastropods
(introsperm-producing taxa [15, 25, 26]), cephalopods [16] and polyplacophorans [3], The
presence of this sperm type in the Tellinoidea and Corbiculoidea raises important questions
concerning the relationship between these superfamilies (see Systematic considerations below). A
report (based on SEM and light microscopy) that spermatozoa of Corbicula fluminea are both
dimorphic (head region either slender or wide) and biflagellate [28], requires confirmation using
transmission electron microscopy.
Group E: Carditoidea (Carditidae, Crassatellidae) [18]. (Fig. IE)
Acrosomal vesicle elongate conical, almost totally invaginated, with the presumed
homologue of the basal ring evident as a dense inner layer. Subacrosomal material differentiated
into axial rod. Nucleus rod-shaped with short but distinct apical depression (partly
accommodating subacrosomal material). Midpiece characterized by 8 (rarely 7 or 9) tightly
adpressed mitochondria surrounding a dense centriolar rod (a metamorphosed proximal centriole)
and distal centriole.
This is a highly distinctive sperm type, and its presence in the Carditidae and Crassatellidae
has wide taxonomic and phylogenetic implications within the Veneroida (see below). The
transformation of the proximal centriole into a rod connecting distal centriole (basal body) to
nuclear fossa is unique among molluscan aquasperm.
Phylogenetic and systematic considerations
According to ALLEN [1] the success of veneroid bivalves has pivoted on their exploitation
of soft sediments, largely as a consequence of their possessing and developing siphons (in
combination, it should be stressed, with an ability to burrow effectively). Although most adaptive
radiation within the Veneroida took place within the Mesozoic, some superfamilies such as the
Lucinoidea and Crassatelloidea extend back to the lower Palaeozoic [1, 6, 31, 32] while several
important living families and genera date only from the late Cretaceous or Tertiary [31]. Veneroid
origins are unclear, and it remains uncertain as to whether the group is truly monophyletic or not.
In the present account, five principal sperm morphologies are recognized within the
Veneroida based on shared ultrastructural features (observed through TEM). Light microscopic
work of KARPEVICH [23] indicates that helical sperm nuclei also occur in several species of
Cardiidae and at least one species of Tellinidae. Present knowledge of comparative sperm
ultrastructure in the Veneroida, despite the absence of data for a number of families and genera,
provides new information relevant to discussions of relationships within the order.
Group A. Despite anatomical specializations in some living representatives, the Lucinoidea
constitute one of the oldest living heterodont superfamilies, being definitely recorded from the
Silurian [1,5] but possibly extending back to the Ordovician if a close relationship with the fossil
genus Babinka is accepted [30, 31, 40], The presence of Group A sperm morphology in this
superfamily, and its widespread occurrence within the Veneroida, suggest that the earliest
members of the order also possessed Group A type spermatozoa. Comparative spermatology in
Group A superfamilies, especially in relation to acrosomal and nuclear features, appears to have
considerable taxonomic potential at the species and generic levels (e.g. in the Veneroidea [10];
Cardioidea [*]; Solenoidea [21]). The disputed superfamily placement of the Hemidonacidae [45],
either among the Cardioidea or the Tellinoidea, could probably be settled by examination of sperm
ultrastructural features, although it should be added that within at least one tellinoidean family
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
163
(Donacidae) the acrosome may be of Group A type ( Donax trunculus only - [47]) or Group C (all
four other investigated species of Donax).
Group B. The Galeommatoidea (= Leptonacea) are small, often hermaphroditic veneroids
which fertilize eggs within the mantle cavity (where young are brooded) and employ a range of
methods to transfer sperm from individual to individual (spermatophores, sperm morulae, usage
of dwarf males) [35-38]. In addition, the only confirmed cases of sperm dimorphism within the
Bivalvia involve galeommatoidean species [35],
The classification of galeommatoidean bivalves (= Leptonacea) is far from being fully
resolved [2, 8], and this seems to be reflected in marked acrosomal differences between examined
genera. For example, in Lasaea, Scintilla and Divariscintilla (Group B), the acrosomal complex is
apically compressed and tilted at a considerable angle. In addition the basal ring is developed
transversely (Fig. IB). By contrast, in My sella (placed in Group C), the acrosomal vesicle tapers
apically and is not tilted. The basal ring is developed longitudinally and accompanied by a
lamellate apical density [35]. Unfortunately the only available TEM micrograph of Pseudopythina
spermatozoa [37] is not detailed enough to determine the substructure of the acrosome. Although
galeommatoideans probably arose through neoteny [1], the actual source (? or sources) of these
bivalves has not been identified with any certainty. Available sperm data suggest that all
galeommatoideans could have arisen from the Tellinoidea or the Arcticoidea, or possibly from
both if the superfamily proves not to be monophyletic.
Group C. Spermatozoa of Group C could have been easily derived from those of Group A
through lengthening and an increase in complexity of the basal ring component of the acrosomal
vesicle. Within the Tellinoidea, spermatozoa of the Donacidae are typical of Group C, while those
of Tellina rostrata (Tellinidae) appear to bridge the gap between Groups C and D by having
significant mitochondrial overlap with the nucleus. It is unfortunate that so few tellinoidean
families have been examined for sperm ultrastructure (e.g. no available data for the Solecurtidae,
Semelidae, Psammobiidae). Such information would be of considerable value not only in
evaluating relationships within the Tellinoidea, but also in exploring possible connections with the
Arcticoidea, Galeommatoidea and freshwater Dreissenoidea. At the species level, comparative
sperm morphology within the Donacidae may prove to be of considerable taxonomic use.
Spermatozoa of the Australian species Donax deltoides are structurally similar to, although not
identical with, those of the South African species D. sordidus and D. madagascariensis but differ
markedly in acrosomal shape from another South African species D. serra (compare Fig. 1C, 2F
with results by HODGSON et al. [20]). Spermatozoa of all four of these species of Donax differ
from those of D. trunculus , which has a more elongate nucleus and an acrosome essentially of the
Group A type [47],
Group D. Particularly interesting is the similarity between spermatozoa of the tellinoidean
Scrobicularia plana (the only investigated member of the Scrobiculariidae) (Fig. ID) and the
freshwater Corbiculoidea. Is it possible that the Corbiculoidea have been derived from the
Tellinoidea, or are the observed sperm similarities between Scrobicularia and Corbicula merely the
result of convergence (that is, a similar fertilization biology)? In the absence of comprehensive
data for a range of tellinoidean and corbiculoidean species this must remain an intriguing but
presently insoluble problem. BOSS [2] considered that the Scrobiculariidae were perhaps
unnecessarily split from the large tellinoidean family Semelidae. Further research on the
spermatozoa of semelids and scrobiculariids (Tellinoidea) will undoubtedly throw further light
onto this issue.
Group E. A close relationship between the Carditidae and Crassatellidae is clearly indicated
by sperm morphology providing strong support for YONGE's suggestion [50, 51] that the
Carditoidea and Crassatelloidea should be united into a single superfamily (for further discussion
see HEALY [18]). Although the relationship of Group E (Carditidae + Crassatellidae) to other
Veneroida is extremely uncertain, the preponderance of apomorphic sperm characters in this
164
J. M. HEALY : VENER01DA (MOLLUSCA)
Group effectively excludes it as a source of other veneroid taxa (e.g. origin of Veneroidea,
Tellinoidea, Chamoidea plus Myoida from the “Carditida” suggested by SCARLATO &
STAROBOGATOV [44]; origin of Cardioidea from carditids or crassatellids suggested by KEEN
[24], origin of Tridacnidae from Carditoidea suggested by ALLEN [1 ]).
ACKNOWLEDGEMENTS
I thank Mrs L. Daddow for her generous assistance with aspects of transmission electron microscopy. Mr. L.
Lamprell (Brisbane). Ms G. Brodie (James Cook University. Townsville) and Mr. M. Healy (Brisbane) are thanked for
collecting or assisting in collecting bivalve material this study. Helpful comments by the referees are also gratefully
acknowledged. The project was supported by an Australian Research Fellowship provided by the Australian Research
Council.
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42. Purchon, R. D., 1987. — Classification and evolution of the Bivalvia: an analytical study. Philosophical
Transactions of the Royal Society of London, B., 316: 277-302.
43. Reunov, A. A. & Hodgson, A. N., 1994. — Ultrastructure of the spermatozoa of five species of South African
bivalves (Mollusca), and an examination of early spermatogenesis. Journal of Morphology, 219: 275-283.
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J. M. HEALY : VENEROIDA (MOLLUSCA)
44. Scarlato, O. A., Starobogatov, Y. I., 1978. — Phylogenetic relations and the early evolution of the class
Bivalvia. Philosophical Transactions of the Royal Society of London, B ., 284: 217-224.
45. Schneider, J. A., 1992. — Preliminary cladistic analysis of the bivalve family Cardiidae. American Malacological
Bulletin, 9: 145-155.
46. Sousa, M. & Azevedo, C., 1988. — Comparative silver staining analysis on spermatozoa of various invertebrate
species. International Journal of Invertebrate Reproduction , 13: 1-8.
47. Sousa, M. & Oliveira, E., 1994. — Ultrastructural and cytochemical study of spermatogenesis in Donax trunculus
(Mollusca, Bivalvia). Journal of Submicroscopic Cytology and Pathology, 26: 305-311.
48. Sousa, M. , Corral, L. & Azevedo, C., 1989. — Ultrastructural and cytochemical study of spermatogenesis in
Scrobicularia plana (Mollusca, Bivalvia). Gamete Research, 24: 393-401.
49. VAUGHT, K. C., 1989. — A Classification of the Living Mollusca. (R. T. ABBOTT & K. J. BOSS, Eds). Melbourne,
Florida, American Malacologists: 1-195.
50. Yonge, C. M., 1969. — Functional morphology and evolution within the Carditacea (Bivalvia). Proceedings of the
Malacological Society of London, 38: 493-527.
51. Yonge, C. M., 1978. — Significance of the ligament in the classification of the Bivalvia. Proceedings of the Royal
Society • of London, B., 202: 231-248.
Source : MNHN, Paris
Spermatozoal Morphology of Patellogastropoda and
Vetigastropoda (Mollusca: Prosobranchia)
Alan N. HODGSON
Department of Zoology & Entomology,
Rhodes University, Grahamstown, 6140, South Africa
ABSTRACT
The morphologies of the aquasperm of Patellogastropoda and Vetigastropoda are distinctly different, adding further
support to the view that these two taxa are not closely related. Within the patellogastropods each family examined to date
(Patellidae, Nacellidae, Lottiidae, Acmaeidae) has sperm with distinguishing features and it is therefore possible to
recognize members of a family and differentiate between families using sperm morphology. Similarly sperm morphology
can be used to differentiate between families of vetigastropods. Although there are a few exceptions, the size (length to
breadth ratio) and shape of the nucleus and acrosome of sperm of species within each family are similar. The broad
similarities in the morphology of the spermatozoa of the Pleurotomarioidea, Fissurelloidea, Haliotoidea and Trochoidea
(with the exception of the Skeneidae) indicate that these taxa are phylogenetically closely related. Some vetigastropods,
notably the Lepetodrilidae, Scissurellidae and Skeneidae, have modified spermatozoa which correlates to internal
fertilization or fertilization within the mantle cavity. It is suggested that more extensive studies on the sperm from these
families as well as other archaeogastropod taxa could provide additional clues to the phylogeny of higher gastropods.
RESUME
La morphologie des spermatozoides des Patellogastropoda et des Vetigastropoda (Mollusca:
Prosobranchia)
La morphologie des aquaspermatozoides est differente chez les Patellogastropoda el les Vetigastropoda, ce qui ajoute des
arguments & I' opinion que ces laxons ne sont pas proches. Parmi les Patellogastropoda chacune des families examinees
jusqu’ici (Patellidae, Nacellidae. Lottiidae et Acmaeidae) a des spermatozoides avec des caractdres distinctifs et il est done
possible de reconnaitre les membres d’une famille et de difterencier les families en utilisant la morphologie du
spermatozoide. De meme, la morphologie du spermatozoide peut etre utilisSe pour differencier les families de
Vetigastropoda. En d6pit de quelques exceptions, les dimensions (rapport longueur sur largeur) et la forme du noyau et de
l’acrosome des spermatozoide des esp&ces d’une meme famille sont similaires. La grande ressemblance morphologique des
spermatozoides des Pleurotomarioidea, Fissurelloidea. Haliotoidea et Trochoidea (& 1’exception des Skeneidae) indique que
ces taxons sont tr£s proches phylog£n6tiquement. Quelques Vetigastropoda. en particulier les Lepetodrilidae,
Scissurellidae et Skeneidae, ont des spermatozoides modifies qui sont correles avec la fecondation interne ou la
fecondation & l’interieur de la cavite palleale. II est probable que des etudes plus nombreuses des spermatozoides de ces
families et d’autres Archaeogastropodes pourraient fournir des indices suppl^mentaires pour la phylog^nie des
Gasteropodes 6volues.
Hodgson, A. N., 1995. — Spermatozoal morphology of Patellogastropoda and Vetigastropoda (Mollusca:
Prosobranchia). In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 167-177. Paris ISBN : 2-85653-225-X.
168
A. N. HODGSON : ARCHAEOGASTKOPODA ( MOLLUSCA )
Comparative spermatology has without doubt made significant contributions to a greater
understanding of the taxonomic and phylogenetic relationships within and between many taxa.
Within the molluscan class Gastropoda, the archaeogastropods are a large assemblage of
prosobranchs, the taxonomic status of which has been the subject of considerable debate [5, 6, 9,
16]. Much of this debate is a result of the discovery of new taxa from deep-sea and hydrothermal
vent communities [6-9, 31]. Thus in several recent re-evaluations of the Archaeogastropoda ( s .
lat.) [7-9, 16, 30], the assemblage has been split into numerous new orders.
A number of the recent publications have also attempted to reconstruct phylogenetic
relationships within the archaeogastropods (s. lat.) as well as between archaeogastropods and
higher gastropod taxa (e.g. caenogastropods and euthyneurans) [5-7, 16, 17]. To date most of
these studies have not incorporated detailed information from sperm morphology (or
spermiogenesis), information which may provide valuable insights into archaeogastropod
taxonomy and phylogeny.
Two of the recognized orders within the archaeogastropod assemblage are the
Patellogastropoda (formally Docoglossa) and Vetigastropoda. Whereas virtually nothing is known
about sperm morphology of the recently discovered archaeogastropods, in the last 14 years there
have been a number of publications on the structure of the spermatozoa of patellogastropods [1,
18-21, 25-28, 34] and vetigastropods [10, 13-15, 22, 23], This paper reviews this information,
discusses the systematic and phylogenetic implications of the data and highlights gaps in our
knowledge.
MATERIALS AND METHODS
Information on the ultrastructure of the spermatozoa of Patellogastropoda and Vetigastropoda was obtained either
from published papers (see Table 1 and reference list) or from tissue prepared for transmission electron microscopy. For
TEM, small portions of the testis of each species were fixed in 2.5% glutaraldehyde in filtered sea water for 2-3 hours. The
exception to this was the hydrothermal vent gastropod Lepeiodrilus fucensis. This material spent several weeks in the
glutaraldehyde fixative. Tissues were post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer and filtered sea
water for 90 minutes, dehydrated in a graded ethanol series, and embedded in a TAAB/Araldite resin mixture via propylene
oxide. Thin sections (silver/gold) were stained in 5% aqueous uranyl acetate (30 minutes) and lead citrate (5 minutes) and
examined in a JF.OL JEM CX1I TEM at 80kV.
RESULTS
Patellogastropoda
The spermatozoa of 43 species from four families (Table 2) of Patellogastropoda have been
examined or described and all have aquasperm. Results to date suggest that members of each
family of patellogastropod have spermatozoa with characteristic morphological features (Fig. 1).
Patellidae. In species of Patellidae (Fig. 1 A-E), the acrosome, the contents of which may or
may not be differentiated into electron-dense and electron-lucent regions, constitutes <50% of the
head length and is deeply invaginated posteriorly. The subacrosomal space does not contain an
axial rod. Despite these similarities within the Patellidae five morphological types of spermatozoa
can be recognized within the family (Fig. 1A-E). The first type (Fig. 1A) have heads with
cylindrical nuclei (usually <5 pm long and “bullet-shaped”) which are rounded anteriorly and
small cap-like acrosomes, the contents of which are uniformly electron-dense (i.e.
undifferentiated) (1 1 species). The second type (Fig. IB; 3 species) have very elongate “flask¬
shaped” nuclei (> 1 2 pm long) which are pointed anteriorly and acrosomes with long anterior
extensions. The third type (Fig. 1C; 11 species) have nuclei (about 5 pm long) which have a
square -shaped anterior which intrudes into the subacrosomal space giving the nucleus a “bottle¬
shaped” appearance. Anterior to the nucleus is a relatively large acrosome, the wall of which is
bulbous posteriorly and in addition the contents are differentiated into electron-dense and electron-
lucent regions. The fourth type (Fig. ID), have cylindrical nuclei (5-7 pm long) and an acrosome
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
169
1. — Summary of the morphological features of the spermatozoa of vetigastropod families. * Information available
as published diagram only. ** Deep-sea species.
170
A. N. HODGSON : ARCHAEOGASTROPODA (MOLLUSCA)
Table 2. — The species of four families of patellogastropod for which descriptions of the sperm exist. Information
obtained from [I, 18-21, 25, 28, 35]. The sperm types of Patellidac (I, II, III, IV, VI) are from [19, 25]. 'indicates
species of uncertain taxonomic status but initially identified as P. miniata from Angola; indicates species of
uncertain taxonomic status but initially identified as P. miniata from Namibia.
which is differentiated internally and has a cylindrical posterior lobe. Finally the fifth type (Fig.
IE; 7 species) consists of a nucleus which is elongate and cylindrical but capped by a short,
undifferentiated A-shaped, conical acrosome.
Nacellidae. In the Nacellidae the heads of the spermatozoa are always elongate (length:
breadth >7:1) and the nucleus is distinctly conical in shape (Fig. IF). The acrosome, which
constitutes <50% of the head length, has a complex internal differentiation which can take the
form of electron-opaque striations (e.g. Cellana capensis [19]). An axial rod is always present in
the subacrosomal space.
Lottiidae. Lottiidae spermatozoa have a head with a short cylindrical nucleus which is
rounded anteriorly (Fig. 1G). Anterior to the nucleus is an elongate acrosome which constitutes
>50% of the total head length. The acrosome is invaginated posteriorly, differentiated internally
into an outer electron-lucent and inner electron-dense region and has an elongate posterior lobe
which protrudes into the sub-acrosomal space. Within this space, the fibrous material is
aggregated into an axial rod-like structure. The midpiece has four to five spherical mitochondria
(with well developed cristae), which surround the proximal and distal centrioles. Surrounding the
anterior portion of the axoneme is an elongate (1 (im) cytoplasmic collar (this structure is much
smaller in other patellogastropod taxa).
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
171
Fig. 1. — Semi-diagrammatic longitudinal sections through the spermatozoa of representative Patellogastropoda. a,
acrosome; ar, axial rod; co, cytoplasmic collar; M. mitochondrion; n. nucleus; pa. posterior acrosomal
invagination. Diagrams A-C, F, G are modified from [21]. Scale bar = 2 pm.
Acmaeidae. Only two species, Patelloida profunda albonotata [18] and P. saccharina lanx
[28], have been described from this family, the sperm of the latter species being illustrated
diagrammatically only. The sperm have elongate, cylindrical heads (about 5.6 |im long x 0.3 |im
mid-diameter in P. p. albonotata and 5.4 pm x 0.4 pm in P. s. lanx ) (Fig. 1H). The nucleus is
cylindrical, tapering towards the rounded anterior, and is capped by a short conical acrosome
(about 1 pm long) the contents of which are undifferentiated. The acrosome has a broad posterior
invagination, the subacrosomal space containing an axial rod. The midpiece contains four to five
spherical mitochondria (about 0.5 pm diameter).
Vetigastropoda
With the exception of the Skeneidae which have dimorphic spermatozoa [13],
vetigastropods produce only euspermatozoa. Although the majority of species have aquasperm
(Fig. 2) each family has spermatozoa with characteristic features (summarized in Table 2).
Pleurotomariidae. Only two species from this family, Perotroclms westralis (described as
Pleurotomaria africana) and Perotroclms quoyanus have been examined [10, 15]. Both species
have aquasperm. The sperm head is composed of a cylindrical nucleus (length: breadth about 3:1)
172
A. N. HODGSON : ARCHAEOGASTROPODA (MOLLUSCA)
which is square anteriorly with a small central invagination. Posteriorly the nucleus has a small
but well developed fossa (described as crypt-like by Healy & Harasewych [15]).
Fibrous rootlets extend from the proximal centriole into this fossa. The acrosome is in the
form of a rounded cone the contents of which are not differentiated. In addition the acrosome has
a deep, narrow posterior invagination.
Haliotidae. The sperm of the Haliotidae all have a head which is cylindrical, with a L:B ratio
normally >5:1 (Table 1). The nucleus, which is cylindrical, has a narrow anterior fossa. The
acrosome, which constitutes -40-50% of the total length of the sperm head has a narrow anterior
canal containing an axial rod. The acrosomal contents may or may not be differentiated.
Scissurellidcie. The spermatozoon of only one species of scissurellid, Sinezona sp. has been
described [14] and it is of the ent-aquasperm type [32] . The head consists of a cylindrical nucleus
which has prominent anterior and posterior invaginations. The anterior invagination houses an
axial rod. The small cap-shaped acrosome is undifferentiated internally. The midpiece consists of
an axoneme which is surrounded by a sleeve-like mitochondrion.
Fissurellidae. Most fissurellids have a sperm head with a length to breadth ratio of >4:1.
The nucleus is cylindrical (L:B 3:1; Montfortula conoidea and Scutus antipodes are exceptions
[14]). Except for M. conoidea and S. antipodes in which the acrosome comprises 50% of the
head length, the acrosome of fissurellids is nearly always small, <35% of the total head length
and is deeply invaginated, the invagination penetrating the acrosome as a narrow tube which
widens at the anterior. In the subfamily Fissurellinae the nucleus either has a small peg-like
anterior extension (includes species of Fissurella and Dendrofissurella) or there is a shallow
anterior nuclear fossa ( Amblychilepas ). By contrast the Eumarginulinae (includes species of
Montfortula and Scutus) all have a large V-shaped anterior nuclear invagination which houses an
axial rod.
Trochidae and Plmsianellidae. The sperm of species from these two families have a barrel¬
shaped nucleus (L:B <4:1) which has a U-shaped anterior invagination. In many species the
contents of the broadly conical acrosome are uniformly electron-opaque [22], whereas in other
species a differentiated acrosome has been noted [10]. The acrosome normally constitutes 50% or
less of the total head length and has a narrow posterior invagination. In most trochids there is an
axial rod.
Turbinidae. Turbinid spermatozoa are characterised by a nucleus with a length to breadth
ratio <1.5:1. The nucleus has a very wide anterior invagination and a relatively large (>50% of the
total head length) conical acrosome, the base of which lies within the nuclear invagination. The
acrosomal contents are differentiated internally. Posteriorly the acrosome has a short, narrow
invagination.
Lepetodrilidae. A preliminary examination of one species (Lepetodrilus fucensis) from this
taxon, has revealed that they produce only euspermatozoa which are modified [23]. The sperm
have an elongate head (about 1 1 pm long) comprised of a long cylindrical nucleus (about 9 pm
long x 0.4 pm mid-diameter) and an anteriorly positioned conical acrosome (about 2 pm long).
Between the acrosome and the nucleus is a tube-like structure, the subacrosomal plate (about 0.3
pm long), which is expanded inwards at its base as a small flange. The lumen of this tube is filled
with an amorphous material. The acrosome, which sits on the tube, is deeply invaginated
posteriorly; the subacrosomal space within the invagination contains an axial rod.
Posteriorly the nucleus has a shallow invagination into which the complex centriolar
apparatus protrudes. A single large mitochondrion it sited laterally to the centriolar apparatus. The
anterior portion of the axoneme (which emerges from the centriolar complex) is surrounded by a
cytoplasmic collar which forms a tube about 2.5 pm long. The cytoplasmic collar contains
numerous tubular structures (about 60 nm diameter) which encircle the axoneme.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
173
LEPETODRILOIDEA TROCHOIDEA
Lepetodrilidae
Skeneidae
Fig. 2. — Semi-diagrammatic longitudinal sections through spermatozoa of nine families of Vetigastropoda. Examples
chosen are thought to typify each family, a, acrosome; af, anterior nuclear fossa; ar, axial rod; co, cytoplasmic
collar; M, mitochondrion; n, nucleus; p, proximal centriole; pa, posterior acrosomal invagination; pe.
subacrosomal plate; pf, posterior nuclear fossa, r, radial arm. Within the Fissurellidae type 1 sperm are from the
Fissurellinae and type 2 from the Eumarginulinae. The diagrams are modified from [13, 22, 23]. Scale
bars = 1 jim.
Skeneidae. The sperm of three species of skeneid have been described [13]. All produce
euspermatozoa and one species ( Zalipais laseroni) has paraspermatozoa. The eusperm are
uniflagellate with a long (about 50 pm in Zalipais laseroni ), tubular helically coiled nucleus.
Anterior to the nucleus of Z laseroni there is a small conical acrosome. The midpiece consists of a
mitochondrial sleeve which surrounds an electron-dense rod (about 3 pm long).
The parasperm of Z. laseroni consist of an elongate electron-dense head, a short midpiece of
centriolar rods and mitochondria, and a posterior tuft of flagella.
174
A. N. HODGSON : ARCHAEOGASTROPODA (MOLLUSCA)
DISCUSSION
Patellogastropod- Vetigastropod relationships
LlNDBERG [30] has argued that limpets from the superfamilies Patelloidea, Nacelloidea and
Acmaeoidea have morphological features which differ from those of other archaeogastropods.
These limpets were therefore removed to a separate order, Patellogastropoda and are regarded as
an early holophyletic gastropod offshoot [7, 9]. It is now clear that there are also distinct
differences in sperm morphology between the patellogastropods and vetigastropods.
Patellogastropods always produce aquasperm, the nuclei of which never have an anterior fossa,
whereas in the majority of vetigastropods with aquasperm (the Fissurellinae is an exception) this
nuclear feature is present. Furthermore, in patellogastropods the acrosome always has a wide
posterior invagination. By contrast in vetigastropods the posterior acrosomal invagination is
always in the form of a narrow canal. These small but consistent differences in sperm
morphology between patellogastropods and vetigastropods supports the view [7, 30] that these
two taxa are distinct from each other.
Patellogastropod relationships
Although information on sperm morphology from families of Patellogastropoda is still
incomplete, results to date suggest that each family has a sperm with distinguishing features. As
the evolutionary trends and relationships between and within patellogastropods are not certain
[30], spermatozoon morphology may eventually provide additional clues to limpet phylogeny
when data from outstanding taxa (e.g. Lepetidae) and more detailed information from other taxa
(e.g. Nacellidae and Acmaeidae) are forthcoming. Despite the incomplete data base, the
information available on sperm morphology invites some speculation on limpet phylogeny. It is
suggested that spermatozoa with the most plesiomorphic features are found in the Patellidae, with
some species having a sperm with a short “bullet-shaped” nucleus and small undifferentiated
acrosome (Fig. 1A). Apomorphic features of sperm of other species of Patellidae, as well as the
Nacellidae, Lottiidae and Acmaeidae would therefore include a lengthening of the nucleus (often
accompanied by an elaboration of the shape) and an increase in the size and complexity of the
acrosome (Fig. 1B-G). Sperm morphology therefore adds further support to the view that within
the Patellogastropoda, the patellids are the most primitive taxon [30],
From the few species described to date [18, 21, 28] (Table 2) the spermatozoa of the
Lottiidae and Acmaeidae appear to be very similar. It is proposed that of the two taxa, the sperm
of the Acmaeidae are more plesiomorphic, having smaller acrosomes which are slightly simpler in
morphology (e.g. undifferentiated internally). As the fossil history for Patelloida (Acmaeidae) is
greater than that of the Lottiidae [30], it might be expected therefore that the acmaeids would have
more plesiomorphic sperm.
The morphology of the spermatozoa of most species of the Patellidae is now known [1 , 18-
21, 25, 26, 34] and the data have not only provided the first insights into relationships within this
family of limpets but have also provided clues to their evolutionary radiation. Most species have
one of three unequivocal types of sperm (designated Types I, III and VI by HODGSON et al. [25];
Table 2) and it is possible that they represent three lines of patellid radiation which have occurred
in separate biogeographic areas. HODGSON et al. [25] suggest that species with type I sperm (Fig.
1 A) probably had their centre of radiation in the Indo-Pacific, those with type III sperm (Fig. 1C)
in the South-East Atlantic and finally those with type VI sperm (Fig. IE) in the North East
Atlantic and Mediterranean.
Vetigastropod relationships
The Vetigastropoda comprise the Pleurotomarioidea, Fissurelloidea, Haliotoidea,
Trochoidea, Scissurelloidea and Lepetodriloidea. The broad similarities in the structure of the
spermatozoa of the Pleurotomarioidea, Fissurelloidea, Haliotoidea, and Trochoidea add further
Source .
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
175
support to the contention [7, 10, 22, 24, 28] that these superfamilies are phylogenetically closely
related. In particular, the spermatozoa of the pleurotomarids and the those of the Trochoidea
(particularly the Trochidae) are very similar [10, 15] suggesting that these two superfamilies have
a close relationship. Thus sperm morphology supports similar conclusions based on other
morphological features [7, 8, 15].
It is suggested that within the Trochoidea, the most plesiomorphic sperm are found within
the Trochidae and Phasianellidae whereas the spermatozoa of the Turbinidae have more
apomorphic features. These features include a very wide anterior nuclear invagination and a
conical acrosome (differentiated internally), the base of which lies within the nuclear invagination,
which has lengthened, constituting >50% of the total head length. Sperm morphology therefore
does not accord with other morphological features of the Turbinidae, the turbinids having retained
many primitive pleurotomariacean features [17]. However it should be noted that HICKMAN &
McLean [17] regard the phasianellids as a subfamily of the Turbinidae. Nevertheless
spermatologically the phasianellids are more similar to the trochids than to other turbinids (Fig.
2)-
The Skeneidae are unlike other trochoideans in that the species examined to date (Z.
laseroni) [13] has dimorphic spermatozoa, and in addition the euspermatozoa are of the modified
(after FRANZEN [4]) or introsperm (after ROUSE & JAMIESON [32]) type. Such spermatozoa are
found in skeneid gastropods with internal fertilization and it is highly likely that skeneids have
internal fertilization. The fact that skeneids have introsperm makes it difficult to use sperm
morphology to assess their phylogenetic relationship to most other vetigastropods. However the
similarity in the structure of the spermatozoa of the skeneids and some caenogastropods (notably
the Cerithioidea) has led HEALY [13] to suggest that despite some morphological incongruities,
the vetigastropods are the probable ancestral source of the caenogastropods.
Representative species from two other vetigastropod superfamilies have now been found to
have “modified” spermatozoa, the Scissurellidae [14] and Lepetodrilidae [23], In both cases
modifications to sperm morphology can be linked to a modification in the biology of fertilization.
The scissurellids do not have organs to facilitate internal fertilization and it has therefore been
suggested that fertilization occurs in the mantle cavity [8J. The morphology of the sperm of both
the Scissurellidae and Lepetodrilidae are consistent with an ent-aquasperm form (after ROUSE &
JAMIESON [32]) supporting the idea of fertilization in the mantle cavity.
Studies on spermatozoon morphology of the Patellogastropoda and Vetigastropoda have
provided interesting insights into relationships within and between these taxa. However if
information from sperm morphology is to be incorporated into phylogenetic and cladistic studies,
descriptions from outstanding taxa are required. Although all patellogastropods and most
vetigastropods produce aquasperm which are essentially plesiomorphic, these sperm can yield
apomorphic characters [26] making it possible to incorporate characters from sperm into cladistic
analyses. Within the Patellogastropoda comprehensive data on the morphology of the sperm of
the Patellidae exist, but far less is known about other families. Within the vetigastropods an
examination of the sperm of hydrothermal vent and deep sea taxa, such as the Skeneidae may help
in furthering an understanding of the evolution of higher gastropods. Finally, urgent attention
needs to be given to other archaeogastropod taxa such as the Neolepetopsidae, Cocculiniformia,
Peltospiroidea, Neomphaloidea, Seguenzioidea, Cyclophoroidea and Ampullarioidea. An
examination of sperm from taxa such as these could contribute to a greater understanding of
gastropod phylogeny.
ACKNOWLEDGEMENTS
I thank Dr Vcrcna Tunnicliffe and Andrew McArthur for collecting and providing samples of Lepetodrilus
fucensis.
176
A. N. HODGSON : ARCHAEOGASTROPODA (MOLLUSCA)
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11. Healy, J. M., 1988b. — Sperm morphology and its systematic importance in the Gastropoda. Malacological
Review Supplement, 4: 251-266.
12. Healy, J. M., 1989. — Ultrastructure of spermiogenesis in the gastropod Calliotropis glyptus Watson
(Prosobranchia: Trochidae), with special reference to the embedded acrosome. Gamete Research, 24: 9-20.
13. Healy, J. M., 1990a. — Euspermatozoa and paraspermatozoa in the trochoid gastropod Zalipais laseroni
(Trochoidea: Skeneidae). Marine Biology, 105: 497-507.
14. Healy, J. M., 1990b. — Sperm structure in the scissurellid gastropod Sinezona sp. (Prosobranchia,
Pleurotomarioidea). Zoologica Scripta, 19: 189-193.
15. Healy. J. M. & HarasEWYCH, M. G., 1992. — Spermatogenesis in Perotrochus quoyanus (Fischer & Bernardi)
(Gastropoda: Pleurotomariidae). The Nautilus, 106: 1-14.
16. Hickman, C. S., 1988. — Archaeogastropod evolution, phylogeny and systematics: a re-evaluation. Malacological
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17. Hickman, C. S. & Mclean, J. H., 1990. — Systematic revision and suprageneric classification of Trochacean
gastropods. Science Series, Natural History Museum of Los Angeles County, 35: 1-169.
18. HODGSON, A. N., Unpublished data.
19. Hodgson, A.N. & Bernard, R. T. F., 1988. — A comparison of the structure of the spermatozoa and
spermatogenesis of 16 species of patellid limpet (Mollusca: Gastropoda: Archaeogastropoda). Journal of
Morphology , 195: 205-223.
20. Hodgson, A. N. & Bernard, R. T. F., 1989. — Spermatozoon structure and the taxonomic affinity of Nacella
delesserti (Gastropoda: Patellidae). Journal of Molluscan Studies, 55: 145-147.
21. Hodgson, A. N. & Chia, F.-S., 1993. — Spermatozoon structure of some North American prosobranchs from the
families Lottiidae (Patellogastropoda) and Fissurellidae (Archaeogastropoda). Marine Biology, 116: 97-101.
22. Hodgson, A. N. & Foster, G. G., 1992. — Structure of the sperm of some South African archaeogastropods
(Mollusca) from the superfamilies Haliotoidea, Fissurelloidea and Trochoidea. Marine Biology, 113: 89-97.
23. Hodgson, A. N„ Healy, J. M. & Tunnicliffe, V., 1995. — Spermatogenesis and sperm ultrastructure of the
hydrothermal vent prosobranch gastropod Hepetodrilus fucensis (Lepetodrilidae, Mollusca). Invertebrate
Reproduction and Development, in press.
24. Hodgson, A. N., Heller, J. & Bernard, R. T. F., 1990. — Ultrastructure of the sperm and spermatogenesis in five
South Africa species of the trochid genus Oxy stele (Mollusca, Prosobranchia). Molecular Reproduction and
Development, 25: 263-271.
25. Hodgson, A. N., Ridgway, S., Branch, G. M. & Hawkins, S. J., submitted. — Spermatozoon morphology of 19
species of prosobranch limpet (Patellogastropoda : Patellidae) with a discussion of patellid relationships.
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
177
26. Jamieson, B. G. M., Hodgson, A. N. & Bernard, R. T. F.. 1991. — Phylogenetic trends and variation in the
ultrastructure of the spermatozoa of sympatric species of South African limpets (Archaeogastropoda; Mollusca).
Invertebrate Reproduction and Development, 20: 137-146.
27. KOHNERT, R. & Storch, V., 1983. — Ultrastrukturelle Untersuchungen zur Morphologie und Genese der Spermien
van Archaeogastropoda. Helgoldnder Meeresuntersuchungen , 36: 77-84.
28. Koike, K., 1985. — Comparative ultrastructural studies on the spermatozoa of the Prosobranchia (Mollusca:
Gastropoda). Science Report of the Faculty' Education Guntna University , 34: 33-153.
29. Lewis, C.A., Leighton, D. L. & Vacquier, V. D., 1980. — Morphology of abalone spermatozoa before and after the
acrosome reaction. Journal of Ultrastructure Research , 20: 462-480.
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Review Supplement , 4: 85-87.
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Prosobranchia). Invertebrate Reproduction and Development, 26: 119-126.
Source : MNHN . Paris
Source : MNHN, Paris
Comparative Silver Staining of
Molluscan Spermatozoa
Mario SOUSA, Elsa OLIVEIRA, Jodo CARVALHEIRO
& Victor Oliveira
Laboratory of Cell Biology,
Institute of Biomedical Sciences, University of Oporto, Lg. Prof. Abel Salazar 2, 4050 Porto, Portugal
ABSTRACT
Mollusc spermatozoa were studied by transmission electron microscopy with the application of the silver nitrate
method. Silver stained some components of the acrosomal vesicle and of the cytoskeletal elements of these cells in a
pattern specific for each species. The importance of this staining method in relation to phylogenetic relationships is
presented and discussed.
RESUME
Coloration comparee a 1’argent des spermatozoides de Mollusques
Des spermatozoides de Mollusques ont 6l6 etudies en microscopic eleclronique & transmission grace h la m^thode au
nitrate d’argent. Dans chaque esp&ce, V argent colore certains composants de la vesicule acrosomienne et les elements
cytoplasmiques de ces cellules d’une manicrc spdcifique. L’ importance de cette m6thode de coloration pour les relations
phylogeniques est presentee et discut6e.
The ultrastructural characteristics of mature spermatozoa have been used to examine
phylogenetic relationships and have also been related to the different aspects of reproductive
biology of the organism [18, 38, 39, 50, 51, 54], In this respect, a great deal of work has been
done on the ultrastructure of spermatozoa in the Patellogastropoda and Archaeogastropoda
(Prosobranchia) as well as in the Heterodonta and Pteriomorphia (Bivalvia) [1-3, 6-37, 39-53,
55, 56, 59, 61-66],
In recent years, we have attempted to show that the silver staining method can be a useful
and easy way to better understand specific aspects of spermatogenesis [57, 59, 60], as well as for
comparative phylogenetical studies [58, 61-64], In the present study, we present and critically
review the silver staining characteristics of mature molluscan spermatozoa, with emphasis on its
importance as a tool for studying phylogenetic relationships. We also provide the detailed
technique so as to enable other laboratories to reproduce this staining method.
Sousa, M., Oliveira, E., Carvalheiro, J., & Oliveira, V„ 1995. — Comparative silver staining of molluscan
spermatozoa. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phytogeny and
Taxonomy. Mem. Mus. nain. Hist, ncit., 166 : 179-187. Paris ISBN : 2-85653-225-X.
180
M. SOUSA ETAL : SILVER STAINING (MOLLUSCA)
MATERIALS AND METHODS
Specimens (Table 1) were collected from sands at the Atlantic coast of Portugal and Spain. Small pieces of testis (<
1 mm) were fixed with 2.5% glutaraldehyde buffered w'ith 0.2 M Na Cacodylate, pH 7.4, for 2 h at 4°C, rinsed in the same
buffer for 2 h at 4°C, postfixed in Carnoy (acetic acid: methanol, 1:3) for 5 min at room temperature, gradually rehydrated
in ethanol (10 min in each step), and rinsed in distilled water for 30 min. They were then immersed in 50% aqueous silver
nitrate (filtered by 0.2 pm) and placed in a water-bath under a Philips lamp (500 W) in such a way as to reach 55°C for 10
min. Finally, the specimens were rinsed in distilled w-ater (3x2 min), incubated in an ammoniacal silver-formalin
solution (2 g silver nitrate in 2.5 ml distilled water + 2.5 ml 33% ammonia solution + 5 ml 3% aqueous formalin, 0.2 pm
filtered) for 3 min, rinsed in distilled water (3 x 2 min), dehydrated in an ethanol graded series, embedded in Epon, and
examined by transmission electron microscopy.
Table 1. — Silver staining of mature spermatozoa in the Mollusca.
AV, acrosomal vesicle; SAM, subacrosomal material; CF/MF. centriolar fossa/mitochondrial fossa; PCS, material that
links the proximal centriole to the nuclear base; Ce, centrioles; PCC/An, pcricentriolar complex/annulus.
Figs 1-13. Prosobranchia. a, acrosomal vesicle; b, subacrosomal material; n, nucleus; m, mitochondria; c, centrioles; f,
flagellum; g, glycogen sheath. 1, 2: Helcion pellucidus. Silver stains the centriolar fossa (arrow), and lightly
stains the centrioles (c) and the pcricentriolar complex (arrowhead), x 29 000. 3, 4: Patella rustica . Silver stains
the centriolar fossa (arrow) and the pericentriolar complex (arrowhead), and lightly stains the centrioles (c).
x 24 600. 5: Gibbula umbilicalis. Silver stains the acrosomal vesicle (a), the centriolar fossa (arrow), the
mitochondrial fossa (double arrow) and the pericentriolar complex (arrowhead), and lightly stains the centrioles
(c). x 17 900. 6: Gibbula cineraria. Silver lightly stains the centriolar fossa (arrow), the mitochondrial fossa
(double arrow), the centrioles (c), and the pericentriolar complex (arrowhead), x 17 900. 7-9: Littorina saxatilis.
Source MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHY LOGENY AND TAXONOMY
181
Silver stains the acrosomal vesicle (a). Large arrow, mitochondrial fossa; small arrow, annulus, x 32 000;
x 18 600; x 18 600. 10: Littorina littorea. Silver stains the acrosomal vesicle (a) and the centriolc (c). Results
for the mitochondrial fossa and the annulus are as for L. saxatilis. SC, sertoli cell. x 41 000 11-13: Nucella
lap ill us. Silver stains the acrosomal vesicle (a), the subacrosomal material (b). the mitochondrial fossa (large
arrow), and the annulus (small arrow). x 51 600; x 30 000; x 33 000.
Source :
182
M. SOUSA ETAL. : SILVER STAINING (MOLLUSCA)
RESULTS AND DISCUSSION
Silver staining has been used to demonstrate the argyrophilia of acidic phosphoproteins
contained in nucleolar components and their metaphase counterparts [60]. However, silver
staining has also been shown to stain, in invertebrate species, other nuclear and cytoplasmic
structures of germ cells and thereafter used to show different staining characteristics in different
sperm species [57-64],
In the present work, we update all results obtained in the Mollusca with this silver staining
technique, and discuss its relative importance as a phylogenetical sperm marker (Table 1). In the
Patellogastropoda, Helcion pellucidus and Patella rustica, which are morphologically different,
exhibit a similar pattern of silver staining (Figs 1-4); in the Archaeogastropoda, Gibbula
umbilicalis and G. cineraria differ in nuclear and acrosomal vesicle dimensions as also in their
silver staining characteristics (Figs 5, 6); in the Caenogastropoda, despite the morphological
similarity between Littorina saxatilis, L. littorea and Nucella lapillus, their silver staining
characteristics are very dissimilar (Figs 7-13). In the Veneroida, Donax trunculus, Spisula
solidissima, Cerastoderma edulis and Scrobicularia plana are all very different in their
morphological aspects, and also present very distinctive silver staining characteristics (Figs 14-
22). In the Mytiloidea (Pteriomorphia), only Mytilus edulis has been studied by silver staining
(Figs 23, 24); but in the Ostreoidea, despite the morphological similarity between Ostrea edulis
and Crassostrea angulata, both species can be easily and completely differentiated by silver
staining (Figs 25-27).
In conclusion, the silver staining of mature spermatozoa can enable us to distinguish
different sperm species which present identical ultrastructural characteristics, and it can also help
in phylogenetical studies since spermatozoa of the same group frequently share several silver
staining characteristics (Table 1). However, the validation of this cytochemical marker as a useful
tool in phylogenetical studies awaits further studies on many other molluscan species.
ACKNOWLEDGEMENTS
This work was supported by JNICT, the Arts and Sciences Institute, and the Eng. Antonio de Almeida Foundation.
We would like to thank Dr. John Healy for critically reviewing the manuscript.
Figs 14-22. — Veneroidea. a, acrosomal vesicle; b, subacrosomal material; n, nucleus; m, mitochondria; c, centrioles; f,
flagellum. 14-16: Donax trunculus. Silver stains the acrosomal vesicle (a), the centriolar fossa (arrow), the
material that links the proximal centriole to the centriolar fossa (double arrow), the centrioles (c), and the
pericentriolar complex (arrowhead), x 22 500; x 40 000; x 40 000. 17-19: Spisula solidissima. Silver stains
the dense component of the acrosomal vesicle (a), the insertion of the tip of the perforatorium into the acrosomal
vesicle (b), the centriolar fossa (arrow) and the pericentriolar complex (arrowhead), and lightly stains the
centrioles (c). x 25 700; x 36 200; x 27 600. 20: Cerastoderma edule. Silver stains the acrosomal vesicle (a),
the nuclear base (arrow), and the material that links the proximal centriole to the nuclear base (double arrows).
Arrowhead, pericentriolar complex, x 20 000; inset A, x 46 000; inset B, x 80 000. 21, 22: Scrobicularia
plana. Silver stains the nuclear base (arrow) and the pericentriolar complex (arrowhead), x 16 900; x 47 400.
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
183
Source : MNHN, Paris
184
M. SOUSA ETAL. : SILVER STAINING (MOLLUSCA)
Figs 23-27. — Pteriomorphia. a, acrosomal vesicle; b, subacrosomal material; n, nucleus; m, mitochondria; c, centrioles;
large arrow, ccntriolar fossa; double arrows, material that links the proximal centriole to the centriolar fossa;
arrowhead, pericentriolar complex. 23, 24 : Mytilus edulis. Silver stains the acrosomal vesicle, x 17 600;
x 40 700. 25: Osirea edulis. Silver stains the tip of the acrosomal vesicle (a), the centriolar fossa (large arrow),
and the material that links the proximal centriole to the centriolar fossa (double arrows), x 28 900.
26, 27: Crassostrea angulata. Silver stains the tip of the acrosomal vesicle (a), the subacrosomal material (b),
the centriolar fossa (large arrow), the material that links the proximal centriole to the ccntriolar fossa (double
arrows), the centrioles (c), and the pericentriolar complex (arrowhead), x 30 900; x 39 900.
Source MNHN, Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
185
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12: 1-20.
52. Popham, J. D., Dickson, M. R. & Goddard, C. K., 1974. — Ultrastructural study of the mature gametes of two
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sperm types in relation to reproductive biology. Journal of Submicroscopic Cytology , 19: 573-584.
55. Selmi, M. G. & Giusti, F., 1983. — The atypical spermatozoon of Theodoxus fluviatilis (L.) (Gastropoda,
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56. Smaldon, P. R. & Duffus, J. H., 1985. — An ultrastructural study of gametes and fertilization in Patella vulgata L.
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57. Sousa, M. & Azevedo, C., 1988. — Ultrastructure and silver staining analysis of spermatogenesis in the sea urchin
Paracentrotus lividus (Echinodermata, Echinoidea). Journal of Morphology, 195: 177-188.
58. Sousa, M. & Azevedo, C., 1988. — Comparative silver staining analysis on spermatozoa of various invertebrate
species. International Journal of Invertebrate Reproduction and Development, 13: 1-8.
59. SOUSA, M. & Azevedo, C., 1989. — Silver staining of spermatogenesis in the starfish Marthasterias glacialis.
Invertebrate Reproduction and Development, 15: 105-108.
60. Sousa, M. & Azevedo, C., 1990. — Ultrastructure and silver staining of echinoderm spermatogenesis. In: B. Dale,
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115-129.
61. Sousa, M. & Oliveira, E., 1994. — Ultrastructural and cytochemical study of spermatogenesis in Donax trunculus
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62. SOUSA, M. & Oliveira, E., 1994. — An ultrastructural study of spermatogenesis in Crassostrea angulata (Mollusca,
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Source : MNHN, Paris
Source : MNHN, Paris
The Use of Spermatozoal Ultrastructure
in Phylogenetic Studies of Tubificidae (Oligochaeta)
Chris ter ERSEUS * & Marco FERRAGUTI **
* Department of Invertebrate Zoology,
Swedish Museum of Natural History, Box 50007, S-104 05 Stockholm, Sweden
** Department of Biology,
Universita degli Studi di Milano, 26, Via Celoria, 1-20133 Milano, Italy
ABSTRACT
The influence of patterns of spermatozoal ultrastructure on hypotheses of phylogenetic relationships within the
Tubificidae is examined on the basis of knowledge for species representing 15 different genera. A parsimony analysis of a
combination of spermatozoal and conventional morphological characters supports that the Phallodrilinae,
Limnodriloidinae and Tubificinae are monophyletic taxa, and that the Rhyacodrilinac is a paraphyletic group as currently
defined. The mature spermatozoa of Heterodrilus spp., Pectinodrilus molestus , Coralliodrilus rugosus , Smithsonidrilus
hummelincki and Tubificoides amplivasatus are described for the first time.
RESUME
L’utilisation de Uultrastructure des spermatozoi'des pour les etudes phylogeniques sur les
Tubificidae (Oligochaeta)
L' influence des differentes organisations ultrastructurales des spermatozoides sur les hypotheses concernant les
relations phyletiques a l’interieur des Tubificidae cst examinee, a partir de nos connaissances sur des especes representant
quinze genres differents. Une analyse de parcimonie portant sur une combinaison de caractercs des spermatozoides et de la
morphologic conventionnelle indique que les Phallodrilinae, Limnodriloidinae et Tubificinae sont des taxons
monophyletiques, et que les Rhyacodrilinac dans leur definition actuelle sont un groupe paraphyletique. Les
spermatozoides murs de Heterodrilus spp., Pectinodrilus molestus , Coralliodrilus rugosus, Smithsonidrilus hummelincki
and Tubificoides amplivasatus sont decrits pour la premiere fois.
The ultrastructure of spermatozoa has proved useful for phylogenetic assessment of higher
taxa within the Clitellata (=Euclitellata sensu Jamieson [33]), particularly with regard to family
level relationships in oligochaetes [34-35, 37, 40]. Spermatozoa are fairly uniform and distinctive
within several clitellate groups [41], and their more general appearance supports a close
relationship between clitellates and onychophorans [36, 37]. On the other hand, their
ultrastructure may sometimes be used to distinguish species within the same genus [27, 44],
Evidence for great variation in the sperm ultrastructure of Tubificidae, a speciose group of aquatic
oligochaetes, has been accumulated in recent years [24].
Ers£us, C., & FERRAGUTI, M., 1995. — The use of spermatozoal ultrastructure in phylogenetic studies of
Tubificidae (Oligochaeta). In: Jamieson, B. G. M., Ausio. J.. & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny
and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 189-201. Paris ISBN : 2-85653-225-X.
190
C. ERSEUS & M. FERRAGUTI : TUBIFIC1DAE, OLIGOCHAETA ( ANNELIDA )
The spermatozoa of tubificids, as well as those of all other clitellates, are characterized by a
sequence of acrosome, nucleus, middle piece and tail (Fig. 2e). The acrosome contains an
acrosome tube involving the other acrosome structures. The middle piece contains only the
mitochondria. In the basal body region of the tail there is a prominent basal cylinder from which
the two central tubules of the axoneme start. The axoneme shows a 9+2 arrangement and is
characterized by the presence of some sort of accessory structure of the central apparatus [21],
and a peripheral ring of glycogen granules. In members of the subfamily Tubificinae a double
sperm line produces euspermatozoa and paraspermatozoa. (These different sperm categories have
been called “typical” and “atypical”, respectively, by one of us [2, 23], but here we adopt the
terminology of Healy & JAMIESON [30].)
Conventional morphological characters of tubificids, as well as of other aquatic
oligochaetes, are few and many similarities are due to homoplasy, i.e., convergence or reversal
[3, 13, 15]. This means that the support for monophyly of some groups, e.g. some tubificid
subfamilies, is weak. Additional data, structural as well as molecular, are needed for a better
understanding of the phylogenetic relationships within the Tubificidae.
In the present paper, parsimony analyses of both spermatozoal and conventional characters
of 15 tubificid genera (Table 1), representing four subfamilies, are presented. The two sets of
characters were run separately and in combination, to examine different impacts on the
phylogenetic hypotheses. The majority of the spermatozoal data are from the literature, but for six
marine species (representing the genera Heterodrilus, Pectinodrilus, Coralliodrilus,
Smithsonidrilus, Tubificoides) the spermatozoa are described for the first time. When
euspermatozoa as well as paraspermatozoa are present, both types are described, but in the
parsimony analyses only euspermatozoa are considered.
MATERIAL AND METHODS
The material of Tubificoides amplivasatus was collected in muddy sediments in the Oresund, Denmark, in the
summer of 1982. Specimens of Heterodrilus pentcheffi and H. minisetosus , and a single individual of Pectinodrilus
molestus were found in subtidal sand at Long Key, Florida Keys, Florida, in October 1992. Additional material of P.
molestus , and specimens of Coralliodrilus rugosus and Smithsonidrilus hummelincki were collected at subtidal and
intertidal sites near Carrie Bow Cay, on the barrier reef off Belize in Central America, in March 1993. The material of T.
amplivasatus was fixed in 3 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The other material was fixed in a picric
acid-glutaraldehyde-paraformaldehyde mixture following Ermak & Eakin [5). After washing in the buffer and postfixation
in similarly buffered 1% osmium tetroxide, the worms were dehydrated in a graded ethanol series and embedded in Spurr's
resin. Sections were cut with LKB 111 and V, and observed under a JEOL 100XS electron microscope.
Fitch parsimony [25] analyses (i.e. multistate characters unordered) of tubificid taxa were performed using PAUP
for Macintosh [43]. The branch-and-bound option (addition sequence: furthest) was selected. For analyses resulting in
more than one equally parsimonious tree, strict consensus trees were calculated.
RESULTS
Description of new sperm types
Spermatozoa of Heterodrilus minisetosus and H. pentcheffi (Rhyacodrilinae) (Figs lb, g, I,
2c). Spermatozoa were examined in the spermathecae. The two species have similar spermatozoa
and will thus be described together. Only euspermatozoa are present, and spermatozeugmata are
not formed. In both species, the acrosome is straight, about 0.6 pm long, 0.1 pm wide, with the
vesicle, acrosome rod and secondary tube all withdrawn. The nucleus is apically straight, basally
loosely twisted, and is followed by five twisted mitochondria, 1.4 pm long, 0.4 pm wide. The
tail has two tetragon fibres, one much longer than the other.
Spermatozoa o/Pectinodrilus molestus (Phallodrilinae) (Figs lc,f 2e). Spermatozoa were
examined in the spermathecae. Only euspermatozoa are present, and spermatozeugmata are not
formed. The acrosome is straight, 0.5 pm long, 0.1 pm wide. A secondary tube (if present at all)
Source :
ADVANCES IN SPERMATOZOAL PHYLOCENY AND TAXONOMY
191
is not visible. The nucleus is apically corkscrew-shaped, then gradually becomes twisted and is
basally almost straight. Five twisted mitochondria follow, 2.5 |im long, 0.36 pm wide. The tail
has tetragon fibres.
Spermatozoa of Coralliodrilus rugosus (Phallodrilinae) (Figs la, k, o, 2d). Spermatozoa
were examined in the spermathecae. Only euspermatozoa are present and spermatozeugmata are
not formed. The acrosome tube is corkscrew-shaped with an helical ridge making 1.5 gyres. The
acrosome rod is indistinct. A secondary tube is not present. The nucleus is apically corkscrew¬
shaped and follows the pitch of the acrosome tube, then becomes twisted with a pitch increasing
towards the base. The five mitochondria (1.2 pm long, 0.3 pm wide) are always surrounded by
residual cytoplasm. The tail has tetragon fibres.
Spermatozoa o/Smithsonidrilus hummelincki (Limnodriloidinae) (Figs ld-e, h, j, n, 2a-b).
Spermatozoa were examined at the ciliated male funnels as well as in the spermathecae.
Euspermatozoa and paraspermatozoa are constantly present and easily distinguished. At the
funnels the two types are randomly mixed, whereas in the spermathecae they are grouped in
different spermatozeugmata, each containing only one sperm type. Euspermatozoa have an
acrosome, 0.7 pm long, 0.15 pm wide, formed by a thin-walled, straight acrosome tube with a
limen, a distinct secondary tube, a short rod and a partly withdrawn acrosome vesicle. The
nucleus is apically twisted and basally straight. Four to five subspherical mitochondria form the
middle piece. The tail shows a prominent central sheath. The paraspermatozoa have a shorter
(0.36 pm) acrosome with the vesicle completely external to the tube, but with no other structure; a
thin and irregularly outlined nucleus; two to four mitochondria characteristically swollen when the
sperm are in the spermatheca, but not when at the funnels; a tail with a swollen plasma membrane
when the sperm are at the funnels, but with apparently degenerating axonemes when in the
spermatheca. The paraspermatozoa are fewer than the euspermatozoa.
Spermatozoa o/Tubificoides amplivasatus (Tubificinae) (Fig. li, m). Spermatozoa at the
funnels as well as in the spermathecae were examined. Both euspermatozoa and paraspermatozoa
are constantly present. Spermatozeugmata are found in the spermathecae. They are composed of
the two sperm types grouped together in the typical tubificine way [22], We have no data on the
acrosome of the euspermatozoa. The nucleus is apically twisted and basally straight. Three small
ovoidal mitochondria separate the nucleus from the tail which has, at least in part, a prominent
central sheath. The paraspermatozoa show a straight, empty acrosome tube, 0.5 pm long, 0. 1 pm
wide, with a small acrosome vesicle completely external to it. The nucleus is rectilinear, 2-3 pm
long (which is shorter than that of the euspermatozoa). It has the shape of an elongated cone and
is partly uncondensed. The two mitochondria are longer and larger than those of the
euspermatozoa. The tail has a plasma membrane widely separated from the axoneme.
Parsimony Analyses
Taxa. Sperm ultrastructure has been reasonably well studied for 17 species of Tubificidae,
representing 15 genera (Table 1). These 15 “genera”, as characterized by their representatives
studied, are regarded as the ingroup taxa. The outgroup is an hypothetical ancestor, the
spermatozoal characters of which are in accordance with the plesiomorphic model suggested by
JAMIESON et al. [40], For the morphological characters, the ancestor is coded as a member the
Phreodrilidae, a putative sister group of the Tubificidae [15].
Characters and character states. Two sets of characters are used, referred to as the
“spermatozoal” and “morphological characters”, respectively. All multistate characters are treated
as unordered. The character states of all taxa are coded in Table 1. The character states are
identical for Heterodrilus pencheffi and H. minisetosus, and for Inanidrilus leukodermatus and
I. bulbosus.
192
C. ERSEUS & M. FERRAGUTI : TUBIF1CIDAE, OLIGOCHAETA (ANNELIDA)
Table 1. — List of ingroup taxa included in the parsimony analyses. The genera are grouped according to their current
subfamilial positions. 1 Rhyacodrilus arthingtonae is an enigmatic species, possibly not a natural member ol
Rhyacodrilus , but it does not belong to Rhizodrilus [19] as was suggested belore [1, 24].
Fig. 1. — Character variation in tubificid spermatozoa (for interpretation of ultrastructural details, see Fig. 2).
a: Coralliodrilus rugosus , longitudinal section of acrosome, bar 0.15 pm. b: Heterodrilus minisetosus ,
longitudinal section of acrosome, bar 0.15 pm. c: Pectinodrilus molestus , longitudinal section of acrosome, bar
0.15 pm. d-e: Smilhsonidrilus hummelincki , longitudinal section of eusperm acrosome (d), and of nuclear tip and
acrosome of paraspermatozoon (e), both bars 0.15 pm. f : Pectinodrilus molestus, corkscrew-shaped portion of
nucleus, bar 0.2 pm. g: Heterodrilus minisetosus , apical, straight portion of nucleus, bar 0.2 pm.
h: Smilhsonidrilus hummelincki, twisted portion of eusperm nucleus, bar 0.3 pm. i: Tubificoides amplivasatus ,
twisted portion of two eusperm nuclei, bar 0.5 pm. j: Smilhsonidrilus hummelincki , euspermatozoon, nuclear
base (top), round mitochondria (centre), and basal body area with evident basal cylinder (bottom), bar 0.2 pm.
k: Coralliodrilus rugosus, cross section of midpiece with five mitochondria (some residual cytoplasm present in
the mature spermatozoon), bar 0.2 pm. 1: Heterodrilus minisetosus, longitudinal section of midpiece, bar 0.2 pm.
m: Tubificoides amplivasatus, cross section of midpiece with three mitochondria, bar 0.2 pm.
n: Smilhsonidrilus hummelincki euspermatozoon, cross section of tails at different levels, showing central
apparatus with tetragon fibres (top), and prominent central sheath (bottom), bar 0.2 pm. o: Coralliodrilus
rugosus, cross section of basal body area with basal cylinder, bar 0.2 pm.
Abbreviations in Figs 1 and 2: AR. acrosome rod, AT, acrosome tube; AV, acrosome vesicle; BC, basal cylinder; M,
mitochondria in midpiece; N, nucleus; T. tail.
Source MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
193
Source : MNHN . Pans
194
C. ERSEUS & M. FERRAGUTI : TUBIFICIDAE, OLICOCHAETA ( ANNELIDA )
Spermatozoal characters
1. Spermatozeugmata : absent (0); present with only one type of spermatozoa (1); of
two types present, one type formed by euspermatozoa, the other by paraspermatozoa (2); of one
type formed by both euspermatozoa and paraspermatozoa (3).
2. Acrosome shape: straight (0); twisted or corkscrew-shaped (1).
3. Acrosome slenderness: length/width ratio less than 8 (0); greater than 8(1). The
slenderness and absolute length of the acrosome vary among species; by presenting the states as
ratios the influence of absolute length is eliminated.
4. Acrosome vesicle withdrawal: ratio acrosome vesicle length/length of portion
withdrawn into acrosome tube considerably greater than 2 (0); less than 2 (1). We accept the
assumption [40] that an acrosome vesicle external to the acrosome tube is plesiomorphic. The
ratio eliminates the influence of absolute length.
5. Acrosome tube: thick-walled throughout (0); at least in part thin-walled (1).
6. Secondary acrosome tube: present (0); much reduced or absent (1).
7. Acrosome rod (perforatorium): protuberant (anterior tip outside acrosome tube) (0);
not protuberant (wholly located inside acrosome tube) (1).
8. Shape of nucleus, anterior portion: straight (0); twisted (1); corkscrew-shaped or
flanged (2). Terminology according to FERRAGUTI et al. [24].
9. Shape of nucleus, posterior portion: straight (0); twisted (1). Nuclear shape is
described as two characters, since different combinations of anterior and posterior shapes are
present among tubificid spermatozoa.
10. Number of mitochondria: four or five (0); less than four (1). Four or five is a
common number of mitochondria in “primitive spermatozoa” [26, 40].
11. Mitochondrial shape: straight and roundish-to-oval, length/width ratio not greater
than about 1.5 (0); spiral and elongate, length/width ratio considerably greater than 1 .5 (1).
12. Central axonemal apparatus: with prominent central sheath throughout flagellum
(0); with tetragon fibres throughout flagellum (1); with prominent central sheath in anterior
portion of flagellum, but tetragon fibres in posterior portion of flagellum (2). See [21].
Morphological characters.
13. Hair setae: present (0); absent (1). With reference to the condition in the outgroup
Phreodrilidae, possession of hair setae has been interpreted as a plesiomorphic trait in the
Tubificidae [15].
14. Penial setae: absent (0); present (1).
15. Coelomocytes: absent or few (0); numerous (1).
16. Alimentary > system: present, body wall without symbiotic bacteria (0); absent,
body wall with symbiotic bacteria (1).
17. Oesophagus (in segment IX): unmodified (0); modified, either bearing a pair of
diverticula, or dilated with reticulate blood plexus (1).
18. Prostate glands: diffuse (0); solid, pedunculate, one per atrium (1); lobed, broadly
attached, one per atrium (2); solid, generally pedunculate, two per atrium (3); absent (4).
19. Ciliation of atrial epithelium: weak (cilia restricted to particular, ciliated cells) or
absent (0); dense (atria heavily ciliated throughout) (1). In Smithsonidrilus, the atrial ampullae are
ciliated, whereas the atrial ducts lack cilia; thus this character is coded as (0) for Smithsonidrilus.
20. Penes: poorly developed or absent (0); well developed, pendent within deep
penial sacs (1).
Results of parsimony analyses
Analysis using only morphological characters (Fig. 3a). This analysis resulted in a single,
most parsimonious, tree with 14 steps and a consistency index (Cl) of 0.786. It suggests that
Rhyacodrilinae, Phallodrilinae and Limnodriloidinae are monophyletic taxa, but Clitellio appears
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
195
Fig. 2. — Schematic drawings of some tubificid sperm models, a-b: Smithsonidrilus hummelincki , paraspermatozoon (a)
and euspermatozoon (b). c: Heterodrilus pentcheffi , euspermatozoon. d: Coralliodrilus rugosus ,
euspermatozoon. e: Pectinodrilus molestus , euspermatozoon. The plasma membrane has been omitted from the
acrosome, mitochondria and tail of the Pectinodrilus spermatozoon (e), to show the internal structures. For
abbreviations, see Fig. 1.
Source : MNHN. Paris
196
C. ERSEUS & M. FERRAGUTI : TUBIFIC1DAE, OLIGOCHAETA ( ANNELIDA )
as the sister taxon of the Phallodrilinae rather than a member of the Tubificinae. The position of
Clitellio is determined by its lack of prostate glands (state 4 of character 18), which is shared by
Coralliodrilus, and the dense ciliation of its atria (character 19). For Clitellio, the coding of the
latter character is based on a recent light microscopical study of C. cirenarius [29]. Most other
tubificines (including Tubifex and Tubificoides ) are still assumed to have few cilia (if any at all) in
their atria [15].
a
b
t
Rhyacodrilus (R)
Rhizodrilus (R)
Heterodrilus (R)
Monopylephorus (R)
Bathydrilus (P)
Olavius (P)
Inanidrilus (P)
Thalassodrilus (P)
Pectinodrilus (P)
Coralliodrilus (P)
Clitellio (T)
0-2 20
^ - 1 -
17 18
0-1
—I -
18 20
— Smithsonidrilus (L)
— Thalassodrilides (L)
— Tubifex (T)
— Tubificoides (T)
Fig. 3. — Results of parsimony analysis, a: The (one) most parsimonious tree for 15 taxa of Tubificidae, based on eight
morphological characters (13-20). Length 14 steps; consistency index 0.786; retention index 0.893. Rooting at
an hypothetical ancestor (see text). Subfamilial positions of taxa indicated in parentheses (L, Limnodriloidinae; P,
Phallodrilinae; R, Rhyacodrilinae; T, Tubificinae). Numbers above symbols for multistate character refer to
transformations of character states (e.g., 0-1 means “going from state 0 to state 1”). Filled rectangle , (unique)
autapomorphy; open rectangle , autapomorphy that is followed by reversal further up the tree; two parallel lines ,
convergence; cross , reversal, b: Strict consensus tree of 45 equally parsimonious trees for 15 taxa of Tubificidae,
based on spermatozoal characters (1-12).
Analysis using only spermatozoa l characters (Fig. 3b). These characters yielded 45 equally
parsimonious trees, all with 32 steps and a Cl of 0.500. The congruence between these trees was
very low, i.e, the spermatozoal characters proved more homoplasic than the morphological
Source
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
197
characters. The only unequivocal suggestions from the sperm data are (1) that Rhyacodrilus has
the most plesiomorphic spermatozoa known among the Tubificidae, and (2) that Thalassodrilus
and Coralliodrilus are very closely related.
Analysis using both sets of characters (Fig. 4). When the spermatozoal and morphological
characters were combined, the stability in tree topology increased considerably, although the Cl
(0.519) was barely greater than that of the trees based on only spermatozoal characters. The
combined set gave three equally parsimonious trees, all with 52 steps, of which 36 emanate from
the spermatozoal, 16 from the morphological characters. The trees differ only in the branching
pattern of some phallodrilines, otherwise they all support (1) monophyly of each of
Phallodrilinae, Limnodriloidinae and Tubificinae, (2) that the two latter are sister taxa, and (3) that
the Phallodrilinae are cladistic members of the (otherwise paraphyletic) Rhyacodrilinae.
Rhyacodrilus (R)
Rhizodrilus (R)
Monopylephorus (R)
Heterodrilus (R)
Bathydrilus (P)
Olavius (P)
Inanidrilus (P)
Thalassodrilus (P)
Coralliodrilus (P)
Pectinodrilus (P)
Smithsonidrilus (L)
Thalassodrilides (L)
Tubifex (T)
Tubificoides (T)
Clitellio (T)
Fig. 4. — One of three equally parsimonious trees for 15 taxa of Tubificidae, based on twelve spermatozoal (1-12) and
eight morphological characters (13-20). Length 52 steps; consistency index 0.519; retention index 0.638.
Rooting at an hypothetical ancestor (see text). Branches shared by all three trees are indicated by bold lines; with
another optimization of character 11. the group consisting of Thalassodrilus + Coralliodrilus and Pectinodrilus
collapses. For other explanations, see Fig. 3.
198
C. ERSEUS & M. FERRAGUTI : TUB1F1C1DAE, OLIGOCHAETA (ANNELIDA)
DISCUSSION
The results of the parsimony analyses
The parsimony analyses revealed features of the character sets that were somewhat
unexpected. The larger (spermatozoal) set could have been expected to yield the more stable trees,
simply because on average there could be more character states to support each node in the trees.
However, the poor congruence within the spermatozoal set made the morphological characters
superior in terms of stabilizing tree topology.
With the two sets of characters combined, there is a trade-off between the congruence of the
morphological data and the homoplasy of the spermatozoal data. Some traits of the tree based on
morphology (Fig. 3a) are retained in the three trees based on all characters (Fig. 4), but there are
also some changes. Clitellio now returns to the “tubificine” clade where it is normally classified,
as the spermatozoal apomorphies (characters 1, 4, 8, 12) shared by Clitellio and
Tubifex/Tuhificoides (and partly by the limnodriloidines) outnumber the morphological ones
shared by Clitellio and the phallodriline genus Coralliodrilus (characters 18, 19). The trees of the
combined data set neither contradict the view that the Phallodrilinae, Limnodriloidinae and
Tubificinae are monophyletic, nor that the Rhyacodrilinae are paraphyletic [15], although it should
be noted that only a few selected genera of each subfamily are included in this analysis.
Two different hypotheses of subfamilial relationships within the Tubificidae, both based on
conventional, morphological, characters, have recently been published. ERSEUS [15] regarded the
Phallodrilinae as an advanced subgroup within the “rhyacodriline” clade (a clade which also
appears to contain the family Naididae, and possibly also the Opistocystidae), and this clade as a
sister group of the rest of the family (Limnodriloidinae, Tubificinae and Telmatodrilinae). None of
this is contradicted by the present study using the combined character sets; although the
Telmatodrilinae are excluded from the present study. In the hypothesis presented by BRINKHURST
[3], the Phallodrilinae is separated from the rhyacodriline clade and instead proposed to be the
sister group of Limnodriloidinae-Tubificinae-Telmatodrilinae. This difference emanates from the
fact that BRINKHURST's analysis included most aquatic oligochaete families in the ingroup,
whereas the study by ERSEUS focused on the Tubificidae and Naididae, using Phreodrilidae as
the outgroup. BRINKHURST'S study implies that presence of hair setae is apomorphic when
occurring in tubificid taxa, whereas this state turns out as a plesiomorphy in the present study as
well as in that by ERSEUS [15].
The relevance of sperm ultrastructure in studies of tubificid phytogeny
Both this and earlier studies [22, 24] show that tubificid sperm exhibit great variation in the
ultrastructural details, sperm type differentiation, and formation of spermatozeugmata.
Interestingly, the spermatozoa of species belonging to the same genus (although so far only
investigated for Inanidrilus and Heterodrilus) are virtually identical, which indicates that
spermatozoal characters are useful for the recognition of at least some genera within the
Tubificidae.
Some spermatozoal features seem to be unique (aut)apomorphies of groups of genera (see
Fig. 4): the twisted acrosome (character 2) of Thalassodrilus and Coralliodrilus , the partly thin-
walled acrosome tube (character 5) of Smithsonidrilus and Thalassodrilides, and the double-line
type of spermatozeugmata (character 1, state 3) of Tubifex, Tubificoides and Clitellio. However,
a majority of sperm traits appear homoplasic. Much of the convergence is probably linked with
the adaptive significance of some character states. For instance, very slender acrosomes (character
3) may have evolved at least three times in the Tubificidae; they are interpreted as independent
transformations for Rhizodrilus, Thalassodrilus and Thalassodrilides (Fig. 4). The twisting of the
nucleus (characters 8-9) seems to be convergent too, but here the most parsimonious hypotheses
also imply reversal for these characters for some taxa ( Monopylephorus , Heterodrilus,
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
199
Thalassodrilus and Thalassodrilides). The number of mitochondria has probably been reduced
(character 10) at least twice, independently, for Monopylephorus and Tubifex/Tubificoides.
The withdrawal of the acrosome rod (character 7) is possibly an autapomorphy of the
Tubificidae, but then this has become secondarily protuberant in Thalassodrilus and
Thalassodrilides. Although the two latter genera clearly belong to different subfamilies, the
spermatozoa of them exhibit several (apparently convergent) similarities: very slender acrosome,
secondarily reduced (?) twisting of nucleus, and secondarily(?) protuberant acrosome rod. It is
ironic that the type species of Thalassodrilides ( Limnodriloides gurwitschi Hrabe) was once
placed, for superficial morphological reasons, in Thalassodrilus ; hence the name Thalassodrilides
[see 4].
To conclude, tubificid spermatozoa provide a whole set of new ultrastructural characters,
which are at least partly useful for phylogenetic assessments and, consequently, classification of
taxa at different levels. However, the present study has shown that, in the Oligochaeta, the
spermatozoal character patterns are complex and contain elements of convergence and probably
also reversal, and therefore they should be used in tubificids only in combination with other
information. Spermiocladistics, a term coined by JAMIESON [38], has proved useful for the
reconstruction of phylogenies for many animal groups [37]. In many of the cases spermatozoal
character patterns have been used to test, and indeed often to corroborate, hypotheses of
phylogeny based on non-spermatozoal characters, or to propose new hypotheses of relationships.
Thus, good congruence between spermatozoal and morphological data has been found at higher
taxonomic levels in oligochaetes [34] and in the Clitellata, e.g. [21].
ACKNOWLEDGEMENTS
This paper is contribution no. 436, Caribbean Coral Reef Ecosystems (CCRE) Program, Smithsonian Institution.
We are indebted to the Swedish Natural Science Research Council, and the Ministry of the University and Scientific
Research, Rome (project MURST 40% “Cellular interactions”), for financial support. CE acknowledges a CCRE award from
the Smithsonian Institution enabling work on Carrie Bow Cay, Belize; and thanks Nicole Dubilier (Boston, MA, USA),
Olav Giere (Hamburg, Germany) and Michael R. Milligan (Sarasota, FL, USA), for assistance in the field.
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Source : MNHN. Paris
Source : MNHN . Paris
Comparative Spermatology of Chelicerata:
Review and Perspective
Gerd ALBERTI
Zoologischcs Institut I (Morphologie/Okologie),
Universitat Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany.
ABSTRACT
Chelicerata have evolved separately from other arthropods since the Early Cambrian and thus provide many peculiarities
aside of convergences realized within the framework of the arthropod concept. The present study describes the current
knowledge on sperm cells of chelicerates. The most primitive types are found in the primarily marine taxa, in particular
within the Xiphosura. Pantopoda have already quite derived sperm cells. In both groups, distinct character deviations are
observable between spermatozoa of different taxa. These demonstrate the applicability of comparative spermatology for
phylogenetic or systematic considerations. The most drastic modifications of the basic plan of spermatozoal structure are
seen in the Arachnida. Representatives of all main taxa have now been studied, and their sperm cells are briefly discussed.
In particular those of Araneae (spiders) and Acari (mites and ticks) have been extensively studied. The spider sperm cells
are probably most interesting because of the various transport-forms such as encapsulated individual sperm cells (one cell
per capsule: cleistospermia), capsules including several or numerous individual sperm cells (coenospermia and so called
“spermatophores”), and, probably exceptional in the whole animal kingdom, capsules containing fused, syncytial sperm
(synspermia). Acari all have aflagellate sperm of an extremely differing structure, which allowed the successful application
of sperm ultrastructure for systematic purposes. In a subgroup of Acari it was possible to correlate alterations in the sperm
structure with modifications in the genital systems and sexual behaviour. Thus, an evolutionary concept of sperm
development in this taxon is suggested.
RESUME
Spermatologie comparee des Chelicerata: synthese et perspectives
Les Chelicerata ont evolue ind£pendamment des autres arthropodes depuis le debut du Cambrien et done montrent de
nombreuses particularity ainsi que des convergences r^alisees dans le cadre du concept des arthropodes. Ce travail d£crit
l’etat actuel de nos connaissances sur les spermatozoides des Chelicerates. Les types les plus primitifs sont rencontres
dans les taxons marins de manure primitive, en particular les Xiphosura. Les Pantopoda ont d6j& des spermatozoides tr£s
derives. Dans les deux groupes, des deviations distinctes des caracteres sont observees entre les spermatozoides de
differents taxons. Ceci demontre que la spermatologie comparee peut etre employee pour des considerations
phylogenetiques ou systematiques. Les modifications les plus marquees du plan structural de base des spermatozoides sont
rencontrees chez les Arachnida. Des representants de tous les taxons principaux ont maintenant ete etudies, et leurs
spermatozoides sont rapidement commentes. En particulier, les spermatozoides des Araneae (araignees) et des Acari
(acariens, tiques) ont ete etudies de maniere exhaustive. Les spermatozoides des araignees sont probablement les plus
interessants du fait des formes de transport varies telles que les spermatozoides encapsuies individuellement (une cellule
par capsule: cieistospermie), des capsules contenant quelques ou de nombreux spermatozoides (coenospermie ou pretendus
“spermatophores”), et, probablement exceptionnelles au sein du Regne Animal, des capsules contenant des
spermatozoides fusionnes ou syncytiaux (synspermie). Les Acariens ont tous des spermatozoides aflageltes de structures
Alberti, G., 1995. — Comparative spermatology of Chelicerata: review and perspective. In: Jamieson, B. G. M.,
Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat.,
166 : 203-230. Paris ISBN : 2-85653-225-X.
204
G. ALBERTI : CHELICERATA
extremement differentes, ce qui permet d'utiliser avec succes I’ ultrastructure des spermatozoides pour la systematique. Dans
un sous-groupe des Acariens il a ete possible de correler les alterations de la structure des spermatozoides avec des
modifications du systcme genital et du comportcment sexuel. De ce fait, on suggere V existence d'un concept gvolutif du
d£veloppement des spermatozoides dans ce taxon.
Spermatozoa of Chelicerata are now well known from the comparative point of view.
Several authors have included spermatological characters in their considerations on phylogenetic
systematics of Chelicerata [117, 136, 137], comparative spermatology in general [33], on
comparative spermatology as a tool in phylogenetic systematics [138], on arthropod phylogeny
[32] or on insect spermatology [75], However, since not earlier than 1987 (Schizomida) [21]
sperm cells of all main taxa had been described at least from one representative species, only an
incomplete basis was available to these authors. Moreover, the many remarkable peculiarities
shown by chelicerate spermatozoa require a separate study. This may stimulate further
investigators to focus on spermatology of this interesting, diverse, and ancient group of
arthropods, which has evolved separate from the other arthropods since the Early Cambrian [36,
By far, the greatest structural diversity of spermatozoa is shown among the terrestrial and
most species-rich Arachnida, which thus will dominate the following review.
OBSERVATIONS AND DISCUSSION
General aspects
Spermatozoa ol Chelicerata represent the main categories of sperm structure distinguished
today [32, 33, 35, 66, 74, 75],
The primitive sperm among chelicerates is only found in horse-shoe crabs (Xiphosura) [18,
29, 56, 124] and is characteristic of those taxa exhibiting aquatic fertilization (aquasperm) [sensu
74], This type is classically characterized by a spherical head containing the acrosomal complex
and nucleus with condensed chromatin, a middle piece containing a few relatively large
mitochondria, and an elongate sperm tail representing the flagellum. The distal centriole is
connected by an elaborate anchoring complex to the plasmalemma. It is already distinctly
modified, however, in the Xiphosura [1, 18] (Fig. 1). The acrosomal complex is remarkable
because of its very long acrosomal filament (perforatorium) which runs through the nucleus and is
coiled around it. The chromatin is not as condensed as is usually the case in mature spermatozoa.
The number of rather small mitochondria is quite high and a distinct middle piece is not always
recognizable since the mitochondria may be scattered throughout the cytoplasm surrounding the
nucleus in the spherical head region [18], There is also no prominent anchoring complex [18, 56]
and the axoneme of the flagellum may lack the central tubules (in the Indowest-Pacific species of
Tachypleus and Carcinoscorpius) [18, 147],
The modified (derived) sperm (filiform-flagellate, biflagellate, or aflagellate to mention
some obvious deviations from the primitive type) is characteristic of taxa exhibiting a modified
fertilization. It is found thus in those aquatic (marine) taxa which have developed internal
fertilization or are at least close to it and in terrestrial animals which depend on internal
eitilization. Thus, in Chelicerata modified sperm cells are found in the marine Pantopoda and in
Arachnida, which are primarily terrestrial animals.
In Pantopoda (Pycnogomda), the sperm of Nymphon species are still close to the primitive
type (big. 1). However, an acrosomal complex is lacking and the nucleus is elongated. It is
surrounded by longitudinally arranged microtubules which persist in the mature sperm cell.
Similar to xiphosurans, chromatin condensation is only weak. The proximal centriole is reduced.
!?e 'S/n * f§CUUm m which considerable inter- and intraspecific variation in the axonemal
pattern (9+0 to 18+0) occurs [48, 49, 52], The sperm cells of Nymphon are motile. On the
conti ary, the elongate, immotile spermatozoa of Pycnogonum littorale are highly modified and
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
205
contain numerous longitudinally arranged microtubules (more than 1000) forming complex
patterns. Aside of folded membranes (nuclear derivative?) found in the central part of the cell no
further organelles were seen [49].
Fig. 1. — Diagrams of sperm of the primary marine
cheli cerates, Xiphosura and Pantopoda.
a: Tachypleus gigas (Xiphosura) sperm are still
close to the primitive type of sperm (aquasperm).
The spermatozoon possesses a spherical head, a
pronounced acrosomal complex, and a long
flagellum. Note characters which deviate from the
primitive sperm: very long acrosomal filament
penetrating' the nucleus and coiling around it,
indistinct middle piece. In contrast to Limulus
polyphemus, this species has a flagellum with
9x2+0 axoneme [18]. b: Nymphon sp.
(Pantopoda). The sperm is further derived. It lacks
an acrosomal complex, the head is elongated, the
middle piece is indistinct, there is only one
centriole [49, 52]. AF, acrosomal filament; AV.
acrosomal vacuole; N, nucleus.
In Arachnida the main subtypes of modified sperm cells are distributed through the taxa as
follows: filiform-flagellate (Scorpiones), coiled-flagellate (Pseudoscorpiones, Uropygi,
Amblypygi, Araneae, Ricinulei), and aflagellate spermatozoa (Opiliones, Palpigradi, Solifugae,
Acari) [4-8, 12, 13, 29, 21, 32, 33]. However, this classification gives only a rough impression
of the diversity found within Arachnida: any conventional structure of sperm cells may be varied.
Fig. 2 gives an impression of the various types of sperm depicting an example of each of the
major taxa.
The extraordinary diversity may be first simply expressed by variations in magnitude.
Sperm cells of Arachnida range between less than 2 |im ( Nicoletiella luteal Acari-Actinotrichida;
Siro r/wr/cor/wT/Opiliones-Cyphophthalmi) [5, ALBERTI, unpublished] to 1000 pm in the argasid
tick Omithodorus tholozani (capacitated sperm cell; see below) [63],
206
G. ALBERTI : CHELICERATA
The individual sperm cell may be of a very complex structure (e.g. Pseudoscorpiones;
certain Acari-Anactinotrichida; Fig. 2) or may be rather simple (e.g. Solifugae; certain Acari-
Actinotrichida) [5, 7] (Fig. 2). In the spider mite Tetranychus urticae (Acari-Actinotrichida),
spermatozoa merely are comprised of a chromatin body, some cytoplasm, and a plasmalemma
with tubular indentations [22],
However, primarily arachnid spermatozoa possess the same set of organelles which
characterizes the ground plan of the metazoan sperm cell [33-35] (see above).
BACCETTI [32, 33] several times stressed the major evolutionary alterations in the
arachnids: coiling of sperm cells and loss of flagellum.
In the following, modifications of conventional structures will be described at first, and
secondly, new types of organization will be considered.
Acrosomal complex
The acrosomal complex typically is composed of an acrosomal vesicle (vacuole) and an
acrosomal filament (perforatorium) (Fig. 3). The space between acrosomal vacuole and
plasmalemma is occupied by a more or less distinct substance termed preacrosomal (also
extraacrosomal or periacrosomal) material. The acrosomal filament is part of the subacrosomal
material containing actin filaments which are highly ordered [124], Another component appears as
an amorphous substance and is termed an intermediate substance. This acrosomal complex is
primarily present in all arachnid orders, probably with the only exception of Palpigradi, in which
no acrosomal filament is found (however, only one species has been observed until now) [4],
Sometimes these conventional structures achieve a peculiar organization. In contrast to certain
other scorpions in which the acrosomal complex is at the tip of the elongate nucleus, in the sperm
of Buthus occitanus the very small acrosomal vacuole is located aside of the helical anterior part of
the nucleus. The subacrosomal material (more strictly the intermediate substance) surrounds the
whole tip of the nucleus [8], comparable to the subacrosomal cone found in certain Onychophora
[74, 75]. A peculiar situation is observable in the opilionid Nemastoma lugubre [ALBERTI,
unpublished] where subacrosomal material spreads between nuclear envelope and plasmalemma!
Thus it apparently connects the latter to the nucleus and moulds the cell parallel to chromatin
condensation and nuclear shaping [see also 81], At the first glance, the situation found in certain
Acari-Anactinotrichida (namely in the vacuolated type of sperm) [6, 9, 13] is quite similar as in
the opilionid just mentioned. Here the acrosomal vacuole is developed into a flat cisterna growing
from a central acrosomal plate [6, 41, 42], This flat cisterna is connected to the plasmalemma by
preacrosomal material and the shape of the cell obviously is influenced by this (Fig. 8e).
The preacrosomal material often contains filaments which are probably stabilizing the
anterior region of the cell during the acrosomal reaction [124]. Such filaments were found in
several Araneae [24, 26, 109] and in the micro whip scorpion Schizomus palaciosi [21] (Fig. 3b).
In the latter an intricate paracrosomal lattice structure appears in the late stages of spermiogenesis
as a transient structure (Figs 3d, 5). Regarding the various shapes of the acrosomal vacuole one
extraordinary example was already demonstrated from the Acari-Anactinotrichida (Figs 2, 8a, c,
e). Another well-known example is that of pseudoscorpions. In this group the acrosomal vacuole
continues into a spiral band which surrounds the elongate smooth nucleus [40, 133] (Fig. 2). A
peculiar sickle-shaped acrosomal vacuole was observed in Ricinulei [20] (Fig. 3c).
Finally it may be mentioned that certain taxa of Opiliones and Acari have lost the acrosomal
filament (as Prokoenenia wheeled, Palpigradi) [4] or are completely devoid of an acrosomal
complex. Sometimes this occurs in the same genus: Siro rubens (Opiliones) with all acrosomal
components) [80], Siro duricorius without acrosomal complex [ALBERTI, unpublished].
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
207
Nucleus
Whereas the corkscrew appearance of the nuclear region in Pseudoscorpiones is derived
from the peculiar spiral band (acrosomal vacuole), the nuclei of certain scorpions and of Uropygi
themselves exhibit a helical, in Amblypygi and basically also in Araneae a corkscrew shape (with
sharp edges) [8, 24, 40, 76, 103, 107, 126, .133, 134] (Fig. 5). In the latter and also in the
uropygid Mastigoproctus giganteus, the acrosomal filament spirals around the periphery of the
nucleus enclosed in a nuclear canal [24, 103, 107] (Figs 3e, 4, 5). In Amblypygi and Araneae
there is a tendency toward asymmetry of the nucleus which extends beyond the implantation fossa
into a postcentriolar nuclear elongation [12, 26]. This asymmetry may be extremely developed in
spiders of the genus Tetragnatha (Tetragnathidae) [12], in which the basis of the axoneme comes
close to the acrosomal vacuole. On the other hand, the nucleus of Pholcus phalangioides
(Pholcidae; Araneae) is nearly of radial symmetry, which is a derived characteristic as can be
concluded by comparison with related taxa [24] (Fig. 5).
EURYPELMA
Araneae
CRYPTOCELLUS
k Ricinulei
CHEIRIDIUM
Pseudoscorpiones
SIRO
Opiliones
SCHIZOMUS
Uropygi sf
TARANTULA
Amblypygi
// CYTA
Acari
Actinotrichida
PROKOENENIA
Palpigradi
EUSIMONIA
Solifugae
DRURUS
jrpiones
OPILIOACARUS
Acari Anactinotrichida
Fig. 2. — Synoptic view of spermatozoa of the terrestrial Arachnida (representatives of some taxa have secondarily
invaded limnic and marine habitats). The spermatozoon of the scorpion is drawn in reduced length (full length:
275 pm). Spermatozoa of Siro duricorius and Cheiridium museorum [original], others [4-7, 9, 21, 26. 76, 77].
Opilioacarus : now Neocarus. Scale bar: 5 pm.
A comparable tendency as in Tetragnatha , probably even more peculiar, is observable in the
ricinuleid Cryptocellus boneti [20]. In this species the axoneme is directly connected with the
acrosomal vacuole, a situation - to the author's knowledge - not found anywhere else. However,
the nucleus is not asymmetrical in contrast to Tetragnatha , though of extraordinary shape. The
anterior part is a thin tube containing the acrosomal filament (Fig. 3c).
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G. ALBERTI : CHELICERATA
Even though these are only few examples, it is evident, that the nuclei may exhibit a
considerable variety of shapes. In the flagellate spermatozoa, whether filiform or coiled, it is
basically a large elongate organelle (Scorpiones, Pseudoscorpiones, Uropygi, Amblypygi,
Araneae) [8, 20, 21, 76, 77, 103, 107, 126, 133, 134], which may even extend beyond the
posterior end of the flagellum ( Cryptocellus boneti) [20]. It is of interest, also from the viewpoint
of functional spermatology, that a manchette of microtubules is observed only in some of these
spermatozoa, namely in those of Uropygi, Amblypygi, Araneae and Ricinulei [20, 24, 26, 76,
126]. No manchette is found in Scorpiones and Pseudoscorpiones [40, 72, 77, 106, 133]. At this
point it is not intended to continue the discussion on the possible function of a manchette of
microtubules in nuclear shaping [see 21, 58, 127 for references]. It may only be mentioned that
the microtubules exhibit alterations in their arrangement parallel to nuclear condensation in
Schizomus palaciosi [21], They follow exactly the sometimes aberrant configurations of the
spermatid nuclei (e.g. in Tetragnatha) [12], The manchette microtubules disappear at the end of
spermiogenesis (see the pantopod Nymphon, however).
Another characteristic which largely varies within the Arachnida is the implantation fossa, a
posterior region of the nucleus which usually contains the centrioles or their derivatives. The
fossa may be a shallow, funnel-shaped indentation as in scorpions [8, 77],
Uropygi/Thelyphonida [107], Amblypygi [76, 126] and Pseudoscorpiones [40, 134]. On the
contrary, in Uropygi/Schizomida and many spiders the implantation fossa is deep, sometimes
making the sperm almost a hollow tube (e.g. Schizomus palaciosi- Schizomida, Pholcus
phalangioides- Araneae) [21, 24] (Figs 4, 5). Often dense material occupies the lumen of the
fossa. This material may be homogeneous, granular (e.g. Pholcus) or may contain filaments or
microtubules [92]. Within aflagellate spermatozoa an implantation fossa is observable only in
opilionids. This is connected, in the genus Siro, with the transient appearance of a flagellum
during spermiogenesis and is only a very shallow depression of the nuclear surface [80].
Nevertheless, in other opilionids a deep and broad invagination which contains the centrioles may
occur [78, 81, 125].
In the aflagellate spermatozoa elongate, bulky, spherical, disc-, and bowl-shaped nuclei
may be encountered (Fig. 2). It appears as if, after giving up the “basic plan” in early times,
nearly any possibility of altering the shape of nucleus and cell has been realized. What is
remarkable in particular with regard to the nucleus is the disappearance of the nuclear envelope in
Fig. 3. Details of spermatozoa of various Cheliceraia. a: Acrosomal region of Tachypleus gigas (Xiphosura). Note
acrosomal filament originating in the centre of a slight posterior indentation of the acrosomal vacuole and
showing regularly arranged subfibres (actin) and nucleus with only loosely arranged chromatin [18], x 53 000.
b: Late spermatid of Filisiaia insidiatrix (Araneae). The cap-like acrosomal vacuole is sectioned transversely and
the acrosomal filament composed of subfibres is shown. The acrosomal vacuole is surrounded by thin filaments.
Note manchette microtubules [24]. x 49 500. c: Spermatid of Cryptocellus boneti (Ricinulei). The small, sickle
shaped acrosomal vacuole is sectioned transversely (large arrowhead). It is surrounded by dense streaks. Note
axoneme originating immediately behind the acrosomal vacuole (the nucleus lies parallel to the axoneme and is
not shown in this figure). At left two of the dense plates which later ensheath the spermatozoon are seen (small
arrowheads) (compare Figs. 2 and 5) [20]. x 28 500. d: Spermatid of Schizomus palaciosi (Uropygi-Schizomida).
The basis of the long acrosomal vacuole, which is deeply indented from behind, rests on the nucleus. The
acrosomal filament is rather thick and shows the same pattern of subfibres as in Tachypleus gigas (compare a).
Note the paracrosomal lattice structure, which is only present during a short period in spermiogenesis [21],
x 63 000. e: Part of synspermium, i.e. the product of four fused spermatids, of Segestria senoculata (Araneae)
showing basis of one axoneme and transverse sections through the coils of all four axonemes. Note 9x2 +3
axonemal pattern (synapomorphy of Megoperculata: Uropygi, Amblypygi and Araneae) and the absence of
membranes separating the axonemes. what indicates the complete fusion between the four spermatids. A secretion
sheath surrounds the synspermium [24], AF. acrosomal filament; AV, acrosomal vacuole; N, nucleus; PAL
paracrosomal lattice structure; SH. sheath of secretion.
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G. ALBERTI : CHEL1CERATA
Solifugae and Acari-Actinotrichida at the end of spermiogenesis. In these taxa the acrosomal
filament penetrates through the chromatin body and thus directly contacts the cytoplasmic matrix
[6, 7] (Figs 6d. 8d). A similar situation is to the author's knowledge found only in Xiphosura
[18,56] (Fig. 1).
Midpiece
Since the work of ANDRE [28] the differentiation of the middle piece and chondriome in
scorpions is known in detail. This is an example of extensive reorganization of mitochondria into
elongate tubular structures (compare Fig. 2). No comparable elongation apparently occurs in
Amblypygi and the early derived spider Heptathela kimurai (Mesothelae) [103] in which
numerous mitochondria are arranged helically. Though quite different, in these three taxa a typical
middle piece is established with mitochondria surrounding the flagellum or the axoneme
respectively (Figs 4,5). In pseudoscorpions a different situation is found. The mitochondria are
very elongate, thin tubular structures which are attached to the basis of the axoneme but they are
otherwise situated in the cytoplasm of the coiled spermatozoon (see below) independently from
the axoneme, where they establish a mitochondrial ring [40, 125] (Fig. 2). Contrary to scorpions,
in pseudoscorpions the mitochondria are attached to the bases of the flagellum by an intricate
structure (compare Fig. 2). In Uropygi no middle piece is encountered [21, 107, & PHILLIPS,
1986, pers. com.] (Fig. 5) as is the case in Ricinulei [20]. In Araneae, beside the primitive
condition shown in Heptathela , mitochondria can be located without special arrangement (e.g.
orthognath spiders, several labidognath spiders) [24, 26] or can be completely absent [93, 104] in
the mature spermatozoa. Sometimes mitochondria pass through a “primitive” position in
spermiogenesis being for some time located at the posterior end of the nucleus close to the basis
of the flagellum ( Schizomus palaciosi, several Araneae) [12, 21] (Fig. 5: Schizomus). In the
aflagellate spermatozoa mitochondria are found in various positions sometimes “embedded” in the
nucleus or chromatin body respectively (in the opilionid genus Siro and certain Acari-
Actinotrichida) [6, 27, 64, 80, 142] (Fig. 7). Spermatozoa of some taxa may be devoid of
mitochondria (e.g. Tetranychus w/T/cae/Acari-Actinotrichida) [22].
Axoneme
The flagellum or axoneme respectively, if present, starts with two centrioles (Pantopoda are
different in this respect; see above) arranged coaxially, an arrangement also found in the
Xiphosura [18, 56] (Fig. 1). Thus this derived characteristic may be regarded as a synapomorphy
of the chelicerates [136, 137], Sometimes, however, the centrioles are arranged nearly
orthogonally as in the mature spermatozoa of bird spiders [26]. Spermiogenesis reveals that this
position is a derived arrangement in these spiders, probably caused by the coiling process, and
follows a phase in which the centrioles are in tandem position. Even the original orthogonal
arrangement is observable in the early stages of spermatogenesis [26].
One of the most interesting results from the viewpoint of comparative spermatology in
Arachnology was the discovery of the 9x2 +3 axoneme in Araneae [103, 1 12], Uropygi [21,
107] and Amblypygi [76, 126] demonstrating the close relationship of these taxa with a
spermatological criterion (Fig. 3e). However, even this apparently very stable characteristic
(compared with the variability found e.g. in scorpions) [8, 72, 77] is altered in certain spiders
(Linyphiidae) in which a 9x2 +0 axoneme occurs [12], Thus this axoneme is by convergence
similar to that of the xiphosuran genera Tachypleus and Carcinoscorpius [18, 147] and several
scorpions [72],
Sometimes dense material surrounds the distal centriole thus probably establishing an
anchoring complex ( Pholcus phalangioides, Dysdera sp., Segestria senoculata ; Araneae) [24]
(Figs 3e, 6b). In spiders the number of tubules constituting the centrioles may be reduced and/or
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dense fibres may be attached to the base of the axoneme. In Cryptocellus boneti (Ricinulei) the
proximal centriole disappears during late spermiogenesis [20].
The only case where an aflagellate spermatozoon reveals relicts of an axoneme is
represented by the cyphophthalmid Siro [80], In these tiny opilionids a flagellate stage is
observable during spermiogenesis.
Fig. 4. — Different shapes of nuclei of spermatids of spiders and their relation to acrosomal complex and axoneme.
a: Heptathela kimurai (Liphistiidae, Mesothelae). b: Filisiata insidiairix (Filistatidae. Opisthothelae).
c: Tetragnatha montana (Tetragnathidae, Opisthothelae). d: Pholcus phalangioides (Pholcidae, Opisthothelae).
The Filislata-lype is the most common in spiders and may be close to the plesiomorphic type of Opisthothelae
A peculiarity of scorpions and pseudoscorpions is the flagellar tunnel surrounding the
axoneme and separating it from the middle piece mitochondria (in scorpions) and from the
cytoplasmic matrix respectively (of the coiled cell in the pseudoscorpions) (Fig. 2). Whereas in
the scorpions the flagellar tunnel comprises a distinct extracellular space, this is completely absent
in pseudoscorpions owing to the close apposition of the membranes, which in early stages had
delimited it. In scorpions and in some pseudoscorpions a dense cylinder surrounds the flagellar
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G. ALBERTI : CHEL1CERATA
tunnel. A flagellar tunnel is also observable during spermiogenesis of Uropygi, Amblypygi,
Araneae and Ricinulei but disappears later [20, 21, 76, 107, 125]. Thus the axoneme is not
surrounded by its genuine membrane in the mature spermatozoa of these taxa (Figs 3e, 6a, b).
It is important to note that the flagellar tunnel separates the flagellum from the mitochondrial
sheath (in scorpions) or mitochondrial ring (in pseudoscorpions). In Amblypygi and the early
derived spider Heptathela , in which a middle piece is still existent, the flagellar tunnel disappears
before the mitochondria occupy their definite position (Fig. 5).
A flagellar tunnel appears also in connection with the transient flagellum of Siro. It is
retained after the axonemal tubules have been withdrawn as a peculiar “crypt” containing
numerous microvilli [80, ALBERTI, unpublished].
The flagellar tunnel may be derived from the cytoplasmic collar around the flagellar basis in
xiphosuran sperm. No such structure occurs in Pantopoda (Fig. 1).
In addition to the diversity achieved in arachnids by variations of the basic sperm
components “new” structures are introduced into this cell type resulting in new structural and
functional possibilities and extraordinary peculiarities. These are dealt with in the following.
Vesiculation and vacuoles
Arachnid spermatozoa are very often rich in vesicles and cisternae, especially those
belonging to the coiled-flagellate type. These vesicles - at least partly a result of the coiling
process during which surface membranes are likely internalized - may be arranged very regularly
under the plasmalemma (Uropygi/Thelyphonida, Amblypygi) [76, 107, 126] (Figs 2, 5). In
Araneae and Ricinulei such vesicles and cisternae often contain dense material forming “dense
streaks” (Fig. 3c). In certain Araneae vesicles fuse to large vacuoles [24]. In addition to these
structures further vesicles are derived by an extended activity of the Golgi apparatus. In Ricinulei
dark plates are formed early in spermatogenesis and contribute to an intracellular capsule
enclosing the central components of the mature spermatozoon (Figs 2, 3c) [20]. In the palpigrade
Prokoenenia xvheeleri a huge vacuole dominates the large cell, which was previously thought to
be a spermatophore (Fig. 2). The spermatozoa of certain Acari/Anactinotrichida develop during a
very complex spermatogenesis a conspicuous vacuole bordered by a complex periphery bearing
numerous “cellular processes” [5, 42, 63, 131] (Figs 2, 7, 8a, c). Perhaps the pouches of the
ribbon sperm of related mites are derived from the vesicular precursors of the large vacuole
characterizing the vacuolate type (Figs 8b) [5, 9, 13, 139-141],
Transport forms - sperm aggregates
Another aspect which was newly (or independently from non-arachnids) achieved during
the evolution of spermatozoa of Arachnida is the establishment of secondary events. These
transform elongate spermatozoa into the already mentioned coiled spermatozoa (Figs 2, 6a, b). In
Uropygi and Amblypygi the coiling process starts from the posterior (flagellar) part of the cell
resulting in a spherical mature spermatozoon. It is of interest to note that this coiling process is
incomplete in the early derived spider Heptathela kimurai (Mesothelae) [89, 103], Only the
flagellum and posterior part of the nucleus are involved. In the bird spiders (orthognath spiders)
the coiling is more complete, leaving only the acrosomal region projecting slightly from the
otherwise more or less spherical cell [26]. Finally, in (most) labidognath spiders the whole cell
including the acrosomal region is completely coiled or spherical again [12, 15, 24, 39, 92, 93,
104, 1 09]. . If it is assumed that the spherical shape is the plesiomorphic state within the
“Pedipalpi”-Araneae, the spiders demonstrate, probably, a nice example of a round about way in
the evolution of their sperm cells. In Araneae the coiled cell is surrounded by an extracellular
secretion sheath in different ways (Figs 3e, 6b and see below).
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The coiling of the spermatid of Pseudoscorpions is a process which largely takes place in an
extensive vacuole [134], After completion of coiling the vacuole is transformed into smaller
vesicles or cisternae (Fig. 2).
Extracellular material or secretions may loosely combine several spermatozoa to form
aggregates (Scorpiones, Solifugae, Bdellidae/Acari/Actinotrichida) (7, 8, 10, 23] (Fig. 6c, d). In
Opiliones/Cyphophthalmi the aggregates include dimorphic spermatozoa [80, ALBERTI,
unpublished] (Fig. 6c). Such sperm aggregations in turn may be surrounded by a common sheath
as in spiders of the families Theraphosidae and Filistatidae (coenospermia) [12, 24, 26, 37],
Rarely, such aggregates can deviate from the spherical shape. Thus, in Telemidae an elongate,
rather complex tubular “spermatophore” is found [82]. A peculiarity observed to the author’s
knowledge only in a few Araneae (Dysderidae, Segestriidae, Scytodidae, Sicariidae) is occurence
of syncytial spermatozoa (synspermia) [10, 12, 24] (Figs 3e, 6b). This fusion (or incomplete
separation of spermatids) occurs already within the testis, whereas the coenospermia and
spermatophores (Telemidae) are surrounded by a secretion sheath in the distal vas deferens as is
the case in the majority of spiders which encase single sperm cells (cleistospermia) [12, 15, 24],
Fig. 5. — Late spermatids of: a: Mastigoproctus giganteus (Uropygi-Thelyphonida). b: Schizomus palaciosi (Uropygi-
Schizomida). c: Tarantula marginemaculata. d: Heptathela kimurai (Araneae-Mesothelae). e: Prokoenenia
wheeleri (Palpigradi). Note that the spermatid of the palpigradid deviates completely from those of the
Megoperculata. A synapomorphy of Amblypygi and Araneae may be the poslcentriolar elongation of the nucleus.
The spermatids of Schizomus , Tarantula and Heptathela are drawn in reduced length [12].
Activation , maturation and capacitation
The spermatozoa of Limulus polyphemus remain immotile until they come into contact with
a component which is released from the egg layer [44],
Spermatozoa of many taxa undergo certain modifications after leaving the male by which
they achieve the capacity to fertilize the oocyte (capacitation) [35]. These transformations may be
restricted to the molecular level as in mammals or may involve distinct morphological alterations
[102, 130]. It is evident that these processes have to occur in a taxon-specific way since the sperm
cells have to cope with a specific environment. In terrestrial animals this is usually the female
genital tract. Exceptions are species with dermal insemination such as some Onychophora [94,
214
G. ALBERTI : CH ELI CERA TA
118, 119] in which the sperm cells migrate through the body cavity. In many arachnids the
mentioned transformations occur in a rather drastic way. However, these events are poorly
investigated. The transport form in which the sperm is transferred to the female is changed into
the fertile, active sperm.
From Araneae and Pseudoscorpions it is known that spermatozoa uncoil in the female [37,
40] and only then are fertile [45]. Probably these events are facilitated by the mentioned vesicles
and vacuoles which may be integrated into the plasmalemma when elongation of the cell occurs.
This process is well known and very intricate in the vacuolated sperm cells of ticks and some
mites (Acari/Anactinotrichida). The shape of the cell is completely changed by a tuming-inside-
out-process which brings the cellular processes to the surface of the cell [13, 41, 42, 63, 101,
1 13, 131]. After that, the sperm cells are capable of several kinds of motion [60, 101] (see Figs
2, 7, 8a, c for cells prior to capacitation).
In ticks, and probably also in anactinotrichid mites with vacuolate spermatozoa, a further
transformation of the cell occurs prior to insemination. A posterior portion of the sperm cell which
is invaginated into the cytoplasmic column (inner core), forming a so-called acrosomal canal in
several species, is also evaginated and contains the nucleus [63, 113]. It has been suggested that
only this nucleus-containing part of the capacitated spermatozoon enters the oocyte [101, 113].
In the ribbon spermatozoa of a related mite, Varroa jacobsoni, the spherical cell transforms
into an elongate cell with completely new appearance and structures which are probably related to
the achievement of motility [17].
In actinotrichid mites, e.g. spider mites (Tetranychidae), spherical spermatozoa become
irregular and possess filaments in cell processes, probably enabling amoeboid movements [13,
16, 22, 98],
Acrosomal reaction
The acrosomal reaction of Limulus polyphemus is a classical subject of study in
spermatology and has been described in detail [29, 47, 124], Upon contact of the sperm cell with
the egg, exocytosis of the acrosomal vacuole occurs. The contents of the vacuole ensures
attachment to the egg. In about 4 seconds, a 60 pm long process grows out of the anterior end of
the cell (so called true discharge). The process contains the acrosomal filament (perforatorium)
which is uncoiled and remains covered by a membrane (the posterior membrane of the acrosomal
vacuole to which material derived from the nuclear envelope is continuously added). The
elongating process rotates, which helps the process to penetrate the extracellular layers of the egg.
According to [47, 129] the driving force which creates the elongation of the process is the
consequence of an alteration in the twist of the actin filaments within the acrosomal filament
(compare Figs la, 3a).
No other chelicerate sperm cell has been investigated with regard to its acrosomal reaction.
Because of the structural similarity of the acrosomal complexes of many arachnids it can be
assumed that the main events are similar to those known from the xiphosuran (Fig. 3). However,
the numerous peculiarities indicated above urgently need a detailed investigation.
Fig. 6. Transport forms of spermatozoa in Arachnida. a: Cryptocellus boneti (Ricinulei). The coiled sperm cell is
encysted. The cyst wall consists of two dense plates which are intracellular products (see Fig. 3c). Additional
secretions are added in the vas deferens [20], x 19 000. b: A synspermium of Segesiria senoculaia (Araneae).
Arrowheads point to the bases of three (of four) axonemes [24], x 13 250. c: A sperm ball of Siro duricorius
(Opiliones-Cyphophthalmi) consists of peripherally arranged infertile sperm cells (large arrowheads) and smaller,
cup-like fertile spermatozoa (small arrowheads). In the centre of the ball conspicuous secretions are seen (compare
Fig. 2 and [80]). x 3 350. d: Pile of 32 spermatozoa arranged in 16 pairs in Eusimonia mirabilis (Solifugae) [71.
x 7 500. N, nucleus; SE, secretion.
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216
G. ALBERTI : CHEL1CERATA
Phylogenetic and systematic considerations
Sperm cells of Chelicerata are evidently very diverse and the taxon-specificity of these cells
offers the possibility to use characteristics of these cells for taxonomic purposes [5, 6, 9, 12, 13,
32, 33, 35, 138].
Among the xiphosurans interesting features have been shown already when comparing the
only Atlantic species ( Limulus polyphemus ) with the three Indowest-Pacific taxa (Tachypleus
gigas, T. tridentatus, Carcinoscorpius rotundicauda). Apart from some deviations in the shape of
the acrosomal vesicle, arrangement of mitochondria and acrosomal filament, the most obvious
difference is the lack of the central microtubules in the axoneme of the Indowest-Pacific species.
This is a synapomorphy of these taxa and demonstrates the result of a long independent evolution
[18, 147],
The few studies on Pantopoda have shown considerable differences between the two genera
investigated (see above) which corroborates the view that Pycnogonidae is the more derived taxon
compared with Nymphonidae [84, 97]. The axonemal pattern may be used to ascertain the validity
of certain species (e.g. Nymphon gracile and N. rubrum) as separate taxa [52],
Within the Arachnida, only in the Opiliones [78, 81, 125] and Acari [5, 6, 9, 13] have
systematic considerations been undertaken on the basis of a sufficiently wide range of taxa. Some
encouraging aspects emerged also from the recent studies of “Pedipalpi” and Araneae [14, 21, 24,
26, 76, 82, 92, 126],
All of these sperm cells are, despite their wide range of complexity, capable of transferring
the male's genetic information to the female germ cell. It is very challenging trying to trace the
evolution of the sperm cell through the branching phylogenetic tree looking for a more
comprehensive understanding of the alterations of this cell type. It is obvious that this is a long
lasting project which leaves much work for the future. Arachnida represent a very old taxon, so
there are not only the usual gaps in knowledge (which could be closed by future research) but also
those caused by extinction of large taxa [36, 70, 86, 105, 136, 137], Thus the extant major taxa
of Arachnida are quite isolated from each other. Spermatology may provide a tool to bridge gaps
in taxonomic understanding between these extant taxa. Furthermore it has to be kept in mind that
convergences may also occur at the cellular level. Some examples were already mentioned:
displacement of axonemal base to the anterior (Cryptocellus/ Ricinulei, Tetragnatha! Araneae),
9x2 +0 axoneme ( Tachypleus , Carcinoscorpius/X iphosura, Scorpiones in part,
Linyphiidae/Araneae), deep implantation fossa ( Schizomus / Uropygi, some Araneae such as
Pholcus ), helical or corkscrew appearance of sperm head ( Buthus/Scorpiones , Pseudoscorpiones,
“Pedipalpi”- Araneae). (In Araneae, this corkscrew appearance in Pholcus phalangioides is
profoundly different from all other spiders and is thus probably an autapomorphic peculiarity of
this taxon) [12, 24] (Fig. 4). Most likely another convergence is demonstrated by the shaping of
the spermatid under the influence of “acrosomal” material (Nemastoma/Opiliones, vacuolate type
of sperm/Acari-Anactinotrichida) (Fig. 8e). Different types of “envelopes” are found in the coiled
sperm: extracellular ones (Pseudoscorpiones, “Pedipalpi”, Araneae) and intracellular ones
(Cryptocellus/ Ricinulei) (Figs 3e, 6a, b). Some of these convergences are easily recognized by
fine structural analysis or observation of spermiogenesis, others need comparison with related
taxa on a wider scale.
FiG. 7. — Sperm lypes in Acari. Asterisks indicate genera and corresponding higher taxa [modified from 13]. Spermatozoa
of Antennophorina are probably intermediate between vacuolated type and ribbon type (ALBERTI & Blaszak
unpublished]. Note that spermatozoa of Actinedida exhibit a diversity which can hardly be depicted here (compare
[6, 13]). See also Fig. 9 with respect to Gamasida.
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
217
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Source . MNHN. Paris
218
G. ALBERTI : CHELICERATA
Thus spermatology may at least shed some new light on systematical interpretations and
may induce a reconsideration of certain traditional ideas. Some examples of relationships which
were suggested previously and which could be viewed at in the light of the new spermatological
findings are: Pseudoscorpiones-Scorpiones [148]; Pseudoscorpiones-Solifugae [69, 114, 117,
136, 137]; Uropygi-Acari [111]; Solifugae-Acari/Actinotrichida [67, 149]; Acari/Anactinotrichida
-Acari/Actinotrichida [83, 53, 91, 117, 136, 137]; Opiliones/ Ricinulei-Acari [30, 88]; Opiliones-
Acari [83, 108, 114]; Ricinulei-Acari [53, 91, 117]; Ricinulei-Acari/Anactinotrichida [69];
Pedipalpi-Palpigradi [83, 1 10]; Schizomida-Palpigradi [38, 132]; Palpigradi-Acari/ Actinotrichida
[69]. In some cases it was already possible to bridge some gaps or solve open questions.
The well known example of the 9x2+3 axoneme uniting “Pedipalpi” with Araneae was
already mentioned (Fig. 3e). This finding was not surprising from the viewpoint of the
systematists (who had known for a long time that these taxa are closely related because of other
character states: Megoperculata) [137], but it was important to recognize that spermatology may
function as a taxonomic tool in Arachnida [21, 76, 103, 107, 112], However, the main problem
with these taxa is concerned with the relationships within the group Megoperculata. Are Uropygi
and Amblypygi sister groups and are they together (as Pedipalpi) the sister group of the taxon
Araneae [117]? Are Amblypygi and Araneae sister groups (= Labellata) [137] forming the sister
group to Uropygi? Does the term “Pedipalpi” thus describe a monophyletic or paraphyletic taxon
[71]? Moreover, do the Uropygi really constitute a natural (monophyletic) group? Several authors
have divided this taxon into Thelyphonida (=Uropygi s. strict .) and Schizomida (= micro whip
scorpions) [90, 96, 97]? Are the Palpigradi closely related to the “Pedipalpi” (especially to
Uropygi-Schizomida) as several authors have suggested [38, 83, 110, 132; see also 117]?
Spermatology may at least point the direction of a solution of these questions (Fig. 5). In Araneae
the nucleus of the sperm cell extends with a postcentriolar nuclear elongation beyond the base of
the axoneme making the nucleus and the whole cell asymmetrical. In Amblypygi the same
asymmetry of the nucleus is found, though less pronounced. Araneae and Amblypygi are thus
united by the presence of this nuclear elongation as a synapomorphy (shared derived character).
The presence of a middle piece in the Amblypygi and the early derived spider Heptathela kimurai
(Mesothelae) is, in contrast, considered a symplesiomorphy. On the other hand Thelyphonida and
Schizomida do not possess a middle piece and a shortened central triplet in the axoneme. Both
characteristics are probable synapomorphies. The helical nucleus is not developed in a corkscrew
(with sharpened edges) as in the Amblypygi and Araneae and does not demonstrate a
postcentriolar nuclear elongation (symmetrical nucleus). These characteristics are most likely
symplesiomorphies of the Uropygi. Thus the spermatological results would support the view that
the taxon "Pedipalpi' describes indeed a paraphyletic group. There are no spermatological
indications which would support a close relationship with Palpigradi. Schizomida possess the
Fig. 8. Some characteristics of acarine sperm cells, a: Vacuolated-type sperm in Sejus togatus (Gamasida-Sejina) in
transverse section. Note the wall of the vacuole provided with numerous so called cellular processes (compare c)
fill- x 14 600. b: Ribbon-type sperm of Parasitus berlesei (Gamasida-Parasitina) sectioned transversely. Note
ribbons (arrowheads) with the paired sacs underneath [5], x 16 500. c: Cellular processes in the spermatozoon of
Sejus togatus (Gamasida-Sejina). Note different profiles according to level of section through the individual
processes and filaments within the processes. Large arrowheads indicate microtubules, small arrowheads indicate
flat acrosomal cisterna (compare e). x 84 000. d: Spermatozoon of Cyta latirostris (Actinedida). This type of
sperm may be regarded as representative (plesiomorphic) of at least a great part of the very diverse Actinedida (see
Fig. 7). Arrowheads indicate coils of the acrosomal filament [6]. x 18 500. e: Part of early spermatid of Epicrius
mollis (Gamasida-Epicriina), which will develop into a vacuolated-type sperm. The acrosomal vacuole has
dillerentiated into an acrosomal plate, to which the nucleus is attached and from which the acrosomal filament
arises (arrow), and an extensive flat portion (cisterna) which grows out under the plasmalemma (arrowheads;
compare c) [5]. x 27 000. APL. acrosomal plate; AV, acrosomal vacuole; N. nucleus; V, vacuole.
Source
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
219
Source : MNHN. Paris
220
G. ALBERTI : CHELICERATA
typical 9x2+3 axoneme of “Pedipalpi”- Araneae (synapomorphy of the Megoperculata), Palpigradi
have aflagellate spermatozoa (Figs 2, 5). Further it appears that it is not necessary to separate
Schizomida from the remaining Uropygi because of the mentioned synapomorphies, despite
differences which include the paracrosomal lattice structure appearing in the late spermatid of
Schizomus as a transient structure (Figs 3d, 5). The position of the acrosomal filament and the
deep implantation fossa are most remarkable (Fig. 5) [21, 24, 76, 107, 126].
Within the Araneae it was shown that spermatozoa can be transferred to the female as
numerous individual cells, each surrounded by its own sheath (cleistospermia), as numerous
aggregates of individual cells, surrounded by a common sheath (coenospermia), as one tubular
“spermatophore” (only in Telemidae) or as numerous syncytial spermatozoa (synspermia) (see
above).
Though only few taxa have been studied with respect to these transport-forms it appears that
the coenospermia are plesiomorphic since these have been observed in the early derived
liphistiomorph spiders [HAUPT & KOVOOR, 1988 pers. comm.; ALBERTI, HAUPT &
SCHWENDINGER, unpublished]. Furthermore, representatives of several families of orthognath
spiders (Atypidae, Antrodiaetidae, Nemesiidae, Dipluridae, Theraphosidae) and of the
labidognath spider family Filistatidae [24, 26, ALBERTI & COYLE unpublished, ALBERTI, HAUPT
& SCHWENDINGER, unpublished] possess this type of encapsulated sperm. The “spermatophore”
of Telemidae is regarded by the present author as an autapomorphy of this group of cave dwelling
spiders since it has been observed only in this family (thus representing a rather isolated
phenomenon within spiders) and does not show any relationship with spermatophores of
Amblypygi, the assumed sister group of spiders. It is formed not by accessory glands, which are
usually involved in spermatophore construction, but by secretions of the vasa deferentia [82],
Thus these “spermatophores” are established in the same region of the genital tract as the
coenospermia and are most likely a special modification of these [12, 15, 24],
Synspermia are obviously an apomorphy. This type of sperm aggregation is not found
elsewhere within the Arachnida and probably the animal kingdom. Until now, it has been
observed only in the families Segestriidae, Dysderidae, Scytodidae and Sicariidae. It therefore
may represent a synapomorphy uniting these (and other?) families of so-called haplogyne spiders,
a group of Araneae which offers many systematical problems [46, 115] (Figs 3e, 6b).
The functional significance of the astonishingly diverse spermatozoa of Araneae, regarding
the general uniform mode of insemination with the male palpal organ, has yet to be fully
understood (see also Conclusions). However, the knowledge of spermatology is developed
further in this functional respect and also with regard to systematical interpretations in Acari [5, 6,
9, 11, 13, 17].
The Acari are most often considered to be related to the Opiliones and/or Ricinulei [see 69,
70, 91, 117, 136, 137 for detailed discussions]. Other candidates are Solifugae and Palpigradi
[67, 69, 70, 149]. However, it has long been debated what comprise the Acari. Are the Acari
polyphyletic, diphyletic or monophyletic [see 129]? Aside of some extreme views which for
example regarded the Opilioacarida or the Ixodida as independent arachnid groups or at least far
separate from the (other) Acari [see 111, 116, 123], in the last decades two suggestions have
mainly been discussed. Some authors support the idea that there are three (Opilioacarida,
Anactinotrichida, Actinotrichida) major acarine groups [68, 79, 83] others think of two
(Anactinotrichida including Opilioacarida, Actinotrichida) [53, 55, 69, 70, 85, 91, 97, 150],
Spermatology has contributed to these questions the following: from the sperm morphology of
Opilioacarida, which is almost identical with that of Ixodida (ticks) and certain Gamasida, it is
obvious that these taxa constitute one group: Anactinotrichida (=Parasitiformes). The vacuolated
type of sperm is a synapomorphy of this group (Fig. 7). On the other hand it was demonstrated
that the spermatozoa of Actinotrichida (=Acariformes) do not have anything in common with
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
221
DERMANYSSINA
Phytoseiidae
Rhodacaridae
Pachylaelapidae
Macrochelidae
Varroidae
¥ tubular ovary
paired oviducts
fat body provides
yolk precursors
insemination via
medioventral genital
opening (tocospermy)
spermatozoa migrate
through oviducts
cf genital opening in midstemal
position, unmodified chelicerae
vacuolated spermatozoa
spermatophore is manipulated
with parts of the gnathosoma
OPILIOACARIDA
HOLOTHYRIDA
IXODIDA
Early derived GAMASIDA
9. — Diagram depicting probable evolutionary changes of genital systems and sperm morphology in Gamasida [13].
Only those gamasid families from which fine structural results are available are listed. Tocospermy: insemination
via the primary genital opening; podospermy: insemination via secondary copulation pores (= solenostomes).
Spermatotreme: slit in the mobile digit of the chelicera to hold the neck of the spermatophore during its transfer;
spermatodactyl: slender process at the mobile digit of the chelicera used to transfer the sperm.
Source
222
G. ALBERTI : CHELICERATA
those of Anactinotrichida what could be termed a synapomorphy [5, 6, 9, 13]. Thus, it is at least
a misleading simplification to consider the sperm cells of, for instance, Ixodida, as typical for the
Acari as a whole (Fig. 7).
The problem of systematics of major groups of Acari is reduced by spermatological data to
diphyly versus monophyly, with two different sperm types to be compared with each other and
with those of the candidates outside the Acari (e.g. Ricinulei and Opiliones-Anactinotrichida;
Solifugae and Palpigradi-Actinotrichida). No decision solving these questions for sister group
relationships can yet be made (compare Fig. 2). The evolutionary distance between these taxa is
probably too large [see for details 13]. At present the spermatological data also do not indicate that
one of the major mite groups developed from part of the other, which would then be a
paraphyletic taxon [see 30, 88].
Comparing sperm cells of Acari on a broad basis of taxa it was possible first to detect the
profound difference between spermatozoa of Anactinotrichida and Actinotrichida (Fig. 7).
Further, within the Anactinotrichida the vacuolated type turned out to be the plesiomorphic type
from which other types (ribbon type and its subtypes) have developed [5, 6, 9, 13, 17, 139-141].
From this basis it was, at least in part, possible to trace the evolution of the sperm cell within the
Anactinotrichida (Fig. 9). It was deduced that the vacuolated and ribbon type are correlated with
different modes of insemination. In those taxa possessing vacuolated spermatozoa (Ixodida,
Gamasida in part) sac like spermatophores are transferred to the female (reproductive behaviour of
Opilioacarida and Holothyrida is not known). The gnathosoma manipulates the very complex
spermatophore [53, 61] to the female genital opening located medioventrally. Capacitated
spermatozoa (see above) migrate through the oviducts into the tubular ovary where fertilization of
oocytes, most likely, occurs [43]. In those taxa having the ribbon type (Gamasida in part:
Parasitina and Dermanyssina), the ovary comprises a nutritive tissue probably derived from sister
cells of the oocytes [3, 25], The ovary is a solid organ. It is thus necessary that female tissues are
penetrated by the ribbon spermatozoa during their migration to the oocytes. There is growing
evidence that this penetration results in a migration of spermatozoa through the haemocoele in
Parasitina to the ovary [11, 13]. In Dermanyssina specialized paired sperm induction pores are
found in a peculiar lateral position at the bases of legs III or IV [1 1, 17, 31, 50, 54, 57]. The
question is, how was it possible to change the fertilization site during evolution of these taxa with
spermatozoa apparently extremely derived (vacuolated type) and adapted to special conditions of
the female genital system. However, knowing the. situation in Parasitina it is not so difficult to
understand this dislocation of the site of sperm transfer. Only the penetration site had to be
displaced. Spermatophores were already manipulated with the chelicerae (Parasitina and
Dermanyssina males have transformed chelicerae for this purpose: spermatotrema,
spermatodactyl) and spermatozoa, of the ribbon type, were already adapted to penetration and
migration through female tissues. Further adaptations are found in Dermanyssina regarding a new
sperm access system which leads the sperm cells from the induction pores (solenostomes) to the
ovary, displacement of the male genital opening into a presternal position and reorganization of
the female genital tract and these in turn result in alterations of spermatozoa of the ribbon type to a
less complex structure compared to the vacuolated type or the ribbon type of the Parasitina [11,
13, 17],
These interpretations which are based primarily on spermatological results, combined with
studies on anatomy, histology and oogenesis, may very well play a key role in understanding the
large group of Anactinotrichida and its most successful (species-rich) taxa Parasitina and
Dermanyssina (Fig. 9). Evidently the modifications can be interpreted in the sense of economy,
with the development of a nutritive tissue (so called lyrate organ) [95] in Parasitina and
Dermanyssina as the initial event [11, 13, 17, 25]. It is of interest that comparable differentiations
occurred in the Acari/Actinotrichida within the spider mites (Tetranychidae: direct sperm transfer
with penis, migration of spermatozoa through haemocoele, nutritive cells in the ovary) and
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
223
Acaridida (penis, secondary copulatory pore, sperm access system leading to ovary) bv
convergence [13, 16, 22, 98, 145],
Thus the case of the Acari not only demonstrates the usefulness of comparative
spermatology in solving systematical problems. It also gives an impression how sperm diversity
has been evolved parallel to phylogenetic alterations with their functional/adaptive implications in
a restricted taxon.
Conclusion and perspective
Though in recent years knowledge on sperm structure in Chelicerata has increased
considerably, many questions remain unsolved. Indeed, the growth in knowledge has shown that
yet more fascinating problems remain to be investigated only some of which can be indicated
here.
First, spermatozoa of several taxonomic groups are only known from one (Solifugae.
Ricinulei, Palpigradi) or very few species (e.g. Uropygi, Amblypygi) and it is evidently
necessary to examine further representatives. From Solifugae only mature spermatozoa are known
and studies on spermatogenesis are urgently required to understand the very peculiar sperm
morphology as well as the aggregations which they form (Fig. 6d).
Capacitation processes are only known in (structural) detail from tick spermatozoa and
similar events (e.g. the turning inside out-process, only to mention the most striking event)
probably occur also in the vacuolated sperm of the other taxa. But there are indications of
capacitation of sperm of other acarine taxa (e.g. Anactinotrichida: Varroa jacobsoni;
Actinotrichida: Tetranychus urticae, Bdella septentrionalis, Phytoptus avellanae, Acarus siro and
Tyrophagus putrescentiae) [5, 17, 19, 22, 23, 144],
In spiders, the synspermia of Dysderidae and also the cleistospermia of Oonopidae possess
large vacuoles [see above, 24]. Probably these facilitate the process of uncoiling in the female.
The organelles such as acrosomal vacuole, nucleus and axoneme, are covered with membranes of
the vesicles/vacuole, which could be integrated into the plasmalemma very quickly. Thus these
spermatozoa may be regarded as "precapacitated” (being somewhat similar by convergence to the
vacuolate sperm cells of the mentioned Acari-Anactinotrichida) (see also vesicles at the periphery
of sperm of Uropygi and Amblypygi; Fig. 2). As the female genital tract of spiders differs
considerably [65], these and other specializations (e.g. the lack of mitochondria in some spiders)
may represent adaptations to these various fertilization conditions. These problems evidently offer
a broad field lor exciting future studies which may be considered under the aspects of sperm
competition and securing sperm priority.
Except for Limulus polyphemus sperm, nothing is known about acrosomal reactions in
chelicerate sperm cells [124], In most taxa the exact site where the eggs are fertilized is unknown
or at least speculative. Evidently sperm cells pass through the haemolymphatic space in certain
mites (Acari: Parasitidae, Tetranychidae). Are these exceptions or do further examples exist?
Apparently this passage brought about the development of new genital ducts (sperm access
system in Dermanyssina) as a reaction of the female comparable to the paragenitalia in bed bugs
and their relatives. This in turn indicates the strong selective influence which is imposed by the
females not only on the male genitalia but also on spermatozoa [see e.g. 11, 13, 17, 51],
Only few chelicerate sperm cells have been investigated with regard to their cytochemistry:
Limulus polyphemus [124] and two gamasid mites [143]. The significance of the many enigmatic
components (vacuoles, vesicles, dense streaks, plates, various filaments, inclusion bodies, etc.)
could be better understood using appropriate and available methods.
Even in those groups in which a broad range of taxa has been investigated, such as Araneae
and Acari, much work is still to be done. Araneae offer many questions, for instance: what is the
functional significance of the various transport-forms (coenospermia, cleistospermia, synspermia,
spermatophores )? Is this diversity related to the male and female copulatory organs which are
224
G. ALBERTI : CHEL1CERATA
insufficiently understood [see 65, 73, 87, 121, 122, 128]. How are these encapsulated
spermatozoa activated and how do they then function? What happens to the enigmatic synspermia
in the female?
Sperm aggregates of Solifugae and the enigmatic small opilionids of the genus Siro need
further investigation. The latter taxon is the only one within the Chelicerata known to have
dimorphic spermatozoa, albeit of unknown functional significance. Does this phenomenon also
occur in other Cyphophthalmi?
The peculiar genital tract of “Pedipalpi”, possessing a pair of glands with holocrine
secretion of a very unconventional type, offers very exciting interpretations. If these glands could
be proven to be of testicular origin, the secretory products could represent strongly modified germ
cells too. This interpretation would be of much interest regarding the relationships between
“Pedipalpi” and Araneae [see above and 13].
The adaptations of sperm cells being transferred via various types of secreted containers
(capsules, stalked spcrmatophores of various complexity) [see e.g. with regard to Acari: 27, 61,
64, 146] need to be studied in more detail.
Only few studies have focused on the association of microorganisms with spermatozoa [2,
59, 62]. In particular the enigmatic Adlerocystis sp. which is a regular symbiotic associate of tick
spermatozoa deserves more attention [62].
In Acari a broad range of sperm cells from a very high complexity to very “simple” cells is
observable (Figs 7, 8) All these cells transfer genetic material apparently successfully. Many taxa,
however, reproduce exclusively parthenogenetically [100]. This is in particular remarkable
regarding the oribatid mites, a very common group living in almost all soils. It has been suggested
that in certain taxa a reversion to sexuality may have occurred [99]. An even more striking
example is represented by the Acaridida (flour mites, house dust mites, feather mites, itch and
mange mites etc.), which reproduce bisexually and have probably developed from within a certain
group of oribatids which is known to reproduce exclusively by parthenogenesis. The idea has
been suggested (as the most parsimonious one) that the Acaridida developed bisexuality, and thus
sperm cells, secondarily [99, 100],
Mites demonstrating the most diverse reproductive behaviour are probably one of the most
promising groups to study the complex interrelationships between behavioural, morphological
and cytological evolutionary processes on a comparative basis in Chelicerata. Such a study almost
certainly would give further material for improving systematical concepts.
ACKNOWLEDGEMENTS
The author wishes to express his thanks to the numerous persons who assisted in many respects to make this study
possible. Among all these three played a major role: the late Prof. Dr. A. Remane (Kiel) as the impressive teacher of
animal morphology. Prof. Dr. R. Schuster (Graz, previously Kiel), who introduced the author to the exciting sciences of
Acarology and Arachnology, and Prof. Dr. V. Storch (Heidelberg, previously Kiel), who opened the door into the
fascinating world of comparative ultrastructure research.
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Source :
Spermatozoal Ultrastructure in
Dendrobranchiata (Crustacea, Decapoda):
Taxonomic and Phylogenetic Considerations
Antonio MEDINA
Departamento de Biologia Animal, Biologfa Vegetal y Ecologia,
Facultad de Ciencias del Mar, Universidad de Cadiz, Aptdo. 40, E-l 1510 Puerto Real, Cadiz, Spain
ABSTRACT
This chapter reviews the diverse spermatozoal patterns found to date in- Dendrobranchiata, including the first
ultrastructural descriptions of solenocerid and sergestid spermatozoa. Some characters are analysed from a spermiocladistic
perspective and a tentative phylogram is presented where the dendrobranchiate sperm forms are related to phyletic
arrangements inferred from holomorphological studies. Evolutionary relationships within Eucarida are discussed with
respect to the various sperm morphologies.
RESUME
Ultrastructure du spermatozoide chez les Dendrobranchiata (Crustacea, Decapoda): considerations
taxonomiques et phylogenetiques
Ce chapitre synthetise nos connaissances sur les differentes morphologies des spermatozoides rencontrecs jusqu’ici
chez les Dendrobranchiata, y compris les premieres descriptions ultrastructurales du spermatozoide d’un Solenoceridae et
d’un Sergestidae. Quelques caracteres sont analyses dans une perspective spermiocladistique et un essai de phylogramme
est presente, dans lequel les morphologies des spermatozoides des Dendrobranchiata sont mises en relation avec les
arrangements phylogeniques issus des etudes holomorphologiques. Les relations evolutives chez les Eucarides sont
discut6es en relation avec les differentes morphologies des spermatozoides.
The almost limitless morphological diversity of animal spermatozoa has inspired an
extensive amount of research work in diverse biological fields. Specifically, in recent years sperm
ultrastructure has become a useful tool in studies on taxonomy and phylogeny, since it proves
effective in resolving problems which escape conventional somatic analyses [25, 30]. The shape
and inner organization of a given sperm cell is definitely characteristic of the species that produces
it; that is, the spermatozoal ultrastructure constitutes a distinctive character of identity for every
animal species. Correspondingly, it is obvious that the evolution of a spermatozoon runs parallel
to the evolution of the corresponding species from which it comes. On this basis, an increasing
number of works have been conducted to establish a congruent relationship between the sperm
Medina, A., 1995. — Spermatozoal ullrastructure in Dendrobranchiata (Crustacea, Decapoda): taxonomic and
phylogenetic considerations. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 . 231-242. Paris ISBN : 2-85653-225-X.
232
A. MEDINA : DENDRUBRANCHIA TA I DECAPODA , CRUSTACEA)
ultrastruclure and the evolution and phylogeny of many animal taxa. JAMIESON [25] coined the
terms “spermiocladistics” and “spermiotaxonomy” in reference to the application of sperm
ultrastructure to phylogenetic and taxonomic studies.
The astonishing diversity of forms that animal spermatozoa can adopt is well exemplified by
the crustaceans, where a wide range of sperm types vary from the aquasperm-like (plesiomorphic)
remipedian spermatozoon, through the amoeboid and acrosome-less forms present in several taxa,
to the aflagellate, either “unistellate” or “multistellate”, decapod sperm [31]. The extreme
strangeness of these spermatozoa aroused the curiosity of early spermatologists. Light
microscopical reports on crustacean spermatozoa are relatively abundant and precocious, some of
them including phylogenetic analyses based on the sperm types of decapods (see review by
FELGENHAUER & ABELE [19]). With the development of electron microscopy, the opportunity to
examine in detail subcellular structures opened the range of possibilities to utilize sperm
morphology in reconstruction of crustacean phylogeny. Three recent reviews have painstakingly
updated the knowledge on the sperm ultrastructure in decapods [19, 31, 39]. These revisions
report the traditional tendency to classify the different decapod sperm morphologies into two
categories, following early classifications of the Decapoda into Natantia and Reptantia. Thus, a
supposedly uniform sperm plan, referred to as the “unistellate spermatozoon”, was thought to be
shared by the suborder Dendrobranchiata and the pleocyematan infraorder Caridea (formerly
grouped in the Natantia along with the Stenopodidea), the “multistellate spermatozoon” being
typical of the rest of the representatives of the suborder Pleocyemata (former Reptantia) [6], Most
recent studies, however, have provided additional information which recommends reconsideration
of this view. Actually, the general unistellate sperm pattern appears to encompass two
significantly distinct sperm structures [44], Furthermore, other sperm morphologies present in
species of Dendrobranchiata do not fit into either of the two traditional categories of decapod
spermatozoa ([13], MEDINA, other chapter in this book [45]).
Table 1. — List of Dendrobranchiata for which sperm ultrastructure is known
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
233
Spermatozoal ultrastructure is well known for numerous species of reptant crustaceans,
notably brachyurans. In a series of exemplary works, JAMIESON and co-workers [22, 27-29, 32-
34] (see also JAMIESON, this volume) have applied spermiocladistics in this group with
considerable success. However, the amount of information available on spermatozoal
ultrastructure in Dendrobranchiata is more limited and mostly deals with the genera Penaeus and
Sicyonia (Table 1). Wide gaps in the current knowledge of the ultrastructure of spermatozoa in
Dendrobranchiata render it impracticable to perform a complete parsimony analysis of the various
families and the phylogenetic relationships between them and with other decapod taxa.
Nevertheless, spermiocladistic criteria may well be applied to phylogeny and taxonomy of the
suborder. This chapter summarizes pre-existing information and contributes original
observations, tentatively aiming at establishing valid foundations for spermiotaxonomy and
spermiocladistics in the Dendrobranchiata that may be useful in forthcoming studies on this
crustacean taxon.
Dendrobranchiate spermatozoal patterns
Thus far, ultrastructural descriptions of spermatozoal morphologies are available for
representatives of three of the four families of the superfamily Penaeoidea: Penaeidae, Sicyonidae
and Aristeidae (see Table 1 for list of species investigated). This previous information is here
augmented with observations on the sperm of Solenocera membranacea as a representative of the
family Solenoceridae. A brief ultrastructural description of the sperm of Sergestes arcticus is also
provided in order to include a member of the superfamily Sergestoidea in support of general
phylogenetic considerations. In aspects of systematics, I will here follow the classification
proposed by BOWMAN & ABELE [5], whereas the spelling of taxa, and particularly that of names
derived from Pen(a)eus , will be the one used and recommended by SCHRAM [59].
Family Penaeidae. Within the Dendrobranchiata, penaeid sperm have been the most
extensively studied in terms of the number of species examined. These include five Penaeus
species (P. setiferus, P. vannamei, P. aztecus, P. japonicus and P. kerathurus), Trachypeneus
similis, Parapeneus longirostris and Peneopsis serrata (Table 1). In gross morphology, the peneid
spermatozoon basically consists of a subspheroidal or ovoid main body and a spike. The main
body comprises the central nuclear region, a cytoplasmic band surrounding it posterolaterally, and
the acrosomal cap, which overlies the nuclear region anteriorly and is prolonged into a tapering
spike (Fig. la, b). Both spike and acrosomal cap make up a membrane-bound acrosomal vesicle,
with heterogeneous contents, which is directly invested by the plasma membrane. In particular,
the spike morphology and substructure vary markedly from species to species. The whole
acrosomal complex is completed with the subacrosomal region, which is quite simple in this
family, merely containing a sparse flocculent material between the chromatin and acrosomal cap.
The sperm of Parapeneus longirostris and Peneopsis serrata have a central protuberance at
the concave side of the acrosomal cap immediately opposite the spike (Figs lb, 2h, i). This
supposed synapomorphy is consistent with the close phylogenetic proximity of the genera
Parapeneus and Peneopsis, both grouped together by BURKENROAD [8] within the tribe
Parapeneini, which also includes Artemesia and Metapeneopsis. Confirmation of such a structure
in the latter genera would strengthen phylogenetic unity of this taxon.
As in all dendrobranchiate species whose spermatozoon has been ultrastructurally studied,
the nuclear region of peneid sperm consists of a non-membrane-bound, filamentous chromatin
mass. Posterolaterally, the chromatin is surrounded by a band of cytoplasm which contains
membrane lamellae, vesicles and mitochondria-like bodies, but lacks centrioles and microtubules.
Within the Dendrobranchiata, the sperm of Penaeus japonicus are exceptional in that they exhibit
several microtubule bundles in the cytoplasm [48], The microtubules appear in primary
spermatocytes of P. japonicus and are retained through spermiogenesis to the mature
234
A. MEDINA : DENDROBRA NCHIA TA (DECAPODA. CRUSTACEA)
spermatozoon (personal observation). In other peneid species (e.g. Penaeus kerathurus,
Parapeneus longirostris), microtubules are absent from all spermatogenetic stages.
Recent molecular studies [5 1 ] have revealed extensive genetic differences between species
of Penaeus which have not been accompanied by substantial evolutionary morphological changes.
This is congruent with the occurrence of diverse species-specific dissimilarities leading to
different ultrastructure of sperm in the genus Penaeus and in general in the Penaeidae, and
confirms the taxonomic validity of the sperm morphology in the Dendrobranchiata.
Family Sicyonidae. Ultrastructural data have been reported for three Sicyonia species: S.
brevirostris, S. carinata and S. ingentis (Table 1). The inner morphological organization of the
spermatozoon is very similar in S. ingentis [37] and S. carinata [47]. In general, as in Penaeidae,
the sperm consist of an acrosomal vesicle (formed by the spike and acrosomal cap), subacrosomal
region, and nuclear region surrounded by a cytoplasmic band (Fig. lc). Anteriorly, the acrosome
and plasma membranes are closely joined. As a taxonomically significant difference, the spike of
S. ingentis is spiralled, whereas that of S. carinata is smooth. The plesiomorphies (1) absence ot
nuclear envelope and (2) perinuclear cytoplasmic band (containing small and large vesicles and
lacking microtubules) are also found in this sperm type. Nevertheless, the highly elaborate
subacrosomal region (comprising diverse distinct structures) [37, 47] appear to be a clear
autapomorphy of the family Sicyonidae. Compared to the spermatozoa ol the other
dendrobranchiate families, the acrosomal vesicle shows the apomorphic character that the
posterior membrane of the acrosomal cap is intricately folded in a ring of convoluted membrane
pouches or digitations [37, 47] (Fig. lc).
In a long series of valuable works, CLARK and co-workers have described morphological
details of the acrosome reaction in S. ingentis [10-12, 20, 21, 24, 62], These accounts reveal the
role played by each of the spermatozoal components during fertilization, hence they greatly aid
understanding of the biological significance of the acrosomal structures in dendrobranchiates.
Family Aristeidae. The relatively high ultrastructural homogeneity found within the
Penaeidae and Sicyonidae is not seen in the Aristeidae. Studies of Aristeus antennatus [13, 14,
45] and Aristaeomorpha foliacea [45] indicate the existence in the family of at least two different
ultrastructural sperm plans that are in turn discordant with the peneid-sicyoniid assemblage. The
A. antennatus (Figs Id, 2c) sperm type exhibits diverse peculiarities in comparison with the other
Dendrobranchiata. First, its spherical acrosome does not cap the nuclear region and lacks both
spike and subacrosomal region; the inner arrangement of the acrosomal contents is complex and
different from that of any other known dendrobranchiate spermatozoon. Secondly, the cytoplasm
does not constitute a band around the filamentous chromatin mass, but is accumulated in a collar
between the acrosome and nuclear region, enclosing mitochondria-like vesicles and membrane
lamellae. Consequently, most of the chromatin is bounded directly by the plasma membrane,
since the nuclear region is, as in all dendrobranchiates, not membrane-bound. I agree with
DEMESTRE & FORTUNO [13] that the basic sperm structure of A. antennatus resembles that of
spiny lobsters, Panulirus spp. [61], although with the highly significant absence of the typically
reptantian radial arms, which suggests parallelism rather than a close phylogenetic relationship.
The Aristaeomorpha foliacea sperm type (Figs le, 2b), lacking the acrosome, also differs
from the dendrobranchiate unistellate spermatozoal morphology. It consists of a central nuclear
region entirely surrounded by the plesiomorphic cytoplasmic band, which includes membrane
lamellae, small peripheral vesicles and mitochondria-like bodies. Plesiomorphic features are also
the absences of nuclear envelope, centrioles and microtubules.
Fig. 1. — Transmission electron micrographs of spermatozoa, a: Penaeus japonicus: b: Parapeneus longirostris, c:
Sicyonia carinata ; d: Aristeus antennatus ; e: Aristaeomorpha foliacea ; f: Solenocera memhranacea ; g: Sergestes
arcticus. Scale bars: 1 pm. a: acrosome, ac: acrosomal cap, c: cytoplasm, m: mitochondria-like bodies, n: nuclear
region, p: protuberance of the acrosomal cap, s: spike, *: subacrosomal region.
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
235
Source : MNHN. Paris
236
A. MEDINA : DENDROBRA NCHIATA (DECAPODA, CRUSTACEA)
Family Solenoceridae. The spermatozoon of Solenocera membranacea is similar to that of
Penaeidae in general morphology (Figs If, 2d), though it shows conspicuous differences with
regard to the other spiked dendrobranchiate sperm. The contents of the acrosomal vesicle are
homogeneously electron-dense and the cap appears asymmetrical in sagittal sections, one of its
lateral expansions projecting further than the other. Another distinctive feature of the
5. membranacea spermatozoon is that the plasma membrane becomes separated from the anterior
acrosome membrane, the intervening space being occupied by part of the cytoplasmic mass. The
perinuclear cytoplasm is rather amorphous, though parallel lamellae and mitochondria-like bodies
may be recognized. It is thick under the lateral edges of the acrosomal cap and grows thinner at
the posterior part of the sperm. Anteriorly, it forms a thin band separating the scarce
subacrosomal substance from the finely filamentous chromatin, a feature that recalls that observed
in the penaeid Parapeneus longirostris [44],
Family Sergestidae. The sperm of Sergestes arcticus are simple, spheroidal or slightly
ellipsoidal cells which much resemble those found in Aristaeomorphafoliacea. They consist of a
central, non-membrane bound nuclear region and surrounding cytoplasm (Figs lg, 2a). The
finely filamentous chromatin mass is encircled by a thin cytoplasmic band that mainly contains
densely-packed electron-clear vesicles and a few mitochondria-like bodies. Occasionally, the
cytoplasm encloses lipid-like, highly osmiophilic inclusions. At some points, the cytoplasmic
band may be interrupted, thus allowing a direct contact of the nucleoplasm with the plasma
membrane. Acrosome, microtubules and centrioles are absent.
In eucarids absence of the acrosome had been reported only in Euphausiacea [31] and
Stenopodidea [19]. Indeed, there appear to be striking resemblances between the spermatozoa of
Sergestes arcticus, Aristaeomorpha foliacea (Fig. le, g) and Euphausia sp. (see [31]) which very
probably are indicative of phylogenetic relationship. These are: (1) central nuclear region
consisting of diffuse, finely filamentous chromatin, (2) complete disruption of the nuclear
envelope, (3) vesiculate, thin perinuclear cytoplasmic band, (4) absence of centrioles and
microtubules, and (5) absence of acrosome. Now the question arises as to the evolutionary
meaning of acrosome-less spermatozoa within Dendrobranchiata. Has this condition been
acquired secondarily or is it a primitive one? This subject is discussed below.
Sperm phylogenetic relationships within Dendrobranchiata and between Dendrobranchiata and
other Eucarida
The present survey suggests as clear dendrobranchiate spermatozoal symplesiomorphies:
(1) complete loss of the nuclear envelope, (2) filamentous chromatin, (3) absence of centrioles,
(4) absence of radial (stellate) arms. The plesiomorphic perinuclear distribution of the cytoplasm
does not occur in Aristeus antennatus ; in this species, the cytoplasm forms a collar between the
acrosome and nuclear region. Whether the acrosome-less condition of Aristaeomorpha foliacea is
an apomorphic character or, in contrast, a plesiomorphy, is a matter that remains to be established
when more data are available. Nonetheless, the finding of similar, acrosome-less sperm patterns
in euphausiids ( Euphausia sp.) [31], stenopids ( Stenopus hispidus) [19], sergestids ( Sergestes
arcticus ) and aristeids {Aristaeomorpha foliacea) appears to point to its plesiomorphy. Although
the loss of the acrosome is a repeated event throughout evolution of the crustacean sperm [26], the
assumption of sperm originally endowed with an acrosome would suppose the highly improbable
independent loss of the acrosome in several separate lineages of the eucarid tree (Fig. 2).
According to JAMESON [31], "the malacostracan acrosome is a new development, in view
of evidence that their acrosome originates from the endoplasmic reticulum and not, as is usual,
from the Golgi." Certainly, several studies have demonstrated that the acrosomal structures in
Decapoda derive from, or in association with, cistemae of the endoplasmic reticulum itself or of
its specialized portion constituting the nuclear envelope [1-3, 14, 23, 35, 38, 40, 43, 44, 46, 49,
53, 55, 56, 58, 60]. Consequently, it can be said that the mechanisms involved in differentiation
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
237
of the acrosomal structures are somehow plesiomorphic. It is not known whether similar
mechanisms take place during spermiogenesis in euphausiids, sergestids, stenopids and
acrosome-less aristeids (as they do in fact in Aristeus antennatus). If so, it is to be assumed that
the capacity to build acrosomes with the involvement of endoplasmic reticulum membrane
systems (irrespective of the appearance or not of a distinct acrosome in mature sperm) was present
in ancestors of eucarids before separation of euphausiids and decapods. Such a widely shared
mechanism of acrosome formation is consistent with the occurrence of apparent homologies in the
acrosomes of as distant taxa as the Penaeidae and the Brachyura [46]. In contrast, the caridean
spike, in spite of resulting in a sperm pattern closely resembling the peneid-sicyoniid-solenocerid
one, is not to be phylogenetically related to the dendrobranchiate spike [44], Definitely,
comparative sperm ultrastructural studies argue against a monophyletic “Natantia”, as
BURKENROAD [8] conjectured more than a decade ago.
If, as appears plausible (see above), the absence of an acrosome is plesiomorphic, then the
spiked acrosome of dendrobranchiates would be a synapomorphy of the families Penaeidae,
Sicyonidae and Solenoceridae, whereas the sperm of the Sergestidae and Aristeidae should be
considered as more primitive, that of Aristeus antennatus showing secondary (thus apomorphic)
acquisition of the acrosome independent of the evolutionary line leading to the other acrosome -
possessing dendrobranchiate spermatozoa. Taking into account the report, albeit requiring
confirmation, that euphausiid spermatozoa [31] are similar to those of Sergestes arcticus and
Aristaeomorpha foliacea (Figs le, g, 2a, b), occurrence of a plesiomorphic acrosome-less sperm
is congruent with the statement of BURKENROAD [8] that the ancestors of the Decapoda were
more euphausiid-like than the modem forms. According to this, the primitive eucarids could have
euphausiid-like sperm, euphausiids, sergestids, aristeids and stenopids having retained this
pattern. Among Aristeidae, some representatives (A. antennatus) might well have recreated a
spheroidal acrosome with no ultrastructural resemblance to the acrosome of any of the other
known dendrobranchiates, the sperm becoming arranged into a reptant-like pattern (although
retaining the plesiomorphic absences of anus, microtubules and nuclear envelope, and therefore
with no apparent direct phylogenetic relation to reptants) which represents an independent
evolutionary line (see Fig. 2).
Spermiocladistic support for the statement of FELGENHAUER & ABELE [17] that the Caridea
and Stenopodidea derive from ancestral reptants would necessitate further research. Derivation of
carids from primitive thalassinoids is not congruent with most recent observations on
spermatozoal ultrastructure by TUDGE [this volume], unless important deviations
(= apomorphies) from the reptant ground plan be assumed, namely the loss of the membrane-
bound acrosome and of microtubule-containing radial arms, as well as the independent
development of a non-membrane bound spike [2, 3, 16, 18, 38, 42, 52, 54, 56, 57] that acts in a
distinct and very particular manner during fertilization [4, 41]. These typically caridean
characteristics confirm a sperm pattern that represents a fairly distinct, clearly identifiable
evolutionary trend within the Decapoda.
The occurrence of either a complete or a discontinuous double-membrane nuclear envelope,
partially invested by the plasma membrane, as well as the occasional presence of centrioles at the
base of the acrosome, are shared by carids and reptantians, these features supporting a certain
unity of both groups. However, the supposed reptantian origins of stenopodideans [17] are
disputed by the ellipsoidal, arm-less and acrosome-less form of the spermatozoon of Stenopus
hispidus [19], which is also characterized by having a lamellar body located against the plasma
membrane at one side of the sperm cell (a structure that strongly suggests reminiscence of the well
developed membrane system associated with proacrosomal vesicles in decapods). At first glance,
this sperm morphology would place the stenopodideans close to the euphausiids, hence
suggesting an early separation of Stenopodidea from the reptantian-caridean stem just above the
origin of the Dendrobranchiata and before appearance of the acrosome and of appendages in
decapod spermatozoa (see Fig. 2).
238
A. MEDINA : DENDROBRANCHIATA ( DECAPODA . CRUSTACEA)
PARAPENEINI
h Panaopa/t aarrala
i Parapanaua k>ntfroatna
Panaaus kiathurus
c antral pfOtuba»anca
f Panaaui / apomcus
a SKyoma cannata
SICYONIDAE
PENAEIDAE
complex aubacroaoma
I Uca tangan
REPTANTIA
STENOPODIDEA'’
d Solanocara mambranacaa
SOLENOCERIDAE
cytopaam bayond
acrosomal cap
► Palaamon aanatus
CARIDEA
non-mamtorana bound sptka
ARISTEIDAE
c Ana taut artannatu s
I Stanopoa Naprtua
STENOPODIOEA *>
b Analaaomorpfia fohacaa
mamWarw -bound sptka
PLEOCYEMATA
PENAEOIDEA
a Sargasras arcocus
SEROESTIDAE
DENDROBRANCHIATA
EUPHAUSIACEA
DECAPODA
EUCARIDA
acroaoma-iaaa aparm?
Fig. 2. — Intuitive phylogenetic tree of the sperm of the Eucarida where the different dendrobranchiate spermatozoal
patterns are represented. Figures b, c, e-k have been prepared from drawings or micrographs which appeared in [19.
44-48, 52]; a and d are original. Scale bar: 8 pm for k, 4 pm for the others.
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
239
Figure 2 represents a tentative phylogenetic tree which attempts to reconcile the current
knowledge of spermatozoal ultrastructure in eucarids with phyletic relationships suggested
recently by reputable authors [7, 17, 26, 31, 36, 59]. Although the phylogram is necessarily
provisional owing to the limited number of studies available, the most important spermatozoal
evolutionary trends are represented in it. In the proposed sperm phylogram, separation of the
Euphausiacea is followed by a node grouping the Decapoda, with two distinct evolutionary lines,
one of which leads to Dendrobranchiata and the other to Pleocyemata. It is believed that the
decapod sperm were originally devoid of an acrosome, a condition that was retained in
Sergestidae as well as representatives of the family Aristeidae (Aristaeomorpha foliacea).
However, another aristeid ( Aristeus antennatus ) has a spermatozoon supplied with an apomorphic
membrane-bound acrosome that resembles the sperm of Panulirus spp. owing to concurrence of
several parallelisms rather than to phylogenetically-based shared features. The three other families
of the Dendrobranchiata have in common spermatozoa which share a synapomorphic membrane-
bound acrosomal spike. From the node uniting these non-aristeid sperm, the first branch to
emerge is represented by the spermatozoon of Solenocera membranacea, which shows an
asymmetrical acrosomal cap and separation of the plasma and anterior acrosome membranes,
allowing part of the cytoplasm to “leak” beyond the acrosomal cap. Finally, Sicyonidae and
Penaeidae appear as two aligned groups, the sperm of which are easily distinguishable by the
highly complicated, apomorphic subacrosomal region present in sicyoniids, in contrast to the
simple one of penaeids. In the Penaeidae, two distinct sperm types have been recognized on the
basis of the presence ( Parapeneus longirostris and Peneopsis serrata) or absence ( Penaeus spp.)
of a central protuberance at the concave side of the acrosomal cap. This dendrobranchiate sperm
phylogenetic arrangement is in agreement with the close interrelation that Burkenroad [8] suggests
between penaeids and sicyoniids. However, with our limited information, no spermatozoal
evidence has been found to ally, as he claims, aristeids and solenocerids. On the contrary, the
spermatozoon of S. membranacea resembles the Penaeidae-Sicyonidae sperm rather than any of
the known Aristeidae sperm types.
The Pleocyemata lineage (Fig. 2) would first include acrosome-less sperm forms, such as
those present in Stenopus hispidus. Therefore, a logical phylogenetic sequence would suggest a
first offshoot leading to Stenopodidea in a scheme that is congruent with the phylogram of
FELGENHAUER & ABELE [17]. However, another spermatologically plausible, albeit less
probable, arrangement following the more recent cladograms of SCHRAM [59] and KIM & ABELE
[36], would place the offshoot of Stenopodidea between the branches leading to Caridea and
Reptantia.
ACKNOWLEDGEMENTS
This work was partially supported by grant AGF93-0173 of the CICYT. I wish to express my sinccrest thanks to
Professor B.G.M. Jamieson for the invitation to prepare this paper and for editorial and scientific help. I am also indebted
to many people who lent valuable support in various ways: Dr. Antonio Rodriguez, 1. L6pezde la Rosa and S. Gonzalez
(Instituto de Ciencias Marinas de Andalucia, CSIC), Dr. G. Mourente and A. Santos (Facultad de Ciencias del Mar, UCA),
I. Sobrino, M.P. Jimenez G6mez and F. Ramos (Instituto Espanol de Oceanograffa, Cadiz), Dr. Terry Gosliner (California
Academy of Sciences), and the staff of the Servicio de Informdtica (UCA) and Servicio de Microscopia Electronica (UCA).
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Source : MNHN, Paris
The Atypical Sperm Morphologies of
Aristeus antennatus and Aristae omorpha foliacea
(Crustacea, Dendrobranchiata, Aristeidae)
and Their Phylogenetic Significance
Antonio MEDINA
Departamento de Biologfa Animal, Biologfa Vegetal y Ecologfa,
Facultad de Ciencias del Mar, Universidad de Cadiz, Aptdo. 40, H- 11510 Puerto Real, Cadiz, Spain
ABSTRACT
The spermatozoa of Aristeus antennatus and Aristaeomorpha foliacea show two distinct morphological patterns which
greatly differ from each other and are also distant from the common dendrobranchiate sperm plan. In A. antennatus , the
spermatozoon possesses a conspicuous spherical acrosome which lacks both spike and subacrosomal region. Its
multilayered inner structure is not comparable to the acrosomal arrangement of any other decapod, and is postulated to be
the result of independent evolution. The cytoplasm is concentrated in a collar between the acrosome and the non¬
membrane bound nuclear material; therefore, it displays a subacrosomal (apomorphic) rather than perinuclear distribution.
In spite of the plesiomorphic lack of radial arms, which is common to all dendrobranchiates, the general morphology of
this spermatozoon resembles that of reptant decapods in several respects. The absence of a spike, the spherical (not
capping) acrosome, and the subacrosomal (instead of perinuclear) cytoplasm are considered as apomorphics. The most
significant feature of Aristaeomorpha foliacea spermatozoon is the absence of the acrosome. This simple sperm cell
consists of the central nuclear material encompassed by a cytoplasmic band that is in turn bounded by the plasma
membrane. Typical dendrobranchiate (plesiomorphic) features of A. foliacea sperm are the perinuclear cytoplasm
(containing small peripheral vesicles, mitochondria and membrane lamellae), the non-membrane bound nuclear material,
and the absences of centrioles and radial arms. The two spermatozoa! patterns described hereafter appear to be the result of
distinct evolutionary trends.
RESUME
Les morphologies atypiques des spermatozoides d 'Aristeus antennatus et Aristaeomorpha foliacea
(Crustacea, Dendrobranchiata, Aristeidae) et Ieur signification phylogenetique.
Les spermatozoides de Aristeus antennatus el Aristaeomorpha foliacea possfcdent des morphologies bien distinctes entre
elles et differentes de la morphologic habituelle des Dendrobranchiata. Le spermatozoidc d’A. antennatus possede un
important acrosome spherique depourvu d’epine et de region subacrosomienne. Sa structure interne a plusieurs couches
n’est comparable & l’acrosome d’aucun autre D6capode, et on fait l’hypoth&se qu'elle est le r6sultat d’une evolution
ind6pendante. Le cytoplasme est concentre en un collier entre l’acrosome et le materiel nucleaire non limits par une
membrane. De ce fait, il montre une distribution subacrosomienne (apomorphe) plutot que perinucleaire. En depit de
I’ absence, plesiomorphe, de bras radiaux, qui est commune h tous les Dendrobranchiata, la morphologie g«§n<§rale de ce
spermatozoidc ressemble h celle des Decapodcs Reptantia pour plusieurs aspects. L’absence d’epines, l’acrosome spherique
(pas en capuchon), et le cytoplasme subacrosomien (au lieu de perinucleaire) sont considers comme des apomorphies. La
Medina, A., 1995. — The atypical sperm morphologies of Aristeus antennatus and Aristaeomorpha foliacea
(Crustacea, Dendrobranchiata, Aristeidae) and their phylogenetic significance. In: Jamieson, B. G. M., Ausio, J., &
Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist. nat.. 166 : 243-250.
Paris ISBN : 2-85653-225-X.
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A. MEDINA : ARISTEIDAE (DECAPODA, CRUSTACEA)
caracteristique la plus significative du spermatozoide d'Aristaeomorpha foliacea est F absence d’acrosome. Ce
spermatozoide simple consiste en un materiel nuclcaire central entoure par une bande cytoplasmique, elle-meme limitee par
la membrane plasmique. Les caracteristiques (plesiomorphes) de Dendrobranchiata du spermatozoide d A. foliacea sont un
cytoplasme perinucldaire (contenant des petites vesicules peripheriques, des mitochondries et des lamelles membranaires),
le materiel nucleaire non limite par une membrane, et I’absence de centrioles et de bras radiaux. Les deux morphologies de
spermatozoide decrites ici semblent etre le resultat de deux tendances evolutives distinctes.
The spermatozoa of dendrobranchiate shrimp are typically described as aflagellate cells
consisting of a rounded main body partially encompassed by a cap with a protruding spike [5, 9,
11], The most prominent feature in this sperm type is the presence of a single appendage, the so-
called spike, which characterizes the “unistellate” condition of "natantian spermatozoa,
distinguishing it from the “multistellate” condition of “reptantian” sperm. However, the spike-less
sperm morphologies of Aristeus antennatus and Aristaeomorpha foliacea provide exceptions to the
supposed spermatozoal uniformity in the Dendrobranchiata and recommend a thorough analysis
of the sperm ultrastructure of both species of Aristeidae. Though DEMESTRE & FORTUNO [3]
briefly described the spike-less spermatozoon of A. antennatus from scanning electron
micrographs, an accurate characterization requires further investigation by means of transmission
electron microscopy. Phylogenetic implications of both dendrobranchiate sperm morphologies
will be considered in the context of the decapod sperm configuration.
MATERIALS AND METHODS
Specimens of Aristeus antennatus (Risso, 1816) and Aristaeomorpha foliacea (Risso, 1827) were collected off the
Gulf of Cadiz in October 1993. Small fragments of vasa deferentia including ampullae were fixed in 2.5% glutaraldehyde in
filtered seawater for 4-12 h, postfixed in 1% osmium tetroxide and dehydrated through acetones. For transmission electron
microscopy, the samples were then infiltrated and embedded in ERL [22]. Ultrathin sections were viewed in a JEOL EX
1200 electron microscope operated at 80 kV.
For scanning electron microscopy, following dehydration, the samples were critical-point dried, mounted on
copper stubs and sputter-coated with gold. Observations were made on a JEOL JSM 820 electron microscope operating at
20 kV.
RESULTS
Spermatozoon of Aristeus antennatus
The spermatozoon of Aristeus antennatus basically comprises a conspicuous and prominent
sperical acrosome and the nuclear structure (Figs 1 a, b, 3a), the cytoplasmic mass being fairly
reduced. The acrosome has a smooth surface, whereas the region containing the nucleus is more
irregular in profile (Fig. la, b). The whole cell is 5.23 ± 0.33 pm (n = 16) in length. The
acrosome measures 2.30 ±0.16 pm (n = 19) in diameter and the nuclear region 3.02 ± 0.25 pm
(n = 16) in width.
Fig. 1. — Scanning (A) and transmission (B-J) electron micrographs of Aristeus antennatus spermatozoa.
A: Spermatozoa in the ampulla. The acrosome (arrows), cytoplasmic collar (arrowheads) and nuclear region
(double arrows) are distinguished. B: Sagittal section of spermatozoon. C-E: Longitudinal sections of acrosomes
showing the complex arrangement of their contents consisting of three distinct layers that correspond to the
peripheral layer (1) of small clear vesicles (arrowheads), the marbled-like subperipheral zone (2), and the central
region of concentrically arranged material (3). At the innermost region is a clear area (4) that usually appears
associated with a dense osmiophilic substance (asterisk). F: Tangential section of acrosome through the
peripheral vesicle layer. G, H: Sagittal (G) and oblique (H) sections of cytoplasmic collars containing
mitochondria and membrane lamellae in continuity with osmiophilic dense lamellae. I: The nuclear region of a
spermatozoon in sagittal section.. Dense bodies are embedded in the nucleoplasm. .1: Detail of dense bodies
showing myelin-like structure, a, acrosome; c, cytoplasmic collar; db, dense bodies; dl, dense lamellae; m,
mitochondria; ml, membrane lamellae; n, nuclear region.
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A. MEDINA : AR1STEIDAE (DECAPODA, CRUSTACEA)
The acrosome is formed by a spherical acrosomal vesicle with structurally complex
contents. No distinct subacrosomal region is found. The inner arrangement of the acrosome is
symmetrical, since both longitudinal and cross sections show equivalent structural patterns. At the
periphery is a single layer of electron-lucent vesicular structures which appear to originate from
small invaginations of the acrosome membrane (Fig. lc-e). Tangential sections show them to be
regularly sized (about 80 nm in diameter) and densely packed (Fig. 10- Immediately under the
vesiculate layer, the acrosome materials take on a marbled appearance (Fig.lc, d). The central
region of the acrosome is filled with a material arranged in concentric rows of apparently hollow
spherical particles (Fig. lc, d). In many sections, an electron-clear area is observed at the
innermost region of the acrosome. It may be centrally located (Fig. lc) or eccentric (Fig. Id). A
dense material is usually associated with this electron-clear zone (Fig. lc).
The cytoplasm is reduced to a collar intervening between the acrosome and nucleus which
extends laterally over the anterior third of the nuclear region (Figs la, b, 3a). The central portion
of the cytoplasmic collar shows no noticeable substructure. In contrast, the lateral expansion
contains parallel membrane arrays, osmiophilic lamellae and clusters of mitochondria (Fig. lb, g,
h). In favourable sections, osmiophilic lamellae are seen to be in continuity with membrane
lamellae (Fig. lh). Both elements are orientated in the direction of the lateral expansion of the
cytoplasmic mass. . .
The nuclear region contains a non-membrane bound network of chromatin fibres (Figs lb,
h-j, 3a). Anteriorly, the chromatin is surrounded by the cytoplasm, and laterally and posteriorly
solely by the plasma membrane. Many fibres appear inserted in the cytoplasmic mass, becoming
orientated longitudinally (Fig. lb, g). Dense bodies and occasional vesicles are observed in the
nucleoplasm (Fig. li, j). The dense bodies are of membrane origin, since they sometimes show a
myelin-like structure (Fig. lj).
Spermatozoon of Aristaeomorpha foliacea
The sperm of Aristaeomorpha foliacea are roundish or slightly ellipsoid cells measuring
5.66 ± 0.79 |im (n = 33) at their widest point. Puzzingly, this dendrobranchiate sperm type does
not exhibit any recognizable acrosomal structure (Figs 2a, b, 3b). Thus, the spermatozoon simply
consists of the nuclear region and cytoplasm (Fig. 2c). The cytoplasm forms a peripheral band
around the nucleoplasm and is bounded by the plasma membrane. The thickness of such a
cytoplasmic band is irregular throughout the cell (Fig. 2b), sometimes having discontinuities in
which the nucleoplasm is in direct contact with the plasma membrane. The main cytoplasmic
elements are electron-lucent vesicles, mitochondria and myelin-like bodies (Fig. 2c-f). The
cytoplasmic background mainly consists of granular material and anastomosing membranes (Fig.
2e). Numerous vesicles about 0.3 pm in size occur at the very periphery of the cytoplasmic band,
often causing slight bumps in the sperm surface (Fig. 2c). As a result of this, the vesicle and
plasma membranes are tightly apposed at the zone of contact of both (Fig. 2d). The peripheral
clear vesicles contain a moderately electron-dense substance which either accumulates in a small
core or forms a thin layer on the inner side of their membrane (Fig. 2d). Mitochondria occur in
numbers of approximately 1-3 per section. They show randomly distributed cristae (Fig. 2 e, f).
Fig. 2. — Scanning (A) and transmission (B-J) electron micrographs of Aristaeomorpha foliacea spermatozoa. A, B:
Sperm mass in the ampulla. C: Radial section of spermatozoon showing the peripheral cytoplasmic band and
central nuclear region. D-F: Diverse details of peripheral cytoplasm to show the main cytoplasmic elements:
peripheral electron-clear vesicles, mitochondria, dense myelin-like bodies and anastomosing membranes
(asterisk). G-I: Myelin-like bodies appear to be released from the cytoplasm and occasionally may occur either out
of the cell (I) or in the nucleoplasm (G). J: The nucleoplasm frequently shows nodules (asterisks) where the
chromatin fibres become densely entangled, c; perinuclear cytoplasm; m, mitochondria; mb, myelin-like bodies;
n, nuclear region; v, peripheral vesicles.
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A. MEDINA : ARISTEIDAE (DECAPODA, CRUSTACEA)
Fig. 3. — A: Spermatozoon of Aristeus antennaius. B: Spermatozoon of Aristaeomorpha foliacea. Drawn from
transmission electron micrographs, a, acrosome; cc, cytoplasmic collar; db, dense bodies; m, mitochondria; mb,
myelin-like bodies; n. nuclear region; pc, perinuclear cytoplasm; v, peripheral vesicles.
Myelin-like bodies apparently originate by coalescence of waste cytoplasmic membranes into
concentric layers. Frequently, membrane layers become deposited around (Fig. 2e) or constitute
dense bodies in close association with mitochondria (Fig. 2e, f). Isolated myelin-like bodies can
be encountered not only in the cytoplasmic band, but also within the nucleoplasm (Fig. 2g). Some
images suggest that at least some of them are extruded at the cell periphery (Fig. 2g-i).
As typical for all dendrobranchiates, the chromatin of A. foliacea sperm shows an
uncondensed reticulate pattern and is not bounded by a nuclear envelope. It is characteristic that in
this species nodules where chromatin fibres become more densely entangled are present (Fig. 2j).
DISCUSSION
The sperm morphologies of the Aristeidae Aristeus antennatus and Aristaeomorpha foliacea
are markedly inconsistent with the dendrobranchiate unistellate sperm plan. While the unistellate
spermatozoa of most dendrobranchiates consist of a main body housing the nucleus and an
acrosomal complex formed by cap region and prolonging spike (see reviews by FELGENHAUER &
ABELE [5], JAMIESON [9], KROL et al. [11]), the sperm of A. antennatus have a spike-less
acrosome and those of A. foliacea lack any acrosomal structure at all.
Unlike the other studied dendrobranchiates, the spermatozoa of Aristeus antennatus lack a
perinuclear cytoplasmic band, so that the nucleoplasm is bounded by only the plasma membrane
in most of the nuclear profile. In gross morphology, these features liken the A. antennatus
spermatozoon to that of reptant Pleocyemata, in particular Anomura [7, 8, 18, 25, 26] and
Astacidea [2, 19, 24], in which the acrosome is prominent and lies fairly apart from the posterior
nuclear region, with the cytoplasmic region located in between. Albeit these features are common
to A. antennatus sperm, the spherical acrosome of the latter has no basal invagination as does the
acrosomal vesicles in the above-noted reptants. In such a respect, this spermatozoon would be
similar to that of the Palinuridae [13, 23]. Nevertheless, in spite of the several coincidences, there
exist notable dissimilarities with regard to the general reptant sperm morphology, such as the
absences of nuclear envelope and nuclear arms or the different substructure of the acrosome.
From the above comments, it is concluded that the spermatozoon of Aristeus antennatus
simultaneously blends typically dendrobranchiate and typically reptantian characteristics, whereby
its placement into one of the two traditionally established decapod sperm categories becomes
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249
difficult. Available ultrastructural descriptions of spermatids in this species [4] reveal a
mechanism of acrosome formation resembling the one observed in many species of Pleocyemata
[1, 6, 10, 12, 13, 15-18, 20]. However, it slightly differs from that described for the only two
other Dendrobranchiata in which spermiogenesis has been ultrastructurally investigated, Sicyonia
ingentis (see [21]) and Parapeneus longirostris (see [14]). Actually, the complex ultrastructural
arrangement of the acrosomal vesicle contents in A. antennatus mature sperm is not comparable
with that of other decapods. The absence of either a spike or any other significant structure at the
place where the spike should supposedly lie, as well as the spherical (not capping) acosome with
varied contents, are to be considered as clear autapomorphies within the Dendrobranchiata.
Another assumed apomorphic character is the subacrosomal (instead of the plesiomorphic
perinuclear) cytoplasm.
The most significant feature of the ellipsoid, appendage-less sperm of Aristaeomorpha
foliacea is the amazing absence of any kind of acrosomal structure, which is unique in the
Dendrobranchiata. Despite thorough examination of spermatozoa from both vasa deferentia from
males and seminal receptacles from females, no trace of any structure that may be related to an
acrosome was found. Hitherto, acrosome lack in Eumalacostraca was only known in
Euphausiacea [9] and in Decapoda Stenopodidea [5]. As up to dat c A. foliacea is the only known
case of dendrobranchiate sperm lacking the acrosome, one would be inclined to infer that this
pattern has been reached by simplification from a peneoid-like (spiked) spermatozoon through
evolutionary loss of the acrosome. Nevertheless, the findings of acrosome-less spermatozoa
resembling the one of A. foliacea in Euphausiacea [9], Stenopodidea [5] and Sergestoidea [see
Medina, this volume] suggest that the absence of the acrosome would be a primitive condition in
Eucarida. Therefore, if the absence of the acrosome is plesiomorphic, the acrosome of Aristeus
antennatus sperm represents a secondary acquisition, hence an apomorphy, within the Aristeidae.
On the other hand, the spiked acrosome would be a synapomorphy of Penaeidae and Sicyonidae.
Well-known spermatozoal symplesiomorphic features of the Decapoda are the diffuse
chromatin, partial or total disruption of the nuclear envelope and absence of flagellum. In
addition, the present study confirms as clear plesiomorphies in Dendrobranchiata: a) non¬
membrane bound nuclear region, b) filamentous chromatin, c) perinuclear cytoplasm, d) absence
of centrioles and radial arms. The reduced subacrosomal distribution of the cytoplasm in Aristeus
antennatus should thus be considered as an autapomorphy. In conclusion, two distinct
evolutionary trends appear to have occurred in Aristeidae, leading to sperm morphologies which
are distant from the dendrobranchiate sperm plan exemplified by the assemblage Penaeidae-
Sicyonidae. This suggests that, although ultrastructural studies on crustacean spermatozoa are
generally useful for phylogenetic approaches, one must be cautious in certain instances, since
notable variation may occur in close taxa [5].
ACKNOWLEDGEMENTS
I wish to thank Ignacio Sobrino (Instituto Espanol de Occanografia [IEO] at Cadiz)
Rosa for collecting the specimens of Aristeus antennatus and Aristaeomorpha foliacea.
Aliseda, Juan Gonzalez and Chema GeraldIa (Servicio de Microscopia Electrdnica de
technical assistance. This work has been partly supported by the grant #AGF93-0137
Interministerial de Ciencia y Tecnologia.
and Inmaculada LOpez de la
Thanks are also due to Olga
la Universidad de C£diz) for
from the Spanish Comision
REFERENCES
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transformation of mitochondria and development of microtubules. Zeitschrift fiir Zellforschung und
Mikroskopische Anatomie,!! : 80-94.
2. Chevaillier, P. & Maillet, P. L., 1965. — Structure fine et constitution cytochimique du spermatozoi'de de la
langoustine Nephrops norvegicus L. (Crustace Decapode). Journal de Microscopie, 4: 679-700.
3. DEMESTRE, M. & Fortuno, J.-M., 1992. — Reproduction of the deep-water shrimp Aristeus antennatus (Decapoda,
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A. MEDINA : ARISTEIDAE (DECAPODA, CRUSTACEA)
4. Demestre, M., Cortadellas , N. & Durfort, M., 1993. — Ultrastructura de les espermatides de la gamba. Aristeus
antennaius (Cruslaci, Decapoda). Biologia de la Reproduced , 3: 14-17.
5. Felgenhauer, B. E. & Abele, L. G., 1991. — Morphological Diversity of Decapod Spermatozoa. In: R. T. Bauer &
J. W. Martin, Crustacean Sexual Biology. New York, Columbia University Press: 322-341.
6. Haley, S. R., 1986. — Ultrastructure of spermatogenesis in the Hawaiian red lobster, Enoplometopus occidentalis
(Randall). Journal of Morphology , 190: 81-92.
7. HiNSCH, G. W., 1980. — Spermiogenesis in Coenobita clypeatus , I. Sperm structure. International Journal of
Invertebrate Reproduction, 2: 189-198.
8. HiNSCH, G. W.. 1991. — Ultrastructure of the sperm and spermatophores of the anomuran crab Pleuroncodes
planipes. Journal of Crustacean Biology , 11: 17-22.
9. Jamieson, B. G. M., 1991. — Ultrastructure and phylogeny of crustacean spermatozoa. Memoirs of the Queensland
Museum, 31: 109-142.
10. Kaye, G. I., Pappas, G. D., Yasuzumi, G. & Yamamoto, H., 1961. — The distribution and form of the endoplasmic
reticulum during spermatogenesis in the crayfish, Cambaroides japonicus. Zeitschrift fur Zellforschung und
Mikroskopische Anatomie, 53: 159-171.
1 1 . Krol, R. M., Hawkins, W. E. & Overstreet. R. M., 1992. — Reproductive components. In: F. W. Harrison & A.
G. HUMES, Microscopic Anatomy of Invertebrates. Vol. 10. Decapod Crustacea. New York, Wiley-Liss: 295-
343.
1 2. Langreth, S. G., 1969. — Spermiogenesis in Cancer crabs. Journal of Cell Biology , 43: 575-603.
13. McKnight, C. E. & HiNSCH, G. W., 1986. — Sperm maturation and ultrastructure in Scyllarus chacei. Tissue and
Cell, IS: 257-266.
14. Medina, A., 1994. — Spermiogenesis and sperm structure in the shrimp Parapenaeus longirostris (Crustacea,
Dendrobranchiata). Comparative aspects among decapods. Marine Biology, 119: 449-460.
15. Medina, A. & Rodriguez, A., 1992. — Spermiogenesis and sperm structure in the crab Uca tangeri (Crustacea,
Brachyura), with special reference to the acrosome differentiation. Zoomorphology , 111: 161-165.
16. MOSES, M. J., 1961. — Spermiogenesis in the crayfish (Procambarus clarkii). II. Description of stages. Journal of
Biophysical and Biochemical Cytology, 10: 301-333.
17. Pearson, P. J. & Walker, M. H., 1975. — Alteration of cytochrome C oxidase activity during spermatogenesis in
Carcinus maenas. Cell and Tissue Research, 164: 401-410.
18. Pochon-Masson, J., 1968a. — L’ultrastructure des spermatozo'ides vcsiculaires chez les crustaces dCcapodes avant et
au cours de leur devagination experimentale. I. Brachyoures et Anomoures. Annales des Sciences Naturelles,
Zoologie, I2eme serie, 10: 1-100.
19. Pochon-Masson, J., 1968b. — L’ultrastructure des spermatozo'ides vcsiculaires chez les crustacCs decapodes avant
et au cours de leur devagination experimentale. II. Macroures. Discussion el conclusions. Annales des Sciences
Naturelles, Zoologie, 12eme serie, 10: 367-454.
20. REGER, J. F., 1970. — Studies on the Fine structure of spermatids and spermatozoa of the crab Pinnixia sp. Journal
of Morphology , 132: 89-100.
21. Shigekawa, K. & Clark, W. H., Jr, 1986. — Spermiogenesis in the marine shrimp, Sicyonia ingentis.
Development, Growth and Differentiation, 28: 95-112.
22. Spurr, A. R., 1969. — A low viscosity epoxy-resin embedding medium for electron microscopy. Journal of
Ultrastructure Research, 26: 31-43.
23. Talbot, P. & Summers, R. G., 1978. — The structure of sperm from Panulirus , the spiny lobster, with special regard
to the acrosome. Journal of Ultrastructure Research, 64: 341-351.
24. Talbot, P. & Chanmanon, P., 1980. — The structure of sperm from the lobster, Homarus americanus. Journal of
Ultrastructure Research, 70: 275-286.
25. Tudge, C. C., 1992. — Comparative ultrastructure of hermit crab spermatozoa (Decapoda: Anomura: Paguroidea).
Journal of Crustacean Biology , 12: 397-409.
26. Tudge, C. C. & Jamieson, B. G. M.. 1991. — Ultrastructure of the mature spermatozoon of the coconut crab Birgus
latro (Coenobitidae: Paguroidea: Decapoda). Marine Biology, 108: 395-402.
Source : MNHN. Paris
Ultrastructure and Phylogeny of the Spermatozoa
of the Infraorders Thalassinidea and Anomura
(Decapoda, Crustacea)
Christopher C. TUDGE
Zoology Department, University of Queensland
Brisbane, Q 4072, Australia
ABSTRACT
The spermatozoal morphology of 62 species of anomuran and lhalassinidean decapod crab (including many species for
which the spermatozoal ultrastructure. is previously undescribed) is compared at the light and electron microscope level.
Relationships between taxa are postulated on the basis of shared spermatozoal _ characters and comparisons made with
existing ideas about the relationships and evolution of these taxa. Pronounced spermatozoal differences between
representatives of some families in the Infraorder Thalassinidea support the separation and reclassification of this group
into three superfamilies. In contrast, within the anomuran superfamily Paguroidea, the families Paguridae, Parapaguridae,
Diogenidae and Coenobitidae are shown to be united by a suite of ultrastructural spermatozoal characters. Each of these
families, however, can be distinguished by characteristic spermatozoal features. The anatomically diverse family
Diogenidae exhibits a range of sperm morphologies intermediate between the Paguridae and Coenobitidae which may
indicate a diphyletic origin for this family. The families Galatheidae and Porccllanidae, within the superfamily
Galatheoidea, each show a set of distinctive spermatozoal characters, but share very few characters.
RESUME
Ultrastructure et phylogenie des spermatozoides des sous-ordres Thalassinidea et Anomura
(Decapoda, Crustacea).
La morphologie du spermatozoide de 62 esp6ces de Crustac6s Decapodes Anomoures et Thalassinides, (y compris de
nombreuses esp6ces dont le spermatozoide n’avait jamais ete decrit), est compare en microscopie photonique et
£lectronique. Les relations entre les taxons sont postulees sur la base de caracteres spermatologiques partages et des
comparaisons sont faites avec les idees pr£-existantes concemant leurs relations phyl6tiques et leur evolution. Les
differences importantes entre les spermatozoides des representants de quelques families du sous-ordre Thalassinidea
soutiennent la separation et la re-classification de ce groupe en trois super-families. Au contraire, & l’interieur de la super-
famille des Paguroidea, on montre que les families Paguridae, Parapaguridae, Diogenidae et Coenobitidae sont unies par un
ensemble de caracteres ultrastructuraux du spermatozoide. Toutefois, chacune de ces families peut etre distinguee par des
caracteristiques spermatologiques. La famille Diogenidae, qui est anatomiquement diversifiee, montre des morphologies
spermatiques intermediaries entre les Paguridae et les Coenobitidae, qui pourrait indiquer le diphyletisme de son origine.
Les families Galatheidae et Porcellanidae, membres de la super-famille Galatheoidea, montrent chacune un ensemble de
caracteres spermatologiques distinctifs, mais ont peu de caracteres en commun.
Tudge, C. C., 1995. — Ultrastructure and phylogeny of the spermatozoa of the infraorders Thalassinidea and
Anomura (Decapoda, Crustacea). In: Jamieson, B. G. M.. Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist. nat.. 166 : 251-263. Paris ISBN : 2-85653-225-X.
252
C. C. TUDGE : THALASSIN1DEA & ANOMURA ( DECAPODA , CRUSTACEA)
Within the order Decapoda, the infraorder Anomura has undergone considerable revision
since its introduction [27], Classifications of the Anomura such as those of GLAESSNER [17] and
BORRADA1LE [3], recognize the four superfamilies, Thalassinoidea, Paguroidea, Galatheoidea
and Hippoidea. More recently the thalassinoids have been excluded from the Anomura and the
constituent anomuran superfamilies redefined as the Paguroidea, Lomoidea, Galatheoidea and
Hippoidea [30, 31].
Some significant taxonomic changes have been proposed for the infraorder Thalassimdea
and the group now comprises three superfamilies and is considered a monophyletic taxon, distinct
from the Anomura [40]. In the past the thalassinids have been linked with the anomurans on the
basis of larval morphology [18, 26], and adult somatic characters [28]. The thalassinids are
considered to be an important pivotal group in the evolution and phylogeny of the other decapod
infraorders [2, 6],
The superfamily Paguroidea consists of the families, Coenobitidae, Diogenidae, Paguridae,
Parapaguridae. Pylochelidae and Lithodidae [30]. All except the Lithodidae are considered true
hermit crabs [5] but recently it has been shown that the lithodids may have close links with the
genus Pagurus in the Paguridae [13].
The superfamily Lomoidea contains the monospecific genus Lomis in the family Lomidae.
Once considered a symmetrical hermit crab [4], this enigmatic crab has since been elevated to its
own family and superfamily [29, 34] but continues to be problematic in regard to its relationship
to the remainder of the Anomura [28],
The superfamily Galatheoidea contains the families Aeglidae, Chirostylidae, Galatheidae
and Porcellanidae, of which the Aeglidae, containing the single genus Aegta, are ecologically
(restricted to freshwater) and morphologically distinct [28].
The superfamily Hippoidea (the mole crabs) contains only two families, Albuneidae and
Hippidae, which have been variously allied with the other families of the Anomura but appear to
be very distinct. They have been postulated to be more closely related to the thalassinoids than to
the Anomura [28].
The species of thalassinidean and anomuran crabs studied for spermatozoal morphology
(light or transmission electron microscopy) and published up to 1994 are listed in Table 1. This
list comprises 32 species from 17 genera in 9 families. A further 30 species have been
investigated for spermatozoal morphology by the author (Table 2) and this brings the total number
of thalassinidean and anomuran taxa for which the spermatozoal structure is known to 62 species
from 33 genera in 15 families. In addition to the listed publications, the ultrastructure of anomuran
spermatozoa is briefly covered in several general crustacean sperm reviews [16, 23, 25] .
MATERIALS AND METHODS
The crab specimens were collected from a wide range of localities including Australia, the South-West Pacific, the
Mediterranean and European waters. The testes and ducts of the vasa deferentia were removed from the crabs and fixed in
cold (4°C) glutaraldehyde for a minimum of 2 hours, after which the remainder of the standard techniques for transmission
electron microscopy [44] were carried out. This procedure was undertaken for all species except for Thalassina squamifera,
which was deep frozen, thawed and fixed in neutral buffered formalin before removal of the testes and then subjected to the
standard TEM fixation procedure. Micrographs were taken on Hitachi H-300, JEOL 100-s and Hitachi H-600 transmission
electron microscopes at 80, 60 and 75 kV respectively.
RESULTS
Infraorder Thalassinidea
The spermatozoa of the three investigated taxa in this group are each different in
morphology, although the sperm cells of Callianassa australiensis and Axius glyptocercus are
more similar to each other than to those of Thalassina squamifera. The sperm cells of
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
253
Table 1. — Thalassinidean and anomuran taxa previously investigated for spermatozoal morphology
254
C. C. TUDGE : THALASSINIDEA & ANOMURA (DECAPODA, CRUSTACEA)
Table 2. — New thalassinidean and anomuran species investigated by the author for spermatozoal morphology.
Infraorder Thalassinidea
Superfamily Thalassinoidea
Family Thalassinidae
Thalassina squamifera
Superfamily Callianassoidea
Family Callianassidae
Callianassa australiensis
C. arenosa
Dardanus sp.
Diogenes pallescens
G. n. sp. n.(cf. Trizopagurus strigimanus)
Family Paguridae
Pagurus chevreuxi
P. hirtimanus
Porcellanopagurus sp.
Xylopagurus sp.
Family Parapaguridae
Sympagurus sp.
Superfamily Axioidea
Family Axiidae
Axius glyptocercus
Infraorder Anomura
Superfamily Paguroidea
Family Coenobitidae
Coenobita brevimanus
C. perlalus
C. purpureus
Family Diogenidae
Calcinus gaimardii
C. laevimanus
C. minutus
Cancellus sp.
Dardanus lagopodes
D. scutellatus
Superfamily Lomoidea
Family Lomidae
Lomis hirta
Superfamily Galatheoidea
Family Chirostylidae
Eumunida sternomaculata
Uroptychus sp.
Family Galatheidae
Muni da sp.
Munidopsis sp.
Family Porcellanidae
Aliaporcellana suluensis
Petrolisthes armatus
Polyonyx transversus
Superfamily Hippoidea
Family Hippidae
Hippa pacifica
C. australiensis and A. glyptocercus are spherical with prominent microtubular spines (probably
four) radiating from the equatorial region. The sperm cell can be divided into two hemispheres,
with the upper containing the cytoplasmic organelles and an electron-dense acrosome vesicle with
operculum, while the lower hemisphere is composed of the nucleus (Fig. 2a). The microtubular
spines originate in the nucleus and appear to pass completely through it. A. glyptocercus differs
from C. australiensis in having a large columnar invagination (perforatorial chamber) penetrating
the sperm cell from the posterior or lower pole to a subterminal position below the operculum
(Fig. 2a). The spermatozoa of T. squamifera differs markedly in being composed of a large,
ovoid, concentrically zoned acrosome vesicle, capped by a tri-layered operculum and posteriorly
embedded in a ring of cytoplasm and nucleus (Fig. 2b). A number of microtubular arms originate
in the posterior nucleus. The acrosome vesicle is penetrated posteriorly by a columnar
perforatorial chamber which terminates below the operculum and appears to have an electron-
dense thickened ring around its base.
Fig. 1. — A-J: Semidiagrammatic representations of longitudinal sections of hermit crab spermatozoa. Homologous
regions between sperm cells are similarly shaded. Drawings based on tracings of micrographs. Scale bars = 1 pm
(except where indicated).
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHY LOG ENT AND TAXONOMY
255
pertoratorial fingers
subopercular zone
pertoratorial
chamber
outer acrosome
zone
acrosome ray
zone
dense pertoratorial
ring
C. Clibanarius longitarsus
(Diogenidae)
fibrillar acrosome core
B. Calcinus minutus
(Diogenidae)
inner acrosome
zone
A. Coenobita perlatus
(Coenobitidae)
reticulate acrosome
zone
microvillar projections^
E. Diogenes pallescens
(Diogenidae)
F. Pagurus chevreuxi
(Paguridae)
D. Dardanus arrosor
(Diogenidae)
microtubular arm a
.operculum
subopercular
zone
pertoratorial
septum
lacunar sheath
H. Xylopagurus sp.
(Paguridae)
reticulate
acrosome zone
J. Cancellus sp.
(Diogenidae)
G. Porcellanopagurus sp.
(Paguridae)
I. Sympagurus sp.
(Parapaguridae)
Source . MNHN . Paris
256
C. C. TUDGE : THALASSINIDEA & ANOMURA ( DECAPODA , CRUSTACEA)
Infraorder Anomura . . •
Family Coenobitidae. The spermatozoa of Birgus latro and the
thp oenus Coenobita are all very similar in morphology [23, 44, 45J. 1 he concentrically zon<-u
acrosome vesTcte is a large and oblong-ovoid (Birgus) to cylindrical (Coenob.ta) structure
nenetrated posteriorly by a columnar perforatorial chamber in which the walls are diawn out into
SSSSSoL ('Fig. 3a). The anterior pole of the acrosome ad«»d
operculum below which a divided subopercular zone occurs (Fig. l a). The acrosome is largely
composed of an acrosome ray zone which has the appearance of radiating dark and light bands
(Fig.P3b). The acrosome vesicle is cupped posteriorly by the cytoplasm (with three miciotubular
arms) and nucleus. c , . ,
Family Diosenidae. The spermatozoa in the genus Calcinus are composed of a spherica
acrosome vesicle, capped by a domed operculum, and posteriorly penetrated by a perforatoria
chamber (Fig. lb). The acrosome is embedded in the cytoplasm and nucleus and the perforatona
chamber walls project laterally to form short microvillar projections. An autapomorphy for this
2enus is the splitting of the anterior end of the perforatorial chamber into two or more fingers
(Fi0 3c). The genus Cancellus has a spermatozoon with a cylindrical acrosome vehicle with a
conspicuous acrosome ray zone, which is penetrated by a large tapeoHg ^rfor^al chamber
(Fig. lj). Although perforatorial tubules are present in the chamber, theie do not appear to be any
microvillar projections and the operculum is unique among the paguroids in havine a centr
perforation (Fig. 3d). The spermatozoa of the genus Clibananus have an ovoid acrosome vesicle
penetrated by a perforatorial chamber with microvillar projections but the chamber 1S distinctive in
havine a bulbous posterior region and a thin anterior projection. A dense ring (autapomoiphy)
occurs around the bulbous region of the perforatorial chamber (Fig. lc). The sperm cells .oft he
genus Dardanus are similar to the spermatozoa of the coenobitids but the acrosome vesicle (with
conspicuous acrosome ray zone) (Fig. 3b) is generally shorter and more ovoid in shape and he
subopercular zone is not divided into two distinct regions (Fig. Id). The spermatozoa of the
genus Diogenes are characterised by modification of the inner acrosome zone into a fibrillar core
structure. The perforatorial chamber possesses microvillar projections and has the posterior bulb
and anterior projection, like in clibanarids, but there is a prominent acrosome ray zone as in the
coenobitids and dardanids (Fig. le). . .
Family Paguridae. The spermatozoa of the genus Pagurus lack microvillar projections in the
perforatorial chamber, generally have ovoid acrosome vesicles with one or more reticulated
acrosome zones (Fig. 3e) and the perforatorial chamber has a bulbous posterior region and a
tapered anterior projection (Fig. If). Porcellanopagurus spermatozoa have a similarly shaped
acrosome vesicle and perforatorial chamber (without microvillar projections) loPagurus but the
bulbous region of the perforatorial chamber has the walls sculpted into longitudinal septa and is
surrounded by a vesiculated (lacunar) sheath (Fig. lg). The spermatozoa of Xylopagurus differ
from the other pagurids in having the microtubular arms emerging from the cytoplasm in tne
anterior part of the sperm cell and not posteriorly (this also differs from all other paguroids) and in
possessing a larger, more cylindrical acrosome vesicle with unusual zonation (Fig. lh).
Family Parapaguridae. The spermatozoa of Sympagurus share the bulbous shape of the
posterior region of the perforatorial chamber, the lack of microvillar projections and the presence
of a reticulated acrosome zone with members of the family Paguridae but differ in having the
acrosome vesicle extended anteriorly to more than three times the length ol any pagund (Fig. 1).
p1G 2. — A-H: Semidiagrammatic representations of longitudinal sections of thalassinidcan and anomuran crab
spermatozoa. Homologous regions between sperm cells are similarly shaded. Drawings based on tracings ol
micrographs. Scale bars = 1 pm.
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
257
microtubules
— operculum
acrosome vesicle
cytoplasm
A. Axius glyptocercus
(Axiidae)
operculum
perforatorial
chamber
thickened ring
B. Thalassina squamifera
(Thalassinidae)
V * j
^;i»r
dense perforatorial cone
posterior perforatorial
ring
— mitochondria
C. Lomis hirta
(Lomidae)
microtubular-
core
perforatorial
tubules
acrosome ray
zone
-nucleus
D. Eumunida sternomaculata
(Chirostylidae)
inner acrosome
zone
%
F. Aliaporcellana suluensis
(Porcellanidae)
operculum
-perforatorial
septum
perforatorial septum
V <
G. Hippa pacifica Kri&tv*-
(Hippidae)
E. Munida sp.
(Galatheidae)
Source : MNHN, Paris
258
C. C. TUDGE : THALASSINIDEA & ANOMURA ( DECAPODA , CRUSTACEA)
Family Lomidae. The only representative in this family, Lomis hirta , has a spermatozoal
morphology which is different from that of all other anomurans. The sperm cell is irregular, but
basically globular, with one to three vertices which may be extended into microtubular arms (two
arms have been observed in a single sperm but often three vertices are apparent) The acrosome
vesicle is an inverted cup shape, penetrated by a perforatorial chamber, and is completely
embedded in the cytoplasm. A discontinuous, electron-dense zone (interpreted as the operculum)
extends around the entire acrosome vesicle (Fig. 2c).
Family Chirostylidae. The investigated genera in this family, Eumunida and U r op ty chits,
have spermatozoa with a spherical to ovoid acrosome vesicle, capped by a domed operculum and
penetrated by a perforatorial chamber. The walls of the perforatorial chamber lack microvillar
projections, but some perforatorial tubules are present in Eumunida (Fig. 2d). An acrosome ray
zone is present in the posterior region of the acrosome vesicle in both.
Family Galatheidae. The spermatozoal morphology of Allogalathea, Munida and
Munidopsis differs slightly in each species, but there are some characteristic features of the
family. The acrosome vesicles are elongate and generally cylindrical, are capped by a domed
operculum and penetrated for most of their length by a perforatorial chamber. The inner acrosome
zone is divided into two regions of differing electron-density, the outer acrosome zone is situated
posteriorly in the acrosome vesicle, and longitudinally arranged perforatorial septa are present in
the posterior region of the perforatorial chamber (Fig. 2e).
Family Porcellanidae. The spermatozoal morphology found in the investigated members of
this family can be divided into two types. In Petrolisthes the nucleus is globular in shape and the
cytoplasm forms a thin, neck-like region between it and the acrosome while in the remaining
genera, Aliaporcellana, Pisidia and Polyonyx, the sperm cells are elongate, with the cytoplasm
and nucleus forming a veneer over a central microtubular core (Fig. 2f). The microtubular core
splits posteriorly to form several external microtubular arms (Fig. 30- The sperm cells of each
have a complexly arranged, concentrically zoned acrosome vesicle, capped by a perforate
operculum, penetrated by a perforatorial chamber (in which the walls are folded into septa) and
share acrosomal features such as a dense perforatorial cone and posterior ring (Fig. 2f).
Family Hippidae. The sperm cell of Hippa pacifica is composed of a spherical to ovoid
acrosome vesicle, capped by a broad operculum and penetrated by a wide perforatorial chamber.
The unusual features of this spermatozoon are that the anterior end of the perforatorial chamber
forms several tapered points, a membranous septum longitudinally divides the chamber and there
are possibly up to five microtubular arms (Fig. 2g).
DISCUSSION
A recent reclassification of the infraorder Thalassinidea [40] has divided this group into
three superfamilies. The available data from spermatozoal morphology would seem to vindicate
this new arrangement, with Thalassina, Callianassa and Axius (from each of the superfamilies)
Fig. 3. — A-F: Transmission electron micrographs of longitudinal sections (LS) and transverse sections (TS) of
spermatozoa of selected anomuran crabs. A: Coenobita perlatus. LS of perforatorial chamber showing microvillar
projections. B: Dardanus arrosor. TS of acrosome vesicle showing conspicuous acrosome ray zone. C: Calcinus
minutus. TS through upper acrosome vesicle showing splitting of the perforatorial chamber into separate fingers.
D: Cancellus sp. LS of opercular region. E: Pagurus chevreuxi. TS through the acrosome vesicle showing
reticulated acrosome zones. F: Polyonyx transversus. Oblique section through the posterior end of the nucleus
showing the splitting of the microtubular core into discrete microtubular arms. Abbreviations: ar, acrosome ray
zone; ia, inner acrosome zone; ma, microtubular arm; mp, microvillar projection; mtc, microtubular core; n,
nucleus; o, operculum; oa, outer acrosome zone; p, perforatorial chamber; pf, perforatorial fingers; ra, reticulated
acrosome zone; so, subopercular zone. Scale bars = 1 Jim.
Source : MNHN. Paris
o
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
259
Source : MNHN, Paris
260
C. C. TUDGE : THALASSINIDEA & ANOMURA ( DECAPODA . CRUSTACEA)
showing a different spermatozoal ultrastructure. The overall sperm morphology for the
Thalassinidea is sufficiently different from that of the Anomura to support the separation of the
two based on evidence from somatic morphology [28]. Of interest is the fact that the spermatozoa
of Thalassina possess an apparent thickened ring (Fig. 2b) which, so far, has been recorded only
in heterotreme and thoracotreme brachyurans [23].
Within the superfamily Paguroidea, the investigated members of the Coenobitidae and
Diogenidae share many spermatozoal characters, but many genera possess apomorphies which
distinguish them. In the Diogenidae, the genus Calcinus has the splitting of the perforatorial
chamber into separate fingers (Figs lb, 3c), Cancellus sp. has a perforate operculum (Figs lj,
3d), the clibanarids possess the dense perforatorial ring (Fig. lc), and Diogenes species have
modified the inner acrosome zone into a fibrillar core structure (Fig. le). Members of the genus
Pagurus (Figs If, 3e), in the Paguridae, share the shape of the perforatorial chamber, the absence
of microvillar projections and the reticulated acrosome zone with Sympagurus (Fig. li) in the
Parapaguridae, but the other genera ( Porcellanopagurus and Xylopagurus) appear more distinctive
(Fig. lg, h). The light microscope observation of Lithodes (Lithodidae) [41] is the only
spermatozoal information available on this family. It shows a sperm cell with a spherical,
concentrically zoned acrosome vesicle, penetrated by a perforatorial chamber, and three
conspicuous microtubular arms. This description places it within the Paguroidea but an
ultrastructural study is necessary to investigate the claim that the lithodids have close links with
the genus Pagurus in the Paguridae [13].
The superfamily Lomoidea contains the monospecific genus Lomis in the family Lomidae.
The spermatozoon of Lomis possesses spermatozoal characters, such as microtubular arms
(possible three?) and an acrosome vesicle penetrated by a perforatorial chamber (Fig. 2c), which
justify its position in the Anomura but its sperm morphology is distinct enough to confirm
placement in its own family and superfamily [29].
Each of the three investigated families, Chirostylidae, Galatheidae and Porceilanidae, in the
superfamily Galatheoidea shows a particular spermatozoal morphology which appears
characteristic for that family. The chirostylid spermatozoal morphology is more similar to that of
hermit crabs than to any other galatheoid, particularly in the shape of the acrosome vesicle and the
possession of an acrosome ray zone (Fig. 2d). The more elongate (fusiform) acrosome vesicle
shape, division of the inner acrosome zone and the presence of septa in the perforatorial chamber
characterise the members of the Galatheidae (Fig. 2e). The investigated members of the
Porceilanidae all show a particular suite of acrosome vesicle characters which unite them but the
overall sperm cell morphology divides the group. The globular nuclear form in Petrolisthes
species clearly differentiates this genus from the other investigated genera which have an elongate
sperm cell (Fig. 2f), reminiscent of flagellate spermatozoa. This basic division of the
Porceilanidae is supported by larval [42, 47] and adult somatic morphology [19].
In the superfamily Hippoidea only representatives from the family Hippidae have been
investigated for spermatozoal morphology. The spermatozoa observed in the genus Emerita
(Table 1) have a more elongate acrosome vesicle than that described for Hippa. Though showing
spermatozoal characteristics which place them in the Anomura (microtubular arms and acrosome
vesicle structure), it is difficult to ally hippids with any other anomuran superfamily. The broad,
horizontal operculum (Fig. 2g) has its nearest counterpart in members of the Porceilanidae (Fig.
2f) (though perforate in the latter case), or perhaps that seen in the thalassinidean, Thalassina
(Fig. 2b). This last comparison may provide some evidence to support the statement that the
hippids are the nearest relatives to the thalassinoids [28].
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
261
ACKNOWLEDGEMENTS
The author acknowledges the assistance of Mrs Lina Daddow (Zoology Department, University of Queensland) in
all aspects of microscopy. Dr Shane Lavery (Zoology Dept., University of Queensland), Dr Bertrand Richer DE Forges
(ORSTOM, New Caledonia) and Dr Gary Poore (Museum of Victoria) are thanked for collecting many of the specimens and
assistance in identifying the crabs was provided by Mr Peter Davie. Mr John Short (Queensland Museum), Dr Gary
Morgan (Western Australian Museum) and Prof. Jacques Forest (Museum National d'Histoire Naturelle, Paris). The
constant support and guidance of Prof. Barrie Jamieson is gratefully acknowledged.
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Source : MNHN. Paris
Source : MNHN , Pahs
Phylogeny of the Brachyura (Crustacea, Decapoda)
Evidence from Spermatozoa! Ultrastructure
Barrie G. M. JAMIESON * Daniele GUINOT **
& Bertrand RICHER DE FORGES ***
* Zoology Department, University of Queensland,
Brisbane, Q 4072, Australia
** Laboratoire de Zoologie (Arthropodes)
Museum National d’Histoire Naturelle, 61 rue Buffon, F-75231 Paris cedex 05, France
*** ORSTOM. B. P. A5, Noumea Cedex, Nouvelle-Caledonie
ABSTRACT
Spermatozoa of Dynomene aff. devaneyi (Dynomenidae) and Homolodrotnia kai (Homolodromiidae) are described.
Parsimony analyses affirm the classification of the Brachyura by Guinot (1978), notably the groupings Podotremata and
Heterotremata sensu lato, as sister-groups, and Thoracotremata are confirmed. In the Podotremata, association of the
Raninoidea and Cyclodorippoidea is upheld (as sister-groups), each with convincing and unique synapomorphies, but
sperm data considered alone do not support alliance of the Homolidae, (a very clearly defined group) with this couplet and
therefore do not endorse the grouping Archaeobrachyura which is, however, upheld by combined spermatozoal and non-
spermatozoal data. The Dromiacea sensu Guinot (Dromiidae, Dynomenidae and Homolodromiidae) is confirmed
spermatologically as a monophyletic grouping but the discreteness of the three constituent families is not upheld.
Homolodrotnia displays a mixture of dromiid and dynomenid spermatozoal features. The Dynomenidae and Dromiidae are
each found to be paraphyletic. Latreillia sp., considered an homoloid by Guinot (1978) and Guinot & Richer deForges
(1995), forms a polytomy either with Homolidae+Raninoidea-Cyclodorippoidea with the combined, spermatozoal and
non-spermatozoal, data set or with Homolidae+Dromiidae-Dynomenidae-Homolodromiidae, for sperm data only. The
association by Guinot (1978) of the Dorippoidea, Portunoidea, Xanthoidea, and Majoidea in the non-thoracotreme
Heterotremata is fully supported spermatologically. Spermatozoal data give majids the most basal position in the
Heterotremata whereas for the combined data Neodorippe (with carrying behaviour, like most podotremes) appears the
least modified member of the heterotreme-thoracotreme assemblage. The Thoracotremata is unequivocally supported.
RESUME
Phylogenie des Brachyura (Crustacea, Decapoda): le temoignage de l’ultrastructure des
spermatozoides
Les spermatozoides de Dynomene aff. devaneyi (Dynomenidae) et Homolodrotnia kai (Homolodromiidae) sont ddcrits.
Les analyses de parcimonie confirment la classification des Brachyura par Guinot (1978), particulierement les
groupements Podotremata et Heterotremata sensu lato commc groupes-freres, et les Thoracotremata sont confirmes. Chez
Jamieson, B. G. M., Guinot, D. & Richer de Forges, B., 1995. — Phylogeny of the Brachyura (Crustacea,
Decapoda): evidence from spermatozoal ultrastructure. In: Jamieson. B. G. M., Ausio, J.. & Justine, J.-L. (eds), Advances
in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166: 265-283. Paris ISBN : 2-85653-225-X.
266
B. G. M. JAMIESON, D. GUINOT & B. RICHER DE FORGES : BRACHYUKA ( CRUSTACEA )
les Podolremata, I'association des Raninoidea et des Cyclodorippoidea est maintenue (comme groupes-frercs), chacun
avec des synapomorphies originales et convaincantes, mais les donnees spermatologiques utilisees seules ne permettent
pas d’affirmer les relations des Homolidae (groupe ires clairement defini) avec ces deux taxons, et done ne supportent pas
le groupement des Archaeobrachyura. Ce dernier est toutefois maintenu si Ton utilise a la fois les donnees
spermatologiques et non spermatologiques. Les Dromiacea sensu Guinot (Dromiidae, Dynomenidae et Homolodromiidae)
sont confirmds par les donndes spermatologiques comme un groupe monophyletique. mais le caractbre sdpare des trois
families n'est pas prouvd. Homolodromia montre un mdlange de caractbres spermatologiques de Dromiidae et de
Dynomenidae. Les Dynomenidae et les Dromiidae oni tous deux 616 trouves paraphyldtiques. Laireillia sp.. considdrd
comme un Homoloidea par Guinot (1978) et Guinot & Richer de Forges (1995), forme une polytomie ou bien avec les
Homolidae+Raninoidea-Cyclodorippoidea si on utilise les donnees spermatologiques et non-spermatologiques
combines, ou avec les Homolidae+Dromiidae-Dynomenidae-Homolodromiidae en utilisant les donndes
spermatologiques seules. L'association par Guinot (1978) des Dorippoidea, Portunoidea. Xanthoidea et Majoidea dans
les Heterotremata non-thoracotremes est parfaitement confirmde par la spermatologie. Les donndes spermatologiques
donnent aux Majidae la position la plus basale dans les Heterotremata alors que, avec les donnees combinces, Neodorippe
(un •porteur’, comme la plupart des Podotremata) apparalt le membre le moins evolud de 1'assemblage Hdterotremes-
Thoracotrcmes. Les Thoracotremata sont confirmes de maniere non equivoque.
The literature on sperm ultrastructure in Crustacea, and its relevance to phylogeny, a
subject briefly addressed earlier for the Brachyura by BROWN [2], has been reviewed by
JAMIESON [18]. Several papers on brachyuran ultrastructure have since appeared [19, 20, 23-
27] and have culminated in a cladistic, parsimony analysis of brachyuran phylogeny [21] which
is extended in the present chapter. The analyses apply the principles of phylogenetic systematics
propounded by HENNIG [13] and computer procedures for phylogenetic analysis under the
principle of parsimony which are enunciated by SWOFFORD [32].
The internal relationships and classification of brachyuran crabs, and particularly of the
Podotremata, have been the subject of controversy. GUINOT [4-8] divides the Brachyura into
three sections mainly on the basis of the location of the male and female pores: the Podotremata,
the Heterotremata and the Thoracotremata. Nevertheless, GUINOT ([5]: p. 218) recognized that
the coxal positions of male and female pores, with external fertilisation, characterizing the
podotremes, were symplesiomorphies.
The Podotremata sensu GUINOT contain the Dromiacea and Archaeobrachyura. The
Dromiacea consist of the Dromioidea and Homolodromioidea. The Archaeobrachyura contain the
Homoloidea, Raninoidea, and Cyclodorippoidea (= Tymoloidea). In other classifications the
superfamily Homoloidea, which includes three families (Homolidae, Latreilliidae and
Poupiniidae) is often associated with or placed in the Dromiacea (see [5, 6, 12]).
The Heterotremata and Thoracotremata share a sternal location of the female pores and
development of a sternal vulva on sternite 6, in direct communication with the seminal
receptacle, allowing for internal fertilization. The Thoracotremata differ in the additional sternal
location of the male pores. Whereas the Thoracotremata appeared to be a monophyletic group,
the Heterotremata were suspected by JAMIESON to be paraphyletic [18].
In some contrast with the classification of GUINOT, nucleotide sequences of 18S ribosomal
RNA support the exclusion of a mono- or poly-phyletic Dromiidae from the Brachyura, and their
association with the Anomura, but support inclusion of the Raninidae in the Brachyura [1, 30,
31]; homolids were not considered in the molecular analyses.
This chapter adds to the former data matrix [21] new spermatozoal data on two families of
questionable relationships, the Dynomenidae, represented by Dynomene aff. devaneyi, and the
Homolodromiidae, represented by Homolodromia kai. The augmented matrix is subjected to
parsimony analysis. In a second analysis, a minimum of non-spermatozoal characters, defining
the Podotremata, Heterotremata and Thoracotremata and separating these from the Anomura, is
added and effects on the original phylogram observed, pending a more comprehensive inclusion
of non-spermatozoal characters.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
267
MATERIAL AND METHODS
The species examined and sources of material are listed by Jamieson [21]. In addition, the material of Dynomene
aff. devaneyi and Homolodromia kai was obtained on the Bathus 3 cruise in New Caledonian waters, at stations CP 805
and CC 848 respectively, on 22 November 1993.
Electron microscopy. Transmission electron microscopy procedures were as in [27].
Cladistics. Methods employed in the parsimony analysis are given in [21]. Characters employed are given in
Table 1 and the data matrix is shown in Table 2. The parameters and specifications for the phylograms obtained are given
in the legends of Fig. 1 A and B.
Table 1. — Character coding employed
Spermatozoal characters
(1) Acrosome length:width: 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0,
(2) Zonation of the contents of the acrosome vesicle predominantly: horizontal 0, concentric 1. intermediate 2,
(3) Operculum: imperforate 0, perforate, open 1. perforate, closed with apical button 2,
(4) Opercular projections into subopercular material absent 0, present 1,
(5) Operculum: discontinuous with capsule 0, continuous with capsule 1.
(6) Operculum: moderately thick 0, very thin double lamina 1,
(7) Operculum width: not extremely wide 0, extremely wide 1,
(8) Periopercular rim: absent 0, weak 1, well developed 2,
(9) Accessory opercular ring: absent 0, present 1,
(10) Subopercular protuberance through operculum: absent 0, weak 1, well developed 2,
(11) True acrosome ray zone: absent 0, present 1, lost 2.
(12) Outer acrosome zone border with peripheral zone: not ragged 0, ragged 1.
(13) Anterolateral pale zone of acrosome contents: absent 0, present 1,
(14) Flangelike peripheral extension of lower acrosome zone: absent 0, present 1,
(15) Xanthid ring: absent 0, present 1, modified and short 2, modified and elongate 3,
(16) Subacrosomal chamber or perforatorium: postequatorial 0, extending preequatorially 1,
(17) Head of perforatorium: non-capitate 0, amoeboid 1, spiked wheel 2, bilateral 3,
(18) Corrugations of wall of perforatorial chamber: absent 0, simple invaginations 1, branched invaginations 2,
invaginations with filaments 3, filaments only 4, evaginations only 5,
(19) Lateral arms: absent 0, one 1 (not found), two 2, three 3, several 4
(20) Lateral arms: absent 0, microtubular with chromatin 1, nuclear only 2, microtubular only 3,
(21) Centrioles: absent 0, present 1, elongate 2. (Excluded).
(22) Posterior median process of nucleus: absent 0, present 1,
(23) Thickened ring: absent 0, present 1,
(24) Concentric lamellae: absent 0, present 1,
(25) Capsular chambers: absent 0, one chamber 1, several 2,
(26) Capsular projections: absent 0, present 1,
(27) Capsular flange: absent 0, present 1,
Non-spermatozoal characters
(28) Genital pores: all coxal 0, female sternal 1, male and female sternal 2,
(29) Separate spermatheca: absent 0, present 1,
(30) P5, reduction of: absent 0, present 1,
(31) P5, dorsal or subdorsal origin: absent 0, present 1,
(32) P5, subcheliform or cheliform modification: absent 0, weak 1, strong 2,
(33) Sella turcica: absent 0, present 1,
(34) Uropods: present 0, vestigial 1, absent 2
In the present analyses, characters were unordered excepting 1,8, 1 1. 25 and 32 (ordered) and 34 (irreversible, up).
268
B. G. M. JAMIESON, D, GUINOT & B. RICHER DE FORGES : BRACHYURA ( CRUSTACEA )
Table 2. — Data matrix
Taxon
1111111111222222222233333
1234567890123456789012345678901234
Stimdromia lateralis
Dagnaudus petterdi
Calocarcinus africamts
Dromidiopsis edwardsi
Paradynomene tuberculata
Latreillopsis gracilipes
Raninoides sp.
Lyreidus brevifrons
Xeinostoma richeri
Cymonomus sp.
Tymolus sp.
Neodorippe 4 astuta '
Port un us pelagicus
Mictyris longicarpus
Ocypode ceratophthalmus
Uca dussumieri
Macrophthalmus crass ipes
Pilodius areolatus
Ranina ranina
Homola ranunculus
Majids
Potamonautes pertains
Latreillia sp.
Pagurus bemhardus
Clibanarius taeniatus
Homolodromia kai
Dynomene aff. devaneyi
3010000002001001300000000100111211
6011000001000001203211000000111212
8100000210110021004210100001000012
3210000002001001303200000000111211
3010000002001101300070000000111111
6011000001000001203211000000111212
7210100000000001023201002110111012
5210100000000001113201000100111012
5010011000000001033211000100111112
6000001000000101033271000100111112
6010011000000001033211000100111212
1
B100000000100001004200100001011212
A1000000001000010042 10100001000012
C12 00000002 0003 10042 00 1100020000 12
9120000000200031004200110002000012
9120000000200001004200100002000012
A1000000002 000010042 00 110002 000012
9100000010110011004200100001000012
8110100000000000023211001110101012
5011000001000001203271000000111212
A11000000010000 1007111 10000 10000 12
2
9100000200100001004220100001000012
6210000000000001307271000000111212
F10000000000000 1043 111 0000000102 00
C100000000000001043100000000010200
4010000002001101300007000000111111
5010000002001001302200000000111111
Fig 1. — Trees of the Brachyura. A: Heuristic 50% Majority rule consensus tree of 959 shortest and equally parsimonious
trees for spermatozoa! characters only. Heuristic search settings: Addition sequence: simple. One tree(s) held at
each step during stepwise addition. Tree-bisection-reconnection (TBR) branch-swapping performed. MULPARS
option in effect. Steepest descent option not in effect. Branches having maximum length zero collapsed to yield
polytomies. Topological constraints not enforced. Trees rooted by outgroup. Multi-state taxa interpreted as
polymorphism. Character 21 excluded. Character-state optimization: Accelerated transformation (ACCTRAN).
Tree length = 4977 1 . Consistency index (Cl) = 0.665. Homoplasy index (HI) = 0.352. Cl excluding
uninformative characters = 0.647. HI excluding uninformative characters = 0.359. Retention index
(RI) = 0.885. Rescaled consistency index (RC) = 0.588. Clades are supported by 100% of trees unless
otherwise indicated. B: Heuristic strict consensus tree of 36 shortest and equally parsimonious trees, for
spermatozoal and non-spermatozoal characters, using the outgroup method. Setting as for (A). Tree
length = 47210. Consistency index (Cl) = 0.701. Homoplasy index (HI) = 0.317. Cl excluding
uninformative characters = 0.682. HI excluding uninformative characters = 0.324. Retention index
(RI) = 0.902. Rescaled consistency index (RC) = 0.632.
Source :
ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY
269
■o
a
a
(0
3
Q>
ST
CD
3
Q>
O
. zr
c
3
3 | - Stin
: | * — d
! DrnmiHinn
Stimdromia lateralis, Dromiidae
Paradynomene tuberculata, Dynomenidae
Homolodromia kai, Homolodromiidae
Dynomene devaneyi, Dynomenidae
Dromidiopsis edwardsi, Dromiidae
Dagnaudus petterdi
o
S’
fi>
8
cr
■ ■
OJ
o
»<
c
Latreillopsis gracilipes
■ Homola ranunculus
Homolidae, Homoloidea
Lyreidus brevifrons_
Raninoides sp.
Ranina ranina
Raninoidea
Xeinostoma richeri
Tymolus sp. Cyclodorippoidea
Cymonomus sp. _
Latreillia sp., Latreilliidae, Homoloidea
Calocarcinus africanus
Pilodius areolatus
— Pagurus bernhardus
Clibanarius taeniatus
Heterotremata s. Guinot
Mictyris longicarpus
Ocypode ceratophthalma
Macrophthalmus crassipes
Uca dussumieri-
Thoracotremata s. Guinot
Neodorippe astuta
Paguroidea, Anomura
Majids
Potamonautes perlatus
Heterotremata s. Guinot
B Spermatozoal and non-spermatozoal characters
Source
270
B. G. M. JAMIESON. D. GUINOT & B. RICHER DE FORGES : BRACHYURA ( CRUSTACEA )
RESULTS AND DISCUSSION
In the parsimony analysis of spermatozoal data, the heuristic search option was used as
computations under the branch and bound option were not completed in reasonable time.
Nevertheless, the resultant phylograms agreed closely with branch and bound trees previously
obtained [21]. The combined, spermatozoal and non-spermatozoal data yielded a highly
structured strict consensus tree (Fig. IB). Spermatozoal data alone gave an unstructured,
completely pectinate strict consensus tree but the 50% Majority Rule consensus tree (Fig. 1A)
was highly dichotomous and clearly meaningful, despite criticisms which have been levelled at
the validity of majority consensus, in terms of resultant groupings, notably the dromiaceans,
homolids, raninoids, cyclodorippoids, heterotremes sensu lato, and thoracotremes, which are
supportable on other grounds. Conclusions from the two consensus trees are discussed below.
Non-spermatozoal characters will be discussed only where especially relevant but have had more
extensive treatment in the previous analysis [21].
The chief difference between the two trees is that the Homolidae and Latreillidae are
associated with the Raninoidea+Cyclodorippoidea in the anlaysis of combined, spermatozoal and
non-spermatozoal data (hereafter termed the combined analysis) (Fig. IB), but associate with the
Dromiacea in the purely spermatozoal analysis (Fig. 1A). The former assemblage corresponds
with and supports the recognition of a taxon Archaeobrachyura by GUINOT [5], Discussion of
the succession of spermatozoal apomorphies and of group synapomorphies in the following
account will chiefly be derived from the combined analysis but, with the exception noted and
some others to be discussed, there is strong agreement between the two analyses. It is stressed
that a larger and more refined suite of morphological characters is required for a combined
analysis (GUINOT et ai, in preparation).
Brachyura
The Brachyura is a monophyletic taxon relative to the anomuran outgroup, Pagurus
bemhardus and Clibanarius laeniatus. Although the sperm of the Anomura [34] and Brachyura
are distinctive relative to other decapods, the Brachyura have only weak spermatozoal
synapomorphies relative to anomurans despite forming a monophyletic brachyuran clade.
Brachyuran monophyly is supported by shortening of the acrosome to a nearly spheroidal form;
loss of corrugations of the wall of the perforatorial chamber, though these reappear in a different
form in raninoids and cyclodorippoids; loss of microtubules from the lateral arms, a doubtful
synapomorphy in view of their presence in at least some majids [14]; and, somatically,
development of a sella turcica and reduction of the uropods. Although spermatozoal support for
a monophyletic Brachyura is weak, many constituent groups are, in contrast, strongly
supported.
Podotremata
In both the combined and the solely spermatozoal analysis, the Podotremata is a
monophyletic taxon and the sister-group of the heterotreme-thoracotreme assemblage (Fig. 1 A,
B), as also shown previously [11, 21]. Synapomorphies of podotreme spermatozoa, as
indicated in the combined anlaysis, include depression of the acrosome; development of a
predominantly horizontal zonation of the acrosome compared with the concentric zonation of
paguroids and heterotremes; and (ambiguously) a bilaterally symmetrical capitate perforatorial
head (developing from the simple, non-capitate form in paguroids and ancestral crabs), which is
lost in some members. The bilateral perforatorial head is seen in dromiids ( Dromidiopsis
edwardsi and Stimdromia lateralis)', in the two investigated dynomenids ( Paradynomene
tuberculata, [21], and Dynomene aff. devaneyi) and in Homolodromia kai and contrasts with
that of homolid sperm which has the form of a horizontally disposed spiked wheel [21, 27].
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
271
PODOTREMATA
1 Acrosomo depressed
2 Zonabon ol acrosome horizontal
3 Operculum perforate
13 Anterolateral pale zone
(with Dromtdiopsis & Paradynomene)
26 Capsular
projections
.
19 Lateral
arms lost
(with Paradynomene)
(a) Slimdromia lateralis (Dromiidae)
10 Subopercular protuberance
(with Sbmdromia & Paradynomene)
13 Anterolateral pale zone
(</) Latreillopsis gracilipes (Homolidae)
14 Flange
(homoplasic with
Cymonomus sp.)
(b) Dromidiopsis edwardsi (Dromiidae) (e)
10 Subopercular protuberance
2 Zonation intermediate _f ’ 3 Anterolateral pale zone
Latreillia sp. (Laueilliidae)
19 Latoral
arms lost
(with Stimc
(c) Paradynomene tuberculala (Dynomenidac)
( / ) Clibanarius taeniatus ( Diogenidac)
Fig. 2. — Drawings of spermatozoa of some podotremes and an anomuran used in this analysis, a: Slimdromia lateralis
(Dromiidae). b: Dromidiopsis edwardsi (Dromiidae). c: Paradynomene tuberculala (Dynomemdae).
d: Latreillopsis gracilipes (Homolidae). e: Latreillia sp. (Latreilliidae). f: Clibanarius taeniatus (Anomura
Diogenidae). The chief apomorphies are indicated but see text for a more detailed explanation. The section ol
Slimdromia (first described as Petalomera [17]) is not precisely sagittal; in micrographs which arc sagittal,
perforation of the operculum is seen. Scale bar 1 Jim. After [21].
Source : MNHN. Paris
272
B. G. M. JAMIESON. D. GUINOT & B. RICHER DE FORGES : BRACHYURA (CRUSTACEA)
Apical perforation of the spermatozoal operculum is a further synapomorphy of
podotremes, the same condition in majids being, it appears, independently derived (homoplasic).
Monophyly of the Podotremata as deduced from species examined for sperm ultrastructure to
date, does not exclude, nor does it support, the possibility that some supposed dromiids, notably
Hypoconcha [31], have been missclassified and may be closer phylogenetically to anomurans
that they are to other brachyurans.
Dromiacea. The Dromiacea as constituted by GUINOT for the Dromiidae,
Homolodromiidae, and Dynomenidae [5, 10], is confirmed as a monophyletic group in both
analyses (Fig. 1A, B). Its spermatozoal synapomorphies, from the combined analysis, are
further depression of the acrosome, well developed protrusion of subopcrcular material through
the operculum (a lesser protrusion occurs in homolids), and development of an anterolateral pale
zone of the acrosome. Although the Dromiacea forms a monophyletic clade, neither the
constituent Dromiidae nor the Dynomenidae appears monophyletic spermatologically. Thus, in
the combined analysis (Fig. IB) Paradynomene pairs with Homolodromia, and these have
Dynomene as their sister-group, the three being closer to Stimdromia than this is to the other
dromiid, Dromidiopsis, which forms the sister-group of the other dromiaceans. In the purely
spermatozoal analysis (Fig. 1A), Paradynomene again pairs with Homolodromia but sister-
groups, in descending order, are Stimdromia, Dromidiopsis and Dynomene. It can thus be said
that although there is distinctive dromiacean spermatozoal ground plan, sperm structure does not
distinguish the constituent families Dromiidae, Homolodromiidae and Dynomenidae. This does
not necessarily challenge definition of these families on the grounds of non-spermatozoal
morphology (e.g. [10, 29]) and further analysis of non-spermatozoal characters is in progress to
further ascertain the relationships of these families (GUINOT, JAMIESON & RICHER DE FORGES,
and GUINOT & TAVARES, in preparation).
Dromiidae. The Dromiidae (see [29]) are elusive of definition spermatologically as shown
in the previous section (see also [21]), being a paraphyletic group in both analyses. In the
combined analysis (Fig. IB), a monophyletic dromiid clade (including dynomenids and
Homolodromia) is identical with the dromiacean clade. Spermatozoa of Stimdromia
(=Petalomera) lateralis , Dromidiopsis edwardsi and Paradynomene tuberculata are illustrated in
Fig. 2A-C and that of Homolodromia kai in Fig. 6B.
In the combined analysis (Fig. IB), Dromidiopsis edwardsi is the sister-taxon of the other
dromiaceans. The sole, and somewhat subjective, apomorphy of the sperm of Dromidiopsis
edwardsi [28] is a zonation of the acrosome which is intermediate between the horizontal and
concentric conditions. Synapomorphies of the dromiid-dynomenid -Homolodromia melange are
weak, being loss of the three arms basic to the anomuran-brachyuran assemblage, and with them
any microtubules in these arms. As arms are present in Dynomene aff. devaneyi, their basal loss
is questionable, but they may well be labile in occurrence. Stimdromia lateralis (Fig. 2A) is
diagnosed by the presence of capsular projections. Dynomene aff. devaneyi, which computes as
basal relative to these taxa, appears to be unique in the Brachyura, in having only two nuclear
arms. A further apomorphy is slight lengthening of the acrosome. Paradynomene (Fig. 2C) and
Homolodromia (Fig. 6B) have a striking similarity, computing as a synapomorphy: a flange like
lateral extension of the lower acrosome zone. Paradynomene is distinguished (ambiguously) by
slight lengthening of the acrosome whereas Homolodromia shows no individual apomorphy; in
the spermatozoal anlaysis, it is distinguished from Paradynomene only by its slightly more
depressed acrosome.
Centrioles are unknown in dromiid sperm but are present in homolids. The difficulty in
unequivocally demonstrating their presence or absence has led to their exclusion from the
parsimony analyses.
Homolodromiidae This family is placed in a monotypic superfamily Homolodromioidea,
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
273
PODOTREMATA (continued)
Cymonomus sp. (Cymonomidae)
CYCLODORIPPOIDEA
7 Operculum very wiOo
3 Operculum
imperforate
1 Acrosome
Lyreidm brevifrons (Raninidae)
RANINOIDEA
2 Zonauon of acrosome intermediate (ambiguous)*
5 Operculum continuous with capsule
17 'Amoeboid'
6 & 7 Operculum very
wide and thin (with Tymolus)
flange
(with Ramna)
lengthening
(with Ranina)
18 Corrugations'
branched
(b) Raninoides sp. ( Rani nidac. )
(e) Xeinostoma richeri (Cyclodorippidac )
5 Operculum continuous
with capsule
I Acrosome
(with Ranina)
27 Capsular
1 Acrosome
25 Several capsular
chambers
2 Zonation concentric
5 Operculum continuous with capsule
6 & 7 Operculum
(with Raninoides)
16 Subacrosomal
chamber postoquatorial
Ranina ranina (Raninidae)
Tymolus sp. (Cyclodorippidac)
Fig. 3. — Drawings of spermatozoa of further podotremes used in this analysis, a: Lyreidus brevifrons (Raninidae,
Lyreidinae). b: Raninoides sp. (Raninidae, Raninoidinae). c: Ranina ranina (Raninidae, Ranininae).
d- Cymonomus sp. (Cymonomidae). e: Xeinostoma richeri (Cyclodorippidae, Xeinostominae). f: Tymolus sp.
(Cyclodorippidac, Cyclodorippinae). The chief apomorphies are indicated but see text for a more detailed
explanation. Scale bar 1 pm. Sources as listed in Material and methods. After Jamieson [21].
Source : MNHN. Paris
274
B. G. M. JAMIESON, D. GUINOT & B. RICHER DE FORGES : BRACHYURA ( CRUSTACEA )
within the Dromiacea, by GUINOT [5, 10]. She considers that the Homolodromioidea represent,
without doubt, the most primitive [jnembers] of the Podotremata and lists a long series of
characters in support of this contention. It is difficult, therefore, to evaluate the relatively
advanced position which Homolodromia appears to occupy, in terms of spermatozoal
ultrastructure, relative to other dromiaceans (Fig. 1 A, B). It is noteworthy, in view of origin of
Homolodromia in the phylograms between Paradynomene on the one hand and Dynomene, with
or without intervention of dromiids, that GUINOT [5] stated that in some regards it is the
dynomenids which seem closer to the Homolodromiidae than do the Dromiidae. The fact that
Homolodromia lies within a dromiid clade is also of interest with regard to GUINOT’S [5, 10]
statement (drawing on [35] and others) that the level of organization of the fossil Prosopidae, the
most ancient crabs known, survives on the one hand in the form (without doubt little modified)
of the Homolodromioidea, which inhabit deep waters, and on the other hand in the form of the
Dromioidea (Dromiidae and Dynomenidae), much more numerous and diversified, which have
developed special adaptations (in most Dromiidae the carapace is protected by a sponge, an
ascidian or a bivalve shell) [29]. The Homolodromiidae have a unique combinaton of
morphological characters, though mostly plesiomorphic. These are, inter alia, fusion of the
ophthalmic segment to the anterior carapace (in Homolodromia)-, the soft branchiostegite;
endophragmal skeleton with anastomoses; abdominal pleura developed; and retention of
abdominal pleopods in the male on segments 3 to 5. Occurrence of uropods which are not dorsal
and are represented by small lobes on the abdominal segment 6 appears to be a homolodromiid
synapomorphy [10]. The phylograms (Fig. 1A, B) are heuristic for reconsideration of the
validity and relationships of the families Dromiidae, Homolodromiidae and Dynomenidae.
In terms of the ultrastructural characters used in the parsimony analyses, the spermatozoon
of Homolodromia kai has the following characteristics. The ratio of length to width of the
acrosome is 0.4; zonation of the acrosome is predominantly horizontal; the operculum is
perforate and lacks opercular projections such as are diagnostic of homolids; the operculum is
not continuous with the acrosomal capsule, and, in contrast with raninoids, it is moderately thick
and is of moderate width, not thin and occupying much of the width of the acrosome as in
cyclodorippoids; there is no periopercular rim nor an accessory opercular ring; protrusion of
subopercular material through the operculum is well developed; a true acrosome ray zone of the
type seen in paguroids, other anomurans and in brachyurans of the Heterotremata sensu stricto,
is absent although a “finger-print” like zone is possibly homologous with this; the ragged outer
acrosomal zone and the xanthid ring, typical of xanthids and some of their relatives, are absent;
an anterior pale zone of the acrosome, seen also in Stimdromia, Dromidiopsis, Dynomene and
Paradynomene, is present; the subacrosomal chamber extends pre-equatorially in the acrosome
as in all investigated species excepting Ranina ranina\ the head of the putative perforatorium is
bilaterally symmetrical, as in Stimdromia, Dromidiopsis and Paradynomene-, corrugations of the
wall of the perforatorial chamber, a thickened ring, concentric lamellae, capsular chambers,
projections and flanges are absent. Nuclear arms and a definite posterior median process are not
demonstrable.
Dynomenidae. GUINOT [5, 8, 10], and GUINOT, JAMIESON & RICHER DE FORGES [11],
ranked dynomenids as a family in the superfamily Dromioidea, placed with the
Homolodromioidea in the subsection Dromiacea, within the section Podotremata. This placement
of dynomenids is wholly supported in both analyses but as indicated above, the Dynomenidae
does not have spermatological support as a monophyletic group (Fig. 1 A, B).
Separation of the Dynomenidae from the Dromiidae is justified, in non-spermatozoal
characters, by a large number of differences [6] which include complete modification of the coxa
of P5 as a penis. Furthermore, dynomenids show reduction of P5 instead of P4 and P5 as in the
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
275
HETEROTREMATA
719 Several latorai arms T
11 True acrosome ray zone with Thoracotremata
23 Thickened ring present
722 Posterior median
process
(a) Menaethius monoceros ( Majidae)
(</) PoiamonquJes pertains (Poianiidae)
1 Acrosomo length
8 Penopercular rim lost
(/») Neodorippe as lit Id (Dorippidae)
(c) Pilodius areolaius (Xanthidae)
(c) Portunus petagicus (Portunidae)
1 Acrosomo Slightly
15 Modified, short,
x an th id ring
Calocarcinus africanus (Trapeziidac)
Fig. 4. — Drawings of spermatozoa of Heterotremata used in this analysis, a: Menaethius monoceros (Majidae).
b: Neodorippe ' astuta \ now considered close to N. callida (Dorippidae). c: Portunus pelagicus (Portunidae).
d : Potamonautes perlatus (Potamidae). e: Pilodius areolaius (Xanthidae). f : Calocarcinus africanus
(Trapeziidae). The chief apomorphies arc indicated but see text for a more detailed explanation. Scale bar 1 pm.
Sources as listed in Material and methods. After [21].
Source . MNHN. Paris
276
B. G. M. JAMIESON, D. GUINOT & B. RICHER DE FORGES : BRACHYURA (CRUSTACEA)
Dromiidae. Despite the more brachyuran facies of some species, several features of the
Dynomenidae appear to be plesiomorphic and to accord with the earlier appearance of
dynomenids in the fossil record relative to dromiids.
The sperm of Dynomene aff. devaneyi (Fig. 6A) resembles that of Homolodromia kai,
described above, in all features mentioned, with the exception of the following. The ratio of
length to width of the acrosome is 0.5; two nuclear arms are detectable; and a posteromedian
process is absent.
Archaeobrachyura. The phylogram for combined data (Fig. IB), as previously [21],
supports recognition of the Archaeobrachyura of GUINOT [5], containing the superfamilies
Homoloidea, Raninoidea and Cyclodorippoidea (=Tymoloidea). The single spermatozoal
synapomorphy for the Archaeobrachyura is weak: the presence of a posterior median process. It
is, however, reinforced by the somatic character loss of the uropods [21], The grouping
Archaeobrachyura is not, however, supported in the purely spermatozoal analysis (Fig. 1 A) in
which Latreillia and the homolids group with the Dromiacea (Dromiidae, Dynomenidae and
Homolodromiidae) and not with the raninoid+cyclodorippoid assemblage.
Homolidae. Spermatozoal ultrastructure has been examined in seven species of the
Homolidae: Homola ranunculus , Paramola bathyalis and Dagnaudus (=Paramola) petterdi [11,
12] and in Homologenus sp., Latreillopsis gracilipes (Fig. 2D), Homolomannia sibogae, and
Paromolopsis boasi [27].
From spermatozoal ultrastructure, the Homolidae is a convincingly monophyletic entity in
the combined and the spermatozoal analyses (Fig. 1A, B, and [21]). Synapomorphies ol
homolid spermatozoa are the following. The presence of numerous radial arranged extensions of
the acrosomal operculum into the perforatorium has been established as an autapomorphy of the
homolids [27] seen in no other brachyurans. Projection of subacrosomal material into the
opercular perforation occurs but is weaker than the strong protrusion which is apparently
independently developed in dromiaceans. Thirdly, the spiked-wheel form of the anterior
expansion of the perforatorium is restricted to the Homolidae for which it is thus an
autapomorphy. Whether a prexisting bilateral form of the head of perforatorium is a basic
condition of all podotremes or the non-capitate condition is basic computes ambiguously. The
radial spikes, approximately 12 in number, extend far laterally. They are supported by fibrous
cores which radiate from the central core of the perforatorium. The spikes are much longer in
Latreillopsis gracilipes (Fig. 2D) than in the other species, curving around the inner aspect of the
vesicle almost to its base.
Raninoidea and Cyclodorippoidea. The Raninoidea (Fig. 3A-C) and Cyclodorippoidea
(Fig. 3D-F) form a monophyletic (but unnamed) clade in both analyses (Fig. 1A, B, and [21]).
Spermatozoal synapomorphies are not striking and two are ambiguous: reversal from a bilateral
to a non-capitate condition of the perforatorium; and development of simple corrugations of the
wall of the perforatorial chamber. Unambiguous are development of outward projections of the
capsule (present study and [21]), seen homoplasically in Stimdromia\ and, somatically, though
confined to the Raninoidea, loss of the subcheliform development of pereiopods 5.
Raninoidea. Spermatozoal ultrastructure has been investigated in Ranina ranina [16] (Fig.
3C), in the subfamily Ranininae, Raninoides sp. [26] (Fig. 3B), in the subfamily Raninoidinae
(reinstated by GUINOT [9]), and Lyreidus brevifrons Sakai, 1937 [26] (Fig. 3A), in the
subfamily Lyreidinae [9]. These raninoids, as a group, are well defined spermatologically (Fig.
1A, B) by virtual continuity of the operculum with the capsule and alteration of the zonation of
the acrosome vesicle to an intermediate condition, with development of a concentric condition in
Ranina ranina. The intermediate condition is homoplasic with Dromidiopsis and Latreillia.
Somatically raninoids have lost subcheliform modification of pereiopods 5 (this study and [21]),
perhaps correlated with a burrowing or swimming habit.
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
277
Ranina [16] and Raninoides [26] share strong synapomorphies: development of posterior
capsular chambers, one in Ranina (Fig. 3C) increasing to several in Raninoides (Fig. 3B); and
the remarkable lateral flange on the capsule. An ambiguous change, not shown in some
parsimony analyses [21], is development of branched septum-like corrugations of the wall of the
perforatorial chamber from the unbranched form basal to the raninoid-cyclodorippoid clade and
persistent in Lyreidus. There is also a strong trend towards a subspheroidal form of the
acrosome, most developed in Ranina in which zonation becomes concentric; and in which the
perforatorium, apparently secondarily, becomes only postequatorial. In Lyreidus (Fig. 3A), the
acrosome becomes secondarily depressed; and the “amoeboid” form of the head of the
perforatorium is seen as development of a capitate condition independently of that in dromiids
and homolids (This study and [21]).
Cyclodorippoidea. The Cyclodorippoidea form the sister-group of the Raninoidea in both
analyses (Fig. 1 A, B). The sperm of the three cyclodorippoids (Fig. 3D-F) [25] are well defined
by the extreme width of the operculum relative to the acrosome. As an ambiguous change,
corrugations of the wall of the perforatorial chamber are invaginations with filaments. A
synapomorphy of Xeinostoma (Fig. 3E) and Tymolus (Fig. 3F) is the extreme thinness of the
operculum. Xeinostoma is apomorphic in further depression of the acrosome. Cymonomus
(Fig. 3D) is apomorphic for all investigated podotremes in losing the opercular perforation. This
supports erection of a separate family Cymonomidae [33]. It appears to have developed the
flange-like extension of the lower acrosome zone independently of Paradynomene and
Homolodromia but the similarity is striking and cyclodorippoid relationships require further
investigation (This study and [21], GuiNOT & Tavares, in preparation).
Latreilliidae. The position of Latreillia sp. (Fig. 2E) is equivocal, as in the previous
cladistic analyses [21], It forms a polytomy either with Homolidae+Raninoidea-
Cyclodorippoidea with the combined data set (Fig. IB) or with Homolidae+Dromiidae-
Dynomenidae-Homolodromiidae, for sperm only (Fig. 1A). This archaeobrachyuran status of
Latreillia for the combined data is in accordance with placement of the Latreilliidae by GUINOT
[5] near the Homolidae and contradicts the view of Wright AND COLLINS (see [5]) that the
accepted close relationship between the Homolidae and Latreilliidae is based on no more than a
few primitive features. Confirmation of the ultrastructural characteristics of Latreillia sperm is
desirable as many spermatozoa of this species used in the cladistic study appeared malformed.
The sole detected apomorphy of Latreillia is development, homoplasically with Dromidiopsis, of
an intermediate condition of the acrosome vesicle contents from the horizontally zoned condition.
In the combined analysis this condition is an ambiguous apomorphy as it could alternatively be
basal to the Podotremata but it is unequivocal in the purely spermatozoal analysis.
Heterotremata and Thoracotremata
In the cladistic analyses (present study and [21]) (Fig. 1A, B), it is seen that within the
heterotreme-thoracotreme assemblage, the Thoracotremata (Fig. 5) is a monophyletic taxon
whereas the Heterotremata sensu stricto (Fig. 4) is a paraphyletic grouping.
The combined Heterotremata-Thoracotremata, which may be termed the Heterotremata
sensu lato [21], is defined by a convincing synapomorphy, presence of the thickened ring. Other
spermatozoal synapomorphies, although unambiguous, are less convincing. Multiplication of
lateral arms from three, common to paguroids and podotremes, to several is a trend rather than a
diagnostic basal apomorphy as it results from polymorphism, there being three in at least some
majids as in the leucosiid Iliacantha subglobosa [3]. Presence of a true acrosome ray zone
appears to be a synapomorphy but is seen, apparently homoplasically, in paguroids.
Cladistically (present study and [21]), the Heterotremata sensu lato form a grouping
whether or not non-spermatozoal characters are included but the sternal female pores constitute,
as GUINOT [5, 6] suggested, their non-spermatozoal synapomorphy. In the combined analysis
278
B. G. M. JAMIESON, D. GUINOT & B. RICHER DE FORGES : BRACHYURA ( CRUSTACEA )
(Fig. IB) as previously [21], Neodorippe forms the plesiomorphic sister-group of all other
included crabs. Its sole (ambiguous) spermatozoal apomorphy is very slight elongation of the
acrosome beyond a spheroidal shape. It is noteworthy, in view of their relatively plesiomorphic
spermatozoal ultrastructure, that dorippids exhibit carrying behaviour, like most dromiids,
Neodorippe callida attaching to leaves. The dorippid included here, and referred to as
Neodorippe astuta (see [22]), is close to N. callida but definitive identification has not been
made. There are, however, no spermatozoal apomorphies distinguishing the remaining crabs of
the Heterotremata sensu lato from Neodorippe , though somatic synapomorphies are loss of
subcheliform development of pereiopods 5 (and also P4). On the basis of purely spermatozoal
data, as in the former analysis [21], the Majidae occupy this basal position (Fig. 1 A).
1 Acrosome slighn>
1 Acrosomo slightly
Shortens
Jca dtissumieri (Ocypodidac)
15 Independent development ol xanihid ring-like structure (with Mictyns)
(c) Ocypode ceratopluhalma (Ocypodidac)
THORACOTREMATA
3 Operculum with apical Dutton (ambiguous)
1 1 Acrosome ray zone lost
24 Concentric lamellae
1 Acrosome elongates
(b) Macrophtlialmus crassipes (Ocypodidac) id)
Miciyris longicarpa (Mictyridac)
Fig. 5. — Drawings of spermatozoa of Thoracotremata used in this analysis, a: Uca dussumieri. b: Macrophthalmus
crassipes. c: Ocypode ceratopluhalma (all Ocypodidae). d: Mictyris longicarpus (Mictyridae). The chief
apomorphies are indicated but see text for a more detailed explanation. Scale bar 1 |im. Sources as listed in
Material and methods. After [21].
Fig. 6. — Transmission electron micrographs of longitudinal sagittal sections of the sperm of two podotreme species
described in this chapter. A: Dynomene aff. devaneyi. Short diameter of perforatorium in main micrograph,
long diameter right inset. B: Homolodromia kai. Long diameter of perforatorium in main micrograph, short
diameter in right inset, detail of acrosome ray zone (“fingerprint” zone) in left inset, ap. apical protuberance; ar,
acrosome ray zone; cap, capitate region of perforatorium; cm, cell membrane; cy, cytoplasm; dm, degenerating
mitochondrion; ia, inner acrosome zone; 1, lamellae; n, nucleus; o, operculum; oa, outer acrosome zone; p,
perforatorium; pa, anterolateral pale acrosome zone; so, subopercular zone.
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279
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280
B. G. M. JAMIESON. D. GUINOT & B. RICHER DE FORGES : BRACHYURA < CRUSTACEA )
Within the heterotremes above Neodorippe (combined data, Fig. IB), or above the majids
(sperm only, Fig. 1A), Calocarcinus and the xanthid Pilodius group together but there is
ambiguity as to whether development of a simple xanthid ring is basic to the two and is retained
in xanthids but transformed in Calocarcinus (as seems likely), whether the supposedly
transformed condition is basal, or whether each developed a form of the xanthid ring de novo.
Their ancestor may have slightly shortened the acrosome. Other significant synapomorphies,
retained in Calocarcinus and Pilodius, are development of an accessory opercular ring and the
ragged form of the outer acrosome zone. However, too literal an acceptance of the precise
sequence of changes should be avoided as it was found in the previous analysis [21] that
Potamonautes (Fig. 4D), Calocarcinus (Fig. 4F) and the two xanthids included were unified by
a periopercular rim, remaining well developed in Potamonautes (Fig. 4D) and Calocarcinus (Fig.
4F), becoming weak in the xanthid Etisus (excluded from the present analyses), and lost in
Pilodius (Fig. 4E) but that this character is ambiguous. When the character was treated as
ordered, it was unambiguous, being represented weakly in the ancestor of this clade and in
Etisus (excluded from the present study), developing from this state to well developed in
Calocarcinus and Potamonautes, and being lost in Pilodius [21]. From intuitive studies, xanthids
are united by the presence of a ring around the base of the inner acrosome zone, the xanthid ring
[15]. In the present study strong development of a periopercular rim occurred independently in
Potamonautes relative to Calocarcinus. Majids are characterized by development of perforation
of the operculum and of a posterior median process independently of that in podotremes.
Portunus pelagicus shows no apomorphies beyond those of basal heterotremes.
The Thoracotremata (Fig. 5A-D) selected for the cladistic studies (This study and [21])
were found to be monophyletic (Fig. 1A, B) on the basis of two unambiguous characters: loss
of the acrosome ray zone and movement of the male pores (following that of the female pores
basic to heterotremes) onto the sternum. Development of the characteristic apical button in the
perforatorium appears ambiguous owing to its alternative absence or loss in Macrophthalmus
(Fig. 5B). A more detailed investigation of thoracotremes might resolve the issue of whether the
button is basic to thoracotremes. In view of the close relationship generally recognized between
Macrophthalmus and Ocypode (Fig. 5C), it seems likely that the absence in Macrophthalmus is
due to loss of a basic thoracotreme condition.
Concentric lamellae in the acrosome appear to be a development, not seen in Uca (Fig.
5A), basal to the higher thoracotremes, Mictyris (Fig. 5D), Ocypode (Fig. 5C) and
Macrophthalmus (Fig. 5B). Uca differs from the basic thoracotreme condition only in slight
shortening of the acrosome.
An interesting outcome of the cladistic analyses is that the “modified xanthid ring” which
has been recognized as a characteristic of some thoracotreme sperm and considered to suggest
derivation of thoracotremes from a xanthid stock [18] computes as an entirely independent
development not related to the xanthid structure (this study and 21]). This does not completely
rule out the possibility of derivation from the xanthid ring, however.
Concluding remarks
The parsimony analyses, whether using only spermatozoal characters or spermatozoal and
non-spermatozoal characters, provide a remarkable affirmation of the classification of the
Brachyura by GUINOT [4, 5] which differed so markedly from pre-existing and, in some
schools, still current classifications. Thus the validity of, and phylogenetic justification for, the
groupings Podotremata and Heterotremata (though only in sensu lato) and Thoracotremata is
affirmed. Podotremes and Heterotremata sensu lato are confirmed as sister-taxa. Association of
the Raninoidea and Cyclodorippoidea is upheld (as sister-groups), each with convincing and
unique synapomorphies, but sperm data considered alone do not support alliance of the
Homolidae, though equally clearly defined, with this Raninoidea+Cyclodorippoidea couplet and
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
281
therefore do not endorse the grouping Archaeobrachyura. Combined spermatozoal and non-
spermatozoal data do, however, support the Archaeobrachyura. There is, nevertheless,
molecular evidence [3 1 ] that raninoids are more closely related to the heterotreme-thoracotreme
assemblage than they are to other podotrematous crabs. Within the Podotremata, the Dromiacea
sensu GuiNOT (Dromiidae, Dynomenidae and Homolodromiidae) is confirmed
spermatologically as a monophyletic grouping but the discreteness of the three constituent
families is not upheld. Homolodromia displays a remarkable mixture of dromiid and dynomenid
spermatozoal features while lacking any distinctive apomorphy, and does not appear
spermatologically to occupy the basal position in the Dromiacea indicated by GUINOT [5, 10]
(the apparent agreement of the combined analysis, in this respect, is due solely to the
spermatozoal characters.) The Dynomenidae and Dromiidae are each found to be paraphyletic.
An 18S rRNA study [31] also found little support for the Dromiidae as a monophyletic group
but, unlike the present study, excluded one dromiid from the Brachyura; the two dromiids
included in the molecular analysis never formed a clade. In a bootstrap analysis the dromiid
Hypoconcha arcuata grouped with a hermit crab while Dromidia antillensis formed their sister
taxon [31]. Examination of the spermatozoa of Hypoconcha would be very desirable.
Relationships of Latreillia sp„ the sole representative in the present study of the Latreilliidae and
considered an homoloid by GUINOT [5] and GUINOT & RICHER DE FORGES [12], are equivocal.
It forms a polytomy either with Homolidae+Raninoidea-Cyclodorippoidea with the combined
data set or with Homolidae+Dromiidae-Dynomenidae-Homolodromiidae, for sperm only. The
association by GUINOT [5] of the Dorippoidea, Portunoidea, Xanthoidea, and Majoidea in the
non-thoracotreme Heterotremata is fully supported spermatologically (calappoids, corystoids,
parthenopoids, bellioids and leucosioids, also included by GUINOT, were not included in
computations). The Thoracotremata is unequivocally supported as a monophyletic group.
ACKNOWLEDGEMENTS
We are grateful to ORSTOM for supporting collection (by B. R. F.) of much of the material used in this study. Dr.
Chris Tudge is thanked for his careful reading of the manuscript and for drawing many of the illustrations. Mrs.
L. Daddow, Mr. D. Scheltinga and Dr. C. Tudge gave excellent technical assistance. This work was made possible by
Australian Research Council funding and support from the Museum National d’Histoire Naturelle, Paris.
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sibogae and Paramolopsis boasi. Helgolander Meeresuntersuchungen, 47: 323-334.
28. Jamieson, B. G. M., Tudge, C. C. & Scheltinga, D. M.. 1994. — The ultrastructure of the spermatozoon of
Dromidiopsis edwardsi Rathbun, 1919 (Crustacea, Brachyura, Dromiidae): confirmation of a dromiid sperm
type. Australian Journal of Zoology, 41: 537-538.
29. McLay, C. 1993. — The Sponge Crabs (Dromiidae) of New Caledonia and the Philippines with a review of the
genera. In: A. Crosnier, Resultats des Campagnes MUSORSTOM, Volume 10. Memoires du Museum National
d'Histoire Naturelle, Paris, \S6: 111-251.
30. Spears. T. & Abele, L. G.. 1988. — Molecular phylogeny of brachyuran crustaceans based on 18S rRNA
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33. Tavares, M., 1994. — Brachyoures bathyaux recoltes par le "Marion Dufresne" an large du Bresil. Systematique et
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Paris 6, Paris, France: 1-324.
34. Tudge, C. C., 1992. — Comparative ultrastructure of hermit crab spermatozoa (Decapoda: Anomura: Paguroidea).
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NOTE ADDED IN PROOF
In a valuable paper received as this chapter was going to press, SCHOLTZ & RICHTER
(1995) conclude, from a preliminary, mainly morphological analysis, that the Homolodromiidae
are the sister-group of all other brachyurans. If this is so, the similarity of the sperm of
Homolodromia to that of Paradynomene is problematical.
Scholtz, G. & Richter. S., 1995. — Phylogenetic systematics of the reptantian Decapoda (Crustacea,
Malacostraca). Zoological Journal of the Linnean Society, 113: 289-328.
Source : MNHN. Paris
Nuclear Alterations during Spermiogenesis of
Triatoma infestans (Hemiptera, Reduviidae)
Heidi D OLDER
Department of Cell Biology, Institute of Biology,
C. Postal 6109, UNICAMP, 13083-970 Campinas, Sao Paulo, Brazil
ABSTRACT
The pattern of spermiogenesis in insects as described for some orders has been generally accepted as the process
typical for this taxon. However some species depart from this pattern at specific steps and these differences may be used
as taxonomic characteristics. In Triatoma infestans, the most striking difference is the lack of nuclear grooves and
adjacent membranes which are substituted by coated nuclear protrusions occurring during nuclear elongation. Ten stages
of spermiogenesis are recognized and described.
RESUME
Alterations nucleaires pendant la spermiogenese de Triatoma infestans (Hemiptera, Reduviidae)
Le processus de spermiogenese des insectes, tel qu’il a 6t6 decrit pour certains ordres, a ete generalement accepte
comme le processus typique pour ce taxon. Toutefois, certaines especes se demarquent de ce processus dans des etapes
specifiques et ces differences peuvent etre utilisees comme des caracteristiques taxonomiques. Chez Triatoma infestans, la
difference la plus marquante est 1’ absence de gouttifcres nucleaires et de membranes adjacentes qui sont remplacees par des
protuberances nucleaires revetues apparaissant pendant reiongation nucleaire. Dix stades de spermiogenese sont
reconnus et decrits.
The differentiation of spermatids into spermatozoa is a profound transformation in which
all organelles are greatly modified in structure and function, or eliminated when the cell has no
further necessity for their contribution towards maturation. To define this process in an orderly
manner, it was divided into specific stages by various authors. First described [15] in
Drosophila melanogaster, the same steps were found in other dipterans such as Ceratitis capitata
[13] and Chrysomya megacephala [11]. Only small differences were noted in other orders such
as Orthoptera [16], and Coleoptera [10].
Some isolated aspects of hemipteran spermiogenesis were included in review articles [2,
3]. Aspects of this differentiation process have been thoroughly investigated, such as the
formation of the Nebenkern or mitochondrial complex [12]. The nature of the mitochondrial
derivatives filled with a paracrystalline structure was investigated in various insects including
Hemiptera [4, 14].
Dolder, H., 1995. — Nuclear alterations during spermiogenesis of Triatoma infestans (Hemiptera. Reduviidae).
In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem.
Mits. natn. Hist, nat., 166 : 285-289. Paris ISBN: 2-85653-225-X.
286
H. DOLDER : TRIATOMA INFESTANS (HEMIPTERA, INSECTA)
The flagellar characteristics of various hemipteran spermatozoa have also been investigated
[1, 6-8]. These studies show a strong resemblance between the flagella of the spermatozoa of
various genera [7] and even of different families [1].
This is not the case in nuclear and acrosome development. A completely distinct nuclear
and acrosome organization was described for the spermatozoa of Saldula saltatoria and
Chartoscirta cineta, where the acrosome is sucker-shaped and the nucleus extends into the
flagellum [1], The development of Cerris puludum is another variation in hemipteran
spermiogenesis in which complex acrosome and nuclear forms were found in late spermatids
[19]. Also in the giant spermatozoon of Notonecta glauca, the development of its very long
acrosome and nucleus is very different from the pattern known for other Heteroptera [18]. A
report on the last stages of spermiogenesis of Leptocoris trivittatus (Hemiptera, Corizidae) is
more similar to the hemipteran investigated in this study, although acrosome location in a nuclear
canal is also distinct from other known species [9]. The most complete description of the nuclear
region of an hemipteran during spermiogenesis can be found in relation to Carabus catenulatus
and Nepa rubra [17]. Unfortunately the difference in the methodology used makes some
structures difficult to compare.
MATERIAL AND METHODS
Final (5th) stage male nymphs of Triaioma infestans were prepared according to routine methods of electron
microscopy for determining ultrastructure, using glutaraldehyde and osmium tetroxide. In the case of the specimens seen
in figures 2 and 7, the post-fixation in osmium tetroxide was omitted.
RESULTS
The early stages of spermatid development have spherical nuclei, similar to somatic cell
nuclei, with finely granular chromatin which condenses only in contact with the nuclear
membrane. The subdivision into stages is therefore based on cytoplasmic features, such as the
Nebenkern formation in stage 2 (Fig. 1), its separation into two mitochondrial derivatives on
each side of the axoneme (stage 3) and their elongation together with the axoneme (stage 4).
The last spherical nucleus stage (5th) is marked by the fusion of the proacrosome vesicle
onto the nuclear membrane and its transport to the anterior region of the nucleus. Up to this
stage, nucleoli can still be seen, suggesting an active cellular metabolism, necessary for the
intense cytoplasmic modifications occurring at this time. Nuclear pores are limited to the region
surrounding the basal plaque in which the centriole is anchored (Fig. 2). However, nuclear
modification has already begun, as indicated by the first condensations of chromatin into thicker
strands (Fig. 3).
In cross sections, the elongating nucleus of stage 6 has two pronounced projections
covered by layered dense material (Fig. 4). Chromatin is more densely packed in these
Figs. 1-9 — Spermiogenesis in Triaioma infestans. 1: Stage 2 spermatid has a round nucleus (N) in which the chromatin
begins to condense near the nuclear membrane. A large mitochondrial complex or “Nebenkern” forms at the
nuclear base. Bar = 1 pm. 2: In stage 5, nuclear pores (P) are limited to the periphery of the basal plate (B).
3: A stage 5 nucleus begins to form chromatin cords of various thicknesses. The basal plate (B) of dense
granular material marks the centriolar insertion. 4: Nuclear elongation and formation of thick chromatic cords
identify stage 6. The transversely sectioned nucleus is surrounded by parallel microtubules (T) with the exception
of two nuclear projections coated with a dense layer (D). 5: In stage 7. elimination of the nuclear matrix
diminishes the nuclear diameter and the nuclear projections. The dense coating is now limited to small tufts.
6: Fusion of cords into chromatin lamellae marks stage 8. 7: A rearrangement of the lamellae to form a regular
network indicates stage 9. 8: A longitudinal section in stage 9 also shows the regular, parallel, chromatin
arrangement. 9: In the last stage (stage 10) of spermatid development, the nucleus is very dense and no
substructure of the chromatin can be seen. Cytoplasmic microtubules will be discarded with the excess cytoplasm
in the spermatozoon.
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288
H. DOLDER : TRIATOMA INFESTANS ( HEMIPTERA , INSECTA)
protrusions than along the rest of the nuclear membrane which is enclosed externally by a row of
parallel microtubules.
In stage 7, elongation is completed and the chromatin has been completely condensed into
thick cords (Fig. 5). The nuclear projections are progressively reduced and the dense material
which covered them is disorganized into tufts and disintegrates in the cytoplasm.
Stage 8 is identified by lamellar organization of the chromatin (Fig. 6), which rapidly
branches out into a close network which identifies stage 9 (Figs. 7 and 8). A longitudinal section
shows the compact parallel arrangement of the chromatin lamellae (Fig. 8).
Continued condensation results in a very dense nucleus in which the chromatin
organization can no longer be visualized (Fig. 9). Only the sloughing off of the extra cytoplasm
and microtubules separate this stage 10 spermatid from the mature spermatozoal structure.
DISCUSSION
Despite the many studies on hemipteran spermatogenesis, these generally concentrate on
an interesting aspect of their development and have not detailed the whole process. However, the
step by step examination of this process shows that the same subdivision into stages can be
applied to this group as has been described for a dipteran [15], an orthopteran [16] and a
coleopteran [10].
The active nucleus is practically unmodified, accompanied by major reorganization of the
cytoplasmic organelles, during the first stages of spermiogenesis. As nuclear elongation and
chromatin condensation begins, these organelles are formed and are gradually modified as
maturation proceeds. The next subdivisions of this process are, therefore, based on the nuclear
alterations.
Only in stages six and seven are there pronounced differences in relation to the other
insects studied. In these previous studies the nucleus forms deep nuclear grooves which are
externally lined by the “adjacent membranes”, a dense layer of the same thickness as a
membrane, which later separates from the nucleus and curls in the cytoplasm where it remains
until it is discarded together with the excess cytoplasm and microtubules.
In Triatoma infestans, the nuclear protrusions can be described as a new feature,
apparently homologous in function with the nuclear grooves. The dense covering of these
protrusions is very different from the adjacent membranes, not only in structure but also in its
progressive disappearance at the end of stage 7 when the nuclear protrusions are absorbed and
the dense material breaks up, becoming indistinguishable in the cytoplasm.
The greater condensation of the chromatin in the protrusions seems to indicate a role in
chromatin condensation into thick cords and their organization along the length of the nucleus.
Stages 8 and 9 were rarely encountered and this has been interpreted as an indication that
the events of chromatin lamination, network formation and condensation into a very dense,
uniform nucleus occur very rapidly. Considering this fact, it may be more appropriate to unite
the eighth and ninth stages as a single, more frequently encountered phase of spermiogenesis.
REFERENCES
1. Afzelius, B. A., Dallai, R. & Lindskog, P., 1985. — Spermatozoa of saldid bugs (Insecta, Hemiptera,
Leptopodomorpha). Journal of Ultrasiruciure Research 90: 304-312.
2. Baccetti, B., 1972. — Insect sperm cell. Advances in Insect Physiology , 9: 315-397.
3. Baccetti, B. & Afzelius, B. A., 1976. — The Biology of the Sperm Cell. Basel, Karger.
4. Baccetti, B., Dallai, R., Pallini, V., Rosati, F. & Afzelius, B. A., 1977. — Protein of insect sperm
mitochondrial crystals. Journal of Cell Biology, 73: 594-600.
5. BAo, S. N., Quagio-Grassiotto, I. & Dolder. H., 1989. — Acrosome formation in Ceratitis capitata (Diptera,
Tephitidae). Cytobios, 58: 93-100.
6. Dallai, R. & Afzelius, B. A., 1980. — Characteristics of the sperm structure in the Heteropteran (Hemiptera,
Insecta). Journal of Morphology , 164: 301-9.
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
289
7. Dallai, R. & Afzelius, B. A., 1982. — On zipper-lines or particle arrays within the plasma membrane of
hemipteran spermatozoa (Heteroptera, Insecta). Journal of Ultrastructure Research, 80: 197-205.
8. Dolder, H., 1988. — Cytoskcletal bridges between organelles in sperm flagellum of Triatoma infestans
(Hemiptera, Reduviidae). Journal of Ultrastructure and Molecular Structure Research, 101: 159-64.
9. Itaya, P. W., Thompson. S. A. & Heidger Jr. P. M., 1980. — Fine structure of late stages of spermiogenesis in
Leptocoris trivittatus Say. (Hemiptera, Corizidae). International Journal of Insect Morphology and
Embryology, 9: 135-145.
10. Lino Neto, J., 1994. — Estudo ultra-estrutural da espenniogenese e dos espennatozoides de Cosmopolites sordidus
Germar ( Coleoptera , Curculionidae). Tese de Mestrado, Instituto de Biologia, Universidade Estadual de
Campinas, Campinas, Brazil: 15-55.
1 1 . Messias Jr. N. S., 1990. — Aspectos ultra-estruturais da espenniogenese de Chrysomya megaccphala Fab ( Diptera :
Calliphoridae). Tese de Mestrado, Instituto de Biologia, Universidade Estadual de Campinas, Campinas,
Brazil: 1-115.
12. Pratt, S., 1968. — Formation and differentiation of the Nebenkern in spermatids of an hemipteran insect,
Murgantia histrionica. In: B. A. BACCETTI, Comparative Spermatology. Rome, Accademia Nazionale dei
Lincei: 301-310.
1 3. Quagio-Grassiotto, I., 1983. — Citodiferenciagdo ultra-estrutural durante a espenniogenese normal de Ceratitis
capitata Weidmann (Diptera: Tephritidae). Tese de Mestrado, Instituto de Biologia, Universidade Estadual de
Campinas, Campinas, Brazil: 1-93.
14. Rosati, F., Selmi, G. & MAZZINI, M., 1976. — Comparative observations on the mitochondrial derivatives of
insect sperm. Journal of Submicroscopic Cytology, 8: 51-67.
15. Stanley, H. P., Bowman, J. T., Romrell, L. J., Reid, S. C. & Wilkinson, R. F., 1972. — Fine structure of normal
spermatid differentiation in Drosophila melanogaster. Journal of Ultras true lure Research. 41: 433-66.
16. SzOLLOSi, A., 1975. — Electron microscope study of spermiogenesis in Locusta migratoria (Insect, Orthoptera).
Journal of Ultrastructure Research, 50: 322-46.
17. Werner, G., 1966. — Untcrsuchungen iiber die Spermiogcnesc bei einem Laufkufer, Carabus catenulatus Scop..
und der Skorpion-wasserwnaze, Nepa rubra L. Zeitschrift fiir Zellforschung, 73: 576-599.
18. Werner, G., Afzelius, B. A. & Mosler, B., 1988. — Acrosome formation during spermiogenesis in Notonecta
glauca L. (Heteroptera). Journal of Submicroscopic Cytology, 20: 123-135.
19. Young, H. L., 1985. — Spermatogenesis of the water strider, Gerris paludum (Heteroptera, Gerridae). Journal of
Ultrastructure Research, 90: 235-250.
Source . MNHN, Paris
Source : MNHN. Paris
Sperm Ultrastructure of Xenos vesparum (Rossi)
and its Significance in the Taxonomy
and Phylogeny of Strepsiptera (Insecta)
Marcella CARCUPINO*, Giuseppe PROFILI*,
Jeyaraney KATHIRITHAMBY** & Massimo MAZZINI*
*Dipartimento di Scienze Ambientali,
Universita della Tuscia, I - 01 100 Viterbo, Italy.
** Department of Zoology,
University of Oxford, South Parks Road, 0X1 3PS Oxford, England.
ABSTRACT
Sperm ultrastructure of the strepsipteran Xenos vesparum was studied by scanning and transmission electron
microscopy. The data obtained were compared with those of other strepsipteran species, namely Xenos mouloni , Elenchus
tenuicornis, E. japonicus and Halictophagus chilensis. From the spermatological point of view. Strepsiptera appear to
form a uniform group in which the mature sperm consist of: an elongated head with a monolayered acrosome and a nucleus
characterized by an eccentric portion of uncondensed chromatin; and a long tail consisting of a 9+9+2 axoneme flanked by
two mitochondrial derivatives of equal size. The spermatozoon of X. vesparum , however, shows some species-specific
characters such as a longer nucleus which is entirely occupied by condensed chromatin, and two U-shaped mitochondrial
derivatives which are devoid of paracrystalline material. These results confirm the sperm morphology as valuable
taxonomic character, even if it is not yet sufficient to clarify the controversial phylogenetic affinities of Strepsiptera.
RESUME
Ultrastructure du spermatozoide de Xenos vesparum (Rossi) et sa signification pour la taxonomie
et la phylogenie des Strepsipteres (Insecta).
L’ultrastructure du spermatozoide du Strepsipt£re Xenos vesparum a ete etudiee en microscopie 61ectronique h
transmission et & balayage. Les donnees obtenues ont ete compares avec celles des autres Strepsipteres Xenos moutoni,
Elenchus tenuicornis , E. japonicus et Halictophagus chilensis. D'un point de vue spermatologique, les Strepsipt6res
semblent former un groupe homogene dans lequel le spermatozoide mur consiste en une tete allongee comprenant un
acrosome a une seule couche et un noyau caracterise par une portion excentrique de chromatine non condensee, et une
longue queue formee d’un axoneme 9+9+2 flanque de deux derives mitochondriaux de taille 6gale. Toutefois, le
spermatozoide de X. vesparum montre quelques caract&res spScifiques tels qu’un noyau plus long qui est enti&rement occupe
par la chromatine condensee, et deux derives mitochondriaux en forme de U qui sont depourvus de materiel paracristallin.
Ces rSsultats confirment la valeur de la morphologie des spermatozoides comme caractere taxonomique, meme s’ils ne sont
pas actuellement suffisant pour 6claircir la position phylog<$netique controversee des Strepsipteres.
Carcupino, M., Profili, G., Kathirithamby, J., & Mazzini, M., 1995. — Sperm ultrastructure of Xenos vesparum
(Rossi) and its significance in the taxonomy and phylogeny of Strepsiptera (Insecta). In: Jamieson, B. G M., Ausio, J., &
Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. nat. Hist. nat.. 166: 291-296.
Paris ISBN: 2-85653-225-X.
292
M. CARCUPINO ET AL. : XENOS VESPARUM ( STREPSIPTERA , INSECTA)
Strepsiptera constitute a small cosmopolitan order of entomophagous parasitic insects which
are characterized by extreme sexual dimorphism with free-living, short-lived males and
permanently endoparasitic neotenic females (except in the family Mengenillidae).
The ordinal status of the Strepsiptera is still debated. Even up to the present day, several
workers, mainly coleopterists, place Strepsiptera as a family (Stylopidae) in the order Coleoptera
(beetles) [2, 9, 10, 20] or as a sister group of the Coleoptera [13]. Using classical morphological
characters, KlNZELBACH [17] reported the plesiomorphies and apomorphies of Strepsiptera. A
large number of the apomorphic characters are adaptations to an endoparasitic life; the only
apomorphic character uniting Strepsiptera and Coleoptera was found to be the use of the hind
wings as the main organ for flight, with consequent specialization of the metathorax for this
function.
Recently, KRISTENSEN [18] divided the holometabolic insects into two groups, one
consisting of Megaloptera, Raphidioptera, Neuroptera and Coleoptera, the other of Panorpoidea
and Hymenoptera, whereas the position of Strepsiptera remained unsolved.
More recently, on the basis of morphological and genetic analysis, a close relationships
between Strepsiptera and Diptera has been suggested [13, 21],
Examination of other characters, such as the morphology of the sperm, could provide useful
contributions to the phytogeny of Strepsiptera. JAMIESON [11] has coined the term
spermiocladistic for the use of sperm ultrastructure in the reconstruction of the phylogeny. There
are many examples of spermatological characters (such as the organization of the acrosomal
complex and the tail) useful for phylogenetic and evolutionary reconstructions in many animal
groups, particularly in insects [for a review see 3, 4, 11],
Descriptions of sperm ultrastructure and spermiogenesis of Strepsiptera have been reported.
BACCETTI [5] was the first to describe the ultrastructure of the sperm tail of Xenos vesparum
Rossi (Stylopidae). Later [8, 15, 16, 19], the sperm organization was studied in four species,
namely Xenos moutoni De Buysson (Stylopidae), Elenchus tenuicornis Kirby, E. japonicus
Esaki & Hashimoto (Elenchidae) and Halictophagus chilensis Hofmann (Halictophagidae). More
recently, CARCUPINO et al. [9] have examined spermiogenesis in E. tenuicornis, while AFZELIUS
& DALLAI [1], using a new fixation technique, studied the tail organization in Stylops sp.
The present paper aims to provide additional information on the mature sperm and the male
reproductive system in X. vesparum, and to compare this with all the previous dataon the sperm
structure in Strepsiptera, in order to contribute to a better understanding of the phylogenetic
position of this peculiar Insect order.
MATERIALS AND METHODS
Stylopized wasps Potisies dominilus (Christ) were collected in the vicinity of Tubingen (Germany) and brought
back to the laboratory. The internal reproductive systems were dissected from free-living males of X. vesparum and
processed for scanning (SEM) and transmission (TEM) electron microscopy.
Scanning electron microscopy. Specimens were fixed in Karnovsky's fixative [12] for 2 h at 4= C, rinsed overnight
in 0.1 M cacodylate buffer at pH 7.2, post-fixed in similarly buffered 1% osmium tetroxide, dehydrated in a graded ethanol
series, dried with the critical point method, gold coated and observed with a JEOL JSM 5200 electron microscope.
Fig. I. — SEM micrograph of the internal reproductive system of adult male Xenos vesparum showing aedeagus (ae),
ejaculatory duct (ed), sperm pump (sp), seminal vesicles (sv), and testes (t). x 215
Figs 2-15. Longitudinal (2, 8) and cross-sections (3-7, 9-15) at different levels of the spermatozoon of X. vesparum.
Observe the brush-like glycocalyx at each level of the sperm surface (arrows). 2: Longitudinal section of the sperm
head, x 14 400. 3: Cross-section of the acrosome. x 90 000. 4-7: Cross-sections at different level of the
kidney-shaped nucleus, from the apex (4) to the base (7). x 90 000. 8-10: Longitudinal (8) and cross-section
(9,10) ol the neck region ol the sperm tail showing the centriolar area with mitochondrial derivatives (m)
characterized by crescent-like appendages (arrows) encircling the axoneme (ax). 8, x 54 000, 9-10, x 90 000.
11-15: Cross-sections at dilferent levels of the tail with mitochondrial derivatives (m) reducing in size and
axoneme (ax) progressively disorganized, x 90 000.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
293
Source : MNHN. Parts
294
M. CARCUPINO ETAL. : XENOS VESPARUM ( STREPSIPTERA , INSECTA)
Transmission electron microscopy. Specimens were fixed and dehydrated as above, then embedded in Epon-Araldite
mixture. Thin sections were cut in a Reichert Ultracut and a LKB Nova ultramicrotomes, stained with uranyl acetate and lead
citrate and observed with a JEOL JEM 1 200 EX II electron microscope.
RESULTS
The reproductive system of X. vesparum (Fig. 1) includes a pair of elongated testes which
open into two large pear-shaped seminal vesicles. The seminal vesicles in turn open separately
into a single large muscular sperm pump via two thick vasa deferentia. A single ejaculatory duct
emerges from the posterior portion of the sperm pump and terminates in the aedeagus (Fig. 1).
The morphology of the mature sperm is similar to that of the other species of Xenos
previously examined [19]. The sperm is filiform with an elongated head and a long tail in which
three different regions, namely neck region, principal piece and end piece, can be recognized
(Figs 2-15).
The head, about 8.5 pm long, is occupied by a monolayered acrosome and a nucleus (Figs
2, 3). In cross-section, the nucleus, which is narrower in the apical portion, is kidney-shaped and
appears entirely occupied by compact and electron-dense chromatin (Figs 4-7).
The tail has a simple organization with the axoneme flanked by two long mitochondrial
derivatives (Figs 8-13). The axoneme has the common 9+9+2 pattern in which nine accessory
tubules, nine microtubular doublets with two dynein arms, and two central microtubules can be
recognized (Figs 11-13). The mitochondrial derivatives have typical cristae looking like a series of
lamellae which are regularly spaced and aligned at right-angles to one side of the major axis of the
derivatives (Fig. 8). No paracrystalline structure seems to be present in the mitochondrial matrix.
The neck region shows a bilocular head-tail junction in which the centriolar region with the
beginning of the axoneme and the mitochondrial derivatives is located (Fig. 8). Cross-sections of
this region show that the mitochondrial derivatives have a U-shaped appearance with a crescent¬
shaped appendage encircling the axoneme on each side (Figs 9, 10). These appendages
progressively reduce in size until they disappear. Along the principal piece, the axoneme is always
flanked by two U-shaped mitochondrial derivatives from which it is separated by a conspicuous
membranous sheath (Figs 11, 12). Proceeding toward the endpiece, the mitochondrial derivatives
reduce in size and the axoneme becomes disorganized (Figs 13-15). Along the endpiece,
axonemal disorganization occurs with the disappearance of the nine accessory tubules first (Fig.
14), then of the two central microtubules, and later of the nine peripheral doublets (Fig. 15).
The entire surface of the sperm, from the acrosome to the endpiece? is covered by a thin
brush-like glycocalyx (Figs 3-7, 9-15).
DISCUSSION
The spermatozoon of Xenos vesparum is similar to that of other species of Strepsiptera
examined previously. Except for a few peculiarities observed in the sperm ultrastructure of each
species examined [8, 15, 16, 19], such as different shape of the acrosome, nucleus and
mitochondrial derivatives, Strepsiptera were considered a uniform group. The mature sperm is
filiform and consists of a monolayered acrosome at the tip of an elongated nucleus which has a
portion of uncondensed chromatin in an eccentric position, and a bilocular head-tail junction
followed by a 9+9+2 axoneme flanked by two mitochondrial derivatives of equal size.
The mature sperm of X. vesparum shows some characters similar to that of X. moutoni
[19], such as U-shaped mitochondrial derivatives and sperm surface covered by a brush-like
glycocalyx, which could be considered as generic characteristics. However, the spermatozoon of
X. vesparum also shows several species-specific characters. The sperm nucleus is about twice as
long as that of the other species examined and lacks the internal portion of uncondensed
chromatin. The mitochondrial derivatives have an unusual appearance. Like the typical
mitochondrial derivatives, they have cristae with the appearance of a series of lamellae regularly
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
295
spaced and aligned orthogonally to the major axis of the sperm, but they lack the paracrystalline
material. Mitochondria without any sign of crystallization were also reported in Stylops sp. [1],
These data confirm that the morphology of the spermatozoon has real value in providing
taxonomic characters, and that the spermatozoon of Strepsiptera does not resemble that of
Coleoptera. Coleoptera sperm resemble those of Strepsiptera in having the tail with a 9+9+2
axoneme flanked by two mitochondrial derivatives but differ in having a three layered acrosomal
complex (the acrosomal vesicle is located between the periacrosomal cap and the subacrosomal
material) and two accessory bodies (for a review see [6, 11]). However, the phylogenetic
affinities of Strepsiptera remain unsolved.
Recently, AFZELIUS & DALLAI [1], studying insect sperm tails fixed with a glutaraldehyde-
tannic acid mixture, reported that Strepsiptera sperm deviate in several important respects from
those of other components of the neuropteroid superorder (Megaloptera, Raphidioptera,
Neuroptera and Coleoptera). In particular, the incomplete kidney-shaped accessory axonemal
tubules, the lack of the intratubular material and of the accessory bodies distinguished the
strepsipteran spermatozoa from those of any other insect groups [1], The sperm tail of
X. vesparum, however, as well as those of the other strepsipteran species previously examined
[5, 8, 15, 16, 19] shows normal circular accessory tubules. It is not easy to discriminate if this
difference is related to specific characteristics or to the new fixative, or to different stages of
sperm maturation. In fact, by using the glutaraldehyde-tannic acid mixture, the axonemal
structures are better resolved, and therefore the kidney shape might represent the real shape of the
strepsipteran accessory tubules. However, the kidney-shaped accessory tubules showed in
Stylops sp. [1] belong to an immature sperm as demonstrated by the presence of microtubules
flanking the mitochondria (see micrograph number 8 in [1]). On this basis, it could be
hypothesized that the strepsipteran accessory tubules are kidney-shaped during the differentiation
stages and become circular in cross-section at the end of maturation. A survey of mature
spermatozoon of Stylops sp. could clarify this matter.
Whether these unusual features of the strepsipteran sperm reflect an adaptation to an
endoparasitic life, or an isolated systematic position (or both) is still unclear. In fact, related to the
endoparasitic life are a very simple female reproductive system [14] and a peculiar mode of
insemination and fertilization to which the organization of the sperm could be also related. Further
light might be thrown on this problem by a study of sperm morphology in the Mengenillidae, a
primitive strepsipteran family in which both males and females are free-living.
REFERENCES
1. AFZELIUS, B. A. & DALLAI, R., 1994. — Characteristics of the flagellar axoneme in Neuroptera, Coleoptera and
Strepsiptera. Journal of Morphology, 219: 15-20.
2. Arnett, R. H., 1968. — The Beetles of the United States (a Manual for Identification). The American Entomological
Institute: 1-112.
3. Baccetti, B„ 1972. — Insect sperm cells. Advances in Insect Physiology. 9: 315-397.
4. Baccetti, B., 1979. — The evolution of the acrosomal complex. In: D. W. Fawcett & J. M. Bedford, The
spermatozoon. Baltimore & Munchen, Urban & Schwarzenberg: 305-329.
5. Baccetti, B., 1989. — The spermatozoon of Strepsiptera and its value in the systematic position of the group.
Journal of Submicroscopic Cytology and Pathology, 21: 397-398.
6. Burrini, A. G., Magnano, L„ Magnano, A. R„ Scala, C. & Baccetti, B„ 1987. — Spermatozoa and phylogeny
in Curculionoidea (Coleoptera). International Journal of Insect Morphology and Embryology, 17: 1-50.
7. Carcupino, M., Kathirithamby, J. & Mazzini, M., 1994. — Spermiogenesis in Elenchus tenuicornis (Kirby)
(Strepsiptera: Elenchidae). Atti XVII Congresso nazionate di Entomologia, Udine 13-18 Ciugno: 343-346.
8. Carcupino, M., Mazzini, M., Olmi, M. & Kathirithamby, J., 1993. — The spermatozoon of Halictophagus
chilensis Hofmann (Strepsiptera, Halictophagidae). Bolletino di Zoologia, 60: 361-365.
9. CROWSON, R. A., 1960. — The phylogeny of Coleoptera. Annual Review of Entomology, 5: 111-134.
10. Crowson. R. A., 1981. — The Biology of Coleoptera. New York, Academic Press: 1-802.
296
M. CARCUPINO ETAL. : XENOS VESPARUM ( STREPSIPTERA, IN SECT A)
11. Jamieson, B. G., 1987. — The Ultrastructure and Phytogeny of Insect Spermatozoa. Cambridge, Cambridge
University Press: 1-320.
12. Karnovsky, M. J., 1965. — A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron
microscopy. Journal of Cell Biology, 27: 137A-138A.
13. Kathirithamby, J., 1989. — Review of the order Strepsiptera. Systematic Entomology , 14: 41-92.
14. Kathirithamby, J., Carcupino, M., & Mazzint, M. 1990. — Ovarian structure in the order Strepsiptera. Frustula
Entomologica , 13: 1-8.
15. Kathirithamby, J., Carcupino, M. & Mazzini. M., 1992. — Ultrastructure of the spermatozoon of Elenchus
japonicus and its bearing on the phylogeny of Strepsiptera. Tissue and Cell , 24: 437-442.
16. Kathirithamby. J.. Carcupino, M. & Mazzini, M., 1993. — Comparative spermatology of four species of
Strepsiptera and comparison with a species of primitive Coleoptera (Rhipiphoridae). International Journal of
Insect Morphology & Embryology , 22: 459-470.
17. Kinzelbach, R. K., 1971. — Morphologische Befunde an Facherfluglem und ihre phylogenetische Bedeutung
(Insecta: Strepsiptera). Zoologica, 41: 1-256.
18. Kristensen, N. P., 1989. — Insect phylogeny based on morphological evidences. In: B. Fernholm, K. Bremer &
H. JOrnvall, The Hierarchy of Life. Amsterdam, Elsevier: 295-306.
19. Mazzini, M., Carcupino. M. & Kathirithamby, J., 1991. — Fine structure of the spermatozoon of the strepsipteran
Xenos moutoni. Tissue and Cell , 23: 199-207.
20. ROSS, H. T., Ross, A. C. & Ross, J. R. P., 1984. — A Textbook of Entomology, 4th Edition. New York, Wiley: 1-
666.
21. Whiting, M. F. & Wheeler, W. C., 1994. — Insect homeotic transformation. Nature , 368: 696.
Source : MNHN. Paris
Characteristics of the spermatozoon of
Cosmopolites sordidus (Coleoptera: Curculionidae)
Jose LINO NETO * & Heidi DOLDER **
* Department of General Biology,
Universidade Federal de Vi$osa, Vi^osa, MG, 36570-000, Brazil.
** Department of Cell Biology, Institute of Biology,
C. Postal 6109, UNICAMP, 13083-970 Campinas, Sao Paulo, Brazil
ABSTRACT
The ultrastructure of the spermatozoon of Cosmopolites sordidus is similar to that described for other beetles. The
acrosome consists of three structures: the perforatorium, the acrosomal vesicle and an extra-acrosomal layer. It is
embedded in the nucleus which contains dense, homogeneous chromatin. The flagellum has the typical 9+9+2
microtubule arrangement, two mitochondrial derivatives and two accessory bodies. However, the implantation of these
organelles is not in agreement with the description given for the subfamily Rhynchophorinae to which this species
belongs. A revision of these characters is needed.
RESUME
Caracteristiques du spermatozoide de Cosmopolites sordidus (Coleoptera: Curculionidae)
L’ultrastructurc du spermatozoide de Cosmopolites sordidus est similairc & celle ddcrite pour les autres ColSoptfcres.
L’acrosome est compose de trois structures: le perforatorium, la vesicule acrosomienne et une couche extra-
acrosomienne. II est enchassS dans le noyau qui contient une chromatine dense et homogene. Le flagelle a la structure
typique 9+9+2, deux derives mitochondriaux et deux corps accessoires. Toutefois, rimplantation de ces organites ne
correspond pas a la description donnee pour la sous-famille des Rhynchophorinae & Iaquelle cette espbcc appartient. Une
revision de ces caracteres est necessaire.
Ultrastructural investigations have furnished valuable contributions in relation to the
phylogenetic study of many animal groups, including insects. JAMIESON proposed the term
spermiocladistics for the use of spermatozoon ultrastructure for phylogenetic reconstruction [8].
Many publications exist along this line; as examples we can cite those dealing with the
Chrysomelidae [2] and the superfamily Curculionoidea [5].
While spermatozoal characteristics have been used to define the phylogenetic position of
various coleopterans, the position of the Cosmopolites sordidus spermatozoon, with regard to its
ultrastructural characteristics, is not clear.
LINO Neto, J., & DOLDER, H., 1995. — Characteristics of the spermatozoon of Cosmopolites sordidus
(Coleoptera: Curculionidae). In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (cds). Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist, not., 166: 297-300. Paris ISBN : 2-85653-225-X.
298
J. LINO NETO & H. DOLDER : COSMOPOLITES SORDIDUS ( COLEOPTERA , INSECTA)
MATERIAL AND METHODS
Adult males were anaesthesized. dissected and their testes fixed by immersion in 3% glutaraldehyde in 0,1 M
phosphate buffer at pH 7.2, for 3 hours, post fixed in osmium telroxide 2% in the same buffer for 1 h, all solutions
maintained at 4°C. They were dehydrated with an ethanol series, followed by acetone and embedded in Epon 812.
Ultrathin sections were examined with a Zeiss EM 902 electron microscope, after staining with uranyl acetate and lead
citrate.
RESULTS
In Cosmopolites sordidus, the acrosome measures 1 |im in length and presents three
structures: the extra-acrosomal layer, the acrosomal vesicle and the perforatorium. This last
structure has its base resting in a concavity of the anterior nuclear surface (Fig. 1).
The nucleus measures 21 pm in length and is totally filled with homogeneous, compact
chromatin. The nuclear base forms a cavity which is occupied by the anterior tip of the major
mitochondrial derivative (Fig. 2). The flagellum consists of the axoneme, two mitochondrial
derivatives and two accessory bodies. In transverse section, the flagellum is oval, with the
axoneme and major mitochondrial derivative occupying opposite ends. The axoneme follows the
9+9+2 pattern, with nine accessory microtubules filled with dense material surrounding the nine
doublets and two central microtubules, of which, in a perfectly positioned axoneme, the left one
is also dense (Fig. 3).
Figs 1-3 — Spermatozoon of Cosmopolites sordidus. 1: Longitudinal section of the acrosome. The nucleus (n), extra-
acrosomal layer (e), acrosomal vesicle (a) and perforatorium (p) can be identified, x 70 000. 2: Longitudinal
section of a late spermatid with the nucleus (N), major mitochondrial derivative (M), minor mitochondrial
derivative (m), the expansion of accessory bodies (ab), the major expansion (Eb) and minor expansion (eb) of the
accessory bodies. Arrows show the mitochondrial cristae and the arrow heads, the microtubules, x 46 800.
3: Transverse section of the flagellum, including the axoneme (ax), large mitochondrial derivative (M), minor
mitochondrial derivative (m), the major expansion (Eb) and minor expansion (eb) of the accessory bodies (ab).
The arrow head indicates the central microtubule not filled with electron dense material, x 82 000.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
299
The major mitochondrial derivative has a diameter equal to twice that of the minor
derivative and is almost entirely occupied by material in a paracrystalline arrangement (Fig. 3),
although small cristae can still be found (Fig. 2). Cristae are larger in the minor mitochondrial
derivative.
The accessory bodies lie parallel to the axoneme, between this structure and the
mitochondrial derivatives. Each body has a dense region and a less compact expansion, which is
larger for one of the accessory bodies (Fig. 3).
DISCUSSION
The acrosome of Cosmopolites sordidus is similar to those of the majority of the
Curculionoidea [5, 6] in relation to structure and localization but the subacrosomal lamella was
not observed between acrosome and nucleus, as described for other Curculionidae [5].
Crystalline arrangements of acrosomal contents have been found in various insects [4, 13, 15],
but no such formation occurs in the acrosome of C. sordidus.
The nucleus, as in most Curculionoidea, is long and thin, containing densely compact and
homogeneous chromatin. This is contrary to the description for Stophilus oryzae L., on which
the taxonomic characteristics of the subfamily Rhynchophorinae has been established. This
species is claimed not to have a compact nucleus [5],
Mitochondrial derivatives of different sizes have been reported for all known
Curculionoidea [6-9, 16]. The internal structures of the two mitochondrial derivatives are similar
to those described for S. multistriatus [16] and various families of Curculionoidea [5]. The
insertion of the major mitochondrial derivative into a cavity of the nucleus is the condition
accepted for most Curculionoidea, with the exception of 5. oryzae [5]. Again, in relation to this
species, the mitochondrial derivative was described as being lateral in relation to the nucleus.
The axoneme has the typical arrangement of microtubules described for many insects [1,
3-5, 8, 11, 14]. However, nine accessory microtubules and one of the central microtubules
contain dense material in C. sordidus, while other insects have dense material in both central
microtubules, as well as in the nine peripheral elements [3, 11-13, 15].
The accessory bodies of C. sordidus are very similar to these structures in other
Curculionoidea previously described [5], They are not, however, a uniform feature in all
coleopterans, often varying in shape, as in the rounded bodies, subdivided into distinct medullar
and cortical regions, found in Divales bipustulatus [10]. They may even be lacking in Dermestes
frischii [7],
The expansions of these bodies are less dense and of unequal size in most Curculionidae
while in other species they are equal U-shaped as in S. multistriatus [9] and Curculio elephas
[5]. This expansions may also not exist, as in D. bipustulatus [10].
The analysis of C. sordidus has shown that this species is very similar to the other families
of Curculionoidea previously studied [5], However this species differs from S. oryzae which
was analysed as a representative of the Rhynchophorinae. In C. sordidus, the acrosome is
implanted in an anterior depression of the nucleus, while this depression is much shallower or
even non-existent in other Curculionidae. Also, there is no subacrosomal lamella in this species,
as has been described for other members of the subfamily. The nucleus of C. sordidus is
densely compacted and has a concavity at its base in which the larger mitochondrial derivative is
anchored. This arrangement of the flagellar organelles is also in disagreement with the
description of S. oryzae, where the mitochondrial derivative was found laterally placed in
relation to a nucleus with diffuse chromatin.
In view of these conflicting features, we believe that other species should be investigated
so as to confirm the structures which may be adopted as taxonomic characteristics of the
Rhynchophororinae.
300
J. LINO NETO & H. DOLDER : COSMOPOLITES SORDIDUS (COLEOPTERA, INSECTA)
REFERENCES
I . BaCCETTI, B.. 1972. — Insect sperm cell. Advances in Insect Physiology, 9: 315-397.
2. BACCETTI, B & Daccordi, M., 1988. — Sperm structure and phylogeny of the Chrysomelidae. In: P. Jouvet, E.
Petitpierre & T. H. Hsiao, Biology of Chrysomelidae. New York, Kluwer Academic Press: 357-378.
3. BAO, S. N., 1987. — Estudo ullra-estrutural da espermiogenese no mutante “ olho-roseo ” camparativo a linhagem
selvagem de Ceratitis capitata Weidmann ( Diptera , Tephritidae). Tese de Mestrado. Universidade Estadual de
Campinas, Campinas, Brazil: 1-94.
4. BAo. S. N., Quagio-Grassiotto, I. & DOLDER, H., 1989. — Acrosome formation in Ceratitis capitata (Diptera,
Tephritidae). Cytobios 58: 93-100.
5. Burrini, A. G. Magnano, L. Magnano, A. R. Scala, C. & Baccetti, B., 1988 — Spermatozoa and phylogeny of
Curculionoidea (Coleoptera). International Journal of Insect Morpholology, 17: 1-50.
6. Gassner, G., Childress, D. & Klemetson, D. J., 1975. — Spermiogenesis in boll weevil, Anthonomus grandis
(Boheman) (Coleoptera: Curculionidae). International Journal of Insect Morphology and Embryology , 4:
115-125.
7 . Hodges, R. J., 1982. — Ultrastructure of the somatic and germ cells of the testes of Dermestes frischii Kugelann
(Coleoptera: Dermestidae). International Journal of Insect Morphology and Embryology , 11: 235-253.
8. Jamieson, B. G. M., 1987. — Ultrastructure and Phylogeny of Insect Spermatozoa. Cambridge, Cambridge
University Press.
9. Jumper, G. A. & Cannon Jr., W. N., 1975. — Spermatogenesis in the smaller european elm bark beetle, Scolytus
multistriatus. Annals of the Entomological Society of America, 68: 733-740.
10. Mazzini, M., 1976. — Giant spermatozoa in Divales bipustulatus F. (Coleoptera: Cleridae). International Journal
of Insect Morphology and Embryology, 5: 107-115.
1 1 . MESSIAS Jr. N. S., 1990. — Aspectos ultra-estruturais da espermiogenese de Chrysomya megacephala Fab ( Diptera :
Calliphoridae). Tese de Mestrado, Instituto de Bioiogia, Universidade Estadual de Campinas, Campinas,
Brazil: 1-115.
1 2. Phillips, D. M., 1970. — Insect sperm: structure and morphogenesis. Journal of Cell Biology, 44: 243-277.
1 3. Quagio-Grassiotto, I., 1983. — Citodiferenciagao ultra-estrutural durante a espermiogenese normal de Ceratitis
capitata Weidmann ( Diptera.Tephritidae ). Tese de Mestrado, Instituto de Bioiogia, Universidade Estadual de
Campinas, Campinas, Brazil: 1-93.
14. Shay, J. W., Dobson. W. J., Simmons, E. E., Biesele, J. J. & Breland, O. P., 1969. — Subunits of flagellar
accessory tubules. Tissue and Cell, 1: 593-596.
15. Warner. F. D., 1971. — Spermatid differentiation in the blowfly Sarcophaga bullata with particular reference to
flagellar morphogenesis. Journal of Ultrastructure Research , 35: 210-232.
16. Werner, G., 1965. — Untersuchungen uber die Spermiogenese beim Sandlaufer, Cicindela campestris (L.).
Zeitschrift fiir Zellforschung, 66: 255-74.
Source . MNHN. Paris
Phylogenetic Significance of Axonemal Ultrastructure
Examples from Diptera and Trichoptera
Romano DALLAI * & Bjorn A. AFZELIUS **
* Department of Evolutionary Biology,
University of Siena, 1-53100 Siena, Italy
** Department of Ultrastructure Research,
Biology E 4, Stockholm University, S-106 91 Stockholm, Sweden
ABSTRACT
The organization of the axoneme in various insects has been examined by using a tannic acid-containing fixative and
been found to differ in a systematic fashion between different insect orders. Within each order, both protofilament number
and other axonemal characteristics are relatively constant, however. There are two exceptions to this statement: Diptera
and Trichoptera. These two orders were therefore examined in order to see whether the axonemal organization of the
various species reflects their systematic position. About 24 dipteran species and 35 trichopteran species were examined in
this respect. Axonemal structure in Brachycera is similar to that of Tipulomorpha, which indicates a derivation of
Brachycera from that group. Mycetophilidae, rather than Tipulomorpha, seems to be the most primitive dipteran taxon, as
judged from the axonemal organization. Axonemes of an examined bibionid species (but not of other members of
Bibionomorpha) resemble those of culicomorph species. The axoneme in Trichoptera is characterized by a loss of the
outer dynein arms and by the presence of accessory tubules with more than 16 protofilaments. The least modified sperm
axoneme is found in the primitive families Rhyacophilidae and Glossosomatidae, which have motile spermatozoa and
have 17 and 18 protofilaments, respectively. These families, as other members of Integripalpia (but not Hydroptilidac),
have inner dynein arms only and sperm motility, whereas members of Annulipalpia have neither inner nor outer dynein
arms and no progressive motility. The variability in axonemal organization is greater in Annulipalpia than in
Integripalpia.
RESUME
La signification phylogenetique de 1’ultrastructure des axonemes: exemples chez les Dipteres et
les Trichopteres
L’organisation de faxondme a ete etudiee, grace a une fixation & facide tannique, chez dc nombreux insectes et s’est
montree differente parmi les differents ordres d’Insectes, en accord avec la systematique. A finterieur de chaquc ordre,
toutefois, le nombre de protofilaments et les autres caracteristiques de faxoneme sont relativement constants, avec deux
exception & cette regie, les Dipteres et les Trichoptdres. Ces deux ordres ont done ete examines pour determiner si
forganisation dcs axonemes reflete leur position systematique. Environ 24 esp£ces de Dipteres et 35 especes de
Trichopteres ont 6t6 dtudiees. La structure de faxoneme chez les Brachyc&res est similaire ^ celle des Tipulomorphes, ce qui
indique une origine des Brachyceres dans ce groupe. D’apr£s forganisation de faxoneme. les Mycetophilidae, plutot que
les Tipulomorphes, semblent etre le taxon de Dipteres le plus primitif. Les axonemes d’une espece de Bibionidae etudiee
(mais pas des autres membres des Bibionomorphes) ressemblent & ccux des Culicomorphes. L'axoneme des Trichopteres
est caract£ris6 par la perte des bras de dyndine externes et par la presence de tubules accessoires ayant plus de 16
Dallai, R., & Afzelius, B. A., 1995. — Phylogenetic significance of axonemal ultrastructure: examples from
Diptera and Trichoptera. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny
and Taxonomy. Mem. Mus. natn. Hist, nat., 166: 301-310. Paris ISBN: 2-85653-225-X.
302
R. DALLAI & B. A. AFZELIUS: DIPTERA & TR1CHOPTERA (INSECTA)
protofilaments. L’axon£me de spermatozoi'de le moins modify est trouve dans les families primitives Rhyacophilidae et
Glossosomatidae, qui ont des spermatozoides motiles, el respectivement 17 et 18 protofilaments. Ces families, comme les
autres Integripalpia (mais pas les Hydroptilidae), ont seulement des bras de dyn6ine internes et des spermatozoides
motiles, alors que les Annulipalpia n'ont ni bras internes ni bras extemes de dyneine et n’ont pas de motilite permettant la
progression. La variability de l’organisation des axonemes est plus grande chez les Annulipalpia que chez les
Integripalpia.
Most insect species have a sperm flagellum that contains a set of microtubules, which
surround the nine microtubular doublets and which are named accessory tubules [21]. The
axoneme is then said to have a 9+9+2 structure, which is a shorthand notation of 9 accessory
tubules, 9 doublets and 2 central microtubules. It was long believed that 9+9+2 axonemes are of
uniform appearance within the insect class, but this is far from true.
In a study from 1990 [4] we describe the axoneme of 49 insect species, representing 20
insect orders and show that there is a considerable diversity between different insects. Since that
date we have extended the examination to many more species. In fact, axonemes from many
species in most orders have been examined by now and this has been done with a new fixation
technique that makes it meaningful to study the axoneme at high magnifications. In the resulting
micrographs it is possible to count the number of protofilaments in the accessory tubules or in the
microtubular doublets and singlets, and it is possible to see a substructure in the lumen of the
microtubules or in the so called intertubular material (Intertubular material appears as one or two
lumps of electron dense material external to the doublets and between the accessory tubules).
The main findings are summarized in Table 1. It can be concluded that diversity in axonemal
structure is related to the systematic position of the animal. Many apomorphic traits can thus be
found, such as accessory tubules with 17 protofilaments in the order Phasmida, accessory tubules
with 13 protofilaments and no intertubular material in the order Ephemeroptera, and aberrant
flagella in the order Thysanoptera. There are also some synapomorphic traits, such as accessory
tubules with an elliptic cross-section recorded from the three insect orders Psocoptera,
Mallophaga and Anoplura.
Although there is a marked diversity in axonemal structure within the class Insecta, there is
a relative invariability within each insect order. There are two exceptions to this statement: Orders
Diptera and Trichoptera have families and suborders with markedly different axonemal patterns.
The purpose of the present study has been to examine the sperm tail from insects in these two
orders in an attempt to see whether any phylogenetic conclusions can be drawn from the
ultrastructural data.
MATERIAL AND METHODS
Spermatozoa from the insect species listed in Table 2 have been fixed for one or several days in a fixative that
consists of 2 % glutaraldehyde, 1 % tannic acid, and 1.8 % sucrose in a 0.1 M phosphate buffer. This fixative is a
modification of the fixative introduced by Mizuhira & Futaesaku [23], Post-fixation and block-staining was performed
with 1 % uranyl acetate in distilled water. No osmium tetroxide fixation was used. After dehydration in ethanols and
embedding in epoxy resins, sections have been made and examined at a direct magnification of 50 000 or higher.
Tannic acid is thought to act as a mordant that encapsulates proteins; uranyl acetate is thought to contrast the
tannic acid. Globular protein such as a tubulin in the microtubular wall will become visible as a lucid spot surrounded by an
electron dense shell. Fibrous proteins, such as those in the spokes, may however appear as electron dense structures.
Interpretation of the electron micrographs is not trivial.
OBSERVATIONS
Diptera
The axoneme of Orfelia sp. (Family Mycetophilidae) is of the 9+9+2 type. Its doublets have
both inner and outer dynein arms. The accessory tubules have 16 protofilaments and the
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303
intertubular material between two accessory tubules appears as two electron dense masses, i.e. a
smaller unit attached to the accessory tubule and a larger one attached to the doublet; occasionally
the two masses are confluent and form a single mass. In these respects the axoneme resembles
that of examined members of several insect orders, such as Zygentoma, Plecoptera, Embioptera,
Orthoptera, Dermaptera, Megaloptera, Raphidioptera, Planipennia, Coleoptera, Lepidoptera, and
Hymenoptera. The cross-sectioned flagellum of an Orfelia spermatozoon differs from that of the
other insects in having a single small mitochondrial derivative and differs also in other respects
from those within other insect orders, but the axoneme has a morphology that can be regarded as
plesiomorphic for insects.
The axoneme from some other members of the Mycetophilidae family has other patterns:
Tarnania 2+9+2 and Exechia seriata 7+9+2 (both with 16 protofilaments in their accessory
tubules) (Fig. 1), Keroplatus reaumuri 9+2, and Boletina sp. 9+9+2 with accessory tubules
consisting of 15 protofilaments (Fig. 2). Such a variety of different axonemal patterns has not
been described from any other insect family apart from Cecidomyiidae, in which there are highly
aberrant spermatozoa.
The three examined members of family Chironomidae all have an axoneme of the 9+9+2
type and the accessory tubules have 15 protofilaments. The accessory tubules of examined
members of the three families Dixidae, Culicidae (Fig. 3), and Bibionidae (Fig. 4) also have 15
protofilaments. However, their axoneme is of the 9+9+1 type as the two central microtubules are
replaced by a central rod or cylinder in the three families.
In the examined species of families Trichoceridae and Tipulidae (Fig. 5) the axoneme is of
the 9+9+2 type but the accessory tubules have 13 protofilaments. In this respect they resemble all
Brachycera species examined so far [15, 24] (Fig. 6). Another characteristic shared by the
axonemes in Tipulidae, Trichoceridae and Brachycera is the prominence of the spoke head which
usually seems to be double. Within Brachycera the intertubular material contains a straight row of
inclusion bodies [15].
Trichoptera
Most specialists on Trichoptera agree that the order can be divided into two subgroups,
Integripalpia and Annulipalpia, although some uncertainty remains as to whether families
Rhyacophilidae, Glossosomatidae and Hydroptilidae belong to one suborder or the other. These
three families are usually regarded as the most primitive ones within the Trichoptera. In the family
Rhyacophilidae the axoneme has a 9+9+2 structure, the doublets have an inner dynein arm but no
outer one, and the accessory tubules have 17 protofilaments along most of their length (Fig. 7),
although 16 close to the posterior end. In the examined member of the family Glossosomatidae
the axoneme has the same characteristics, except that there are 18 rather than 17 protofilaments
along most of the length of the accessory tubules. Spermatozoa from Rhyacophilidae and
Glossosomatidae show progressive motility, whereas spermatozoa from the examined members
of the family Hydroptilidae lack a flagellum and are immotile.
The integripalpian family Leptoceridae also has a 9+9+2 axoneme, inner dynein arms only,
and accessory tubules with 18 protofilaments (Fig. 8). The axoneme differs from that in
Glossosomatidae, however, in that the two central microtubules always occupy an eccentric
position. Axonemes in the three Integripalpia families Limnephilidae (Fig. 9), Goeridae and
Odontoceridae (Fig. 10), also resemble those in the family Glossosomatidae, although the number
of protofilaments is 19 along most of the length, decreasing to 16 distally. The two members of
the family Sericostomatidae, finally, have 20 protofilaments proximally, the highest number
recorded from any insect or other animal. Posteriorly the number decreases to 16 near the distal
end. An accessory body lies lateral to the axoneme.
304
R. DALLAI & B. A. AFZELIUS: D/PTERA & TRICHOPTERA ( INSECTA )
TABLE 1. — Some characteristics of the sperm tail axoneme in examined members of insects trom the various insect orders
Abbreviations in the Table are: AT = accessory tubule, IM = intertubular material, OA = outer dynein arms, pfs =
protofilaments, the formula 9+9+2 signifies the number of accessory tubules, microtubular doublets, and central
microtubules, respectively.
Figs 1-6. Axonemes from various dipterans: 1: Exechia seriata; 2: Boletina sp.; 3: Culex pipiens; 4: Bibio sp.;
5: Tipula sp.; 6: Scatophaga sp. Scale bar 0.1 pm.
Source : MNHN . Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
305
Source : MNHN. Paris
306
R. DALLAI & B. A. AFZELIUS: D1PTERA & TRICHOPTERA (INSECTA)
Table 2. — Species investigated and sources of data
Group, Species Family Reference
DIPTERA NEMATOCERA
Orfelia sp.
Exechia seriaia (Meigen)
Boletina sp.
Keroplatus reaunturii Dufour
Asphondylia ruebsaameni Kertesz
Sciara sp.
Chironomus (three unidentified species)
Cul ex pipiens L.
Anopheles maculipennis Meigen
Dixa sp.
Bihio sp.
Trichocera hiemalis De Geer
Tipula sp.
DIPTERA BRACHYCERA
Bombylius sp.
Ramphomyia sp.
Drosophila melanogaster Meigen
Bacirocera ( =Dacus) oleae (Gmelin)
Ce rat it is capitata (Wied.)
Scatophaga sp.
Calliphora vomitoria L.
Gasterophilus intestinalis De Geer
TRICHOPTERA INTEGRIPALPIA
Rhyacophila (Rhyacophila) foliacea Moretti
Rhyacophila ( Rhyacophila ) dorsalis (Curtis)
Rhyacophila (Pararhyacophila) italica Moretti
Rhyacophila (Hyporhyacophila) tristis Pictet
Catagapetns ni grans McLachlan
Stactobia caspersi Ulmer
Orthotrichia costalis (Curtis)
Hydroptila aegyptia Ulmer
Hydroptila angulata Mosely
Hydroptila forcipata Eaton
Hydroptila tineoides Dalman
Oecetis furva Rambur
Leptocerus tineiformis Curtis
Mystacides azure a L.
Odontocerum albicorne Scopoli
Sericostoma italicum Moretti
Sericostonia pedemontanum McLachlan
Silo mediterraneus saturniae Moretti
Leptodrusus budtzi Ulmer
Limnephilus bipunctatus Curtis
Linmephilus rhombicus (L.)
Glyphotaelius pellucidus Retzius
Potamophylax cingulatus (Stephens)
Melampophylax melampus McLachlan
Stenophylax permistus McLachlan
Micropterna sequax McLachlan
Chaetopteryx gessneri McLachlan
TRICHOPTERA ANNULIPALPIA
Philopotamus ludificatus McLachlan
Philopotamus montanus Donovan
Wormaldia occipitalis Pictet
Wormaldia copiosa McLachlan
Plectrocnemia geniculata McLachlan
Polycentropus mortoni Moseley
Polycentropus irroratus Curtis
Cyrnus trimaculatus Curtis
Hydropsyche pellucidula Curtis
Figs 7-12. Axonemes of some trichopterans. 7: Rhyacophila foliacea; 8: Leptocerus tineiformis', 9: Potamophylax
cingulatus; 10: Odontocerum albicorne ; 11: Wormaldia copiosa; 12: Philopotamus montanus. Scale bar 0.1 pm.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
307
Source : MNHN . Paris
308
R. DALLAI & B. A. AFZELIUS: DIPTERA <6 TRICHOPTERA ( INSECTA )
Members of the suborder Annulipalpia lack inner and outer dynein arms. Hence they have
no regular, flagellar movements. Weak vibrations have been observed in Philopotamus
spermatozoa, when remaining in the testicular fluid, however. The philopotamid species
Wormaldia copiosa has a unique axonemal pattern, 13+13+v, where v stands for a cylindrical
vesicle along the central axis of the axoneme (Fig. 1 1), whereas another member of the same
genus, W. occipitalis , has a similar axoneme although with the formula 9+9+v. In examined
members of Philopotamus the axonemal pattern is 9+9+7 and the seven central microtubules
consist of 13 or 14 protofilaments (Fig. 12).
The family Polycentropodidae is related to the Philopotamidae. Its members have lost the
accessory tubules and have a 9+7 axoneme, in which the central microtubules have 13 or 14
protofilaments. Also related to these families are the Hydropsychidae, whose members have a
highly aberrant sperm morphology. Whereas a section just distal to the centriole shows nine
axonemal doublets, sections posterior to that level show a great number of microtubular singlets
and doublets, some enclosed in the cell body, others contained in finger-like rodlets projecting
from the cell body.
DISCUSSION
Diptera
The suborder Nematocera is generally considered to be more primitive than the Brachycera
and its subgroup Tipulomorpha (= Tipulidae + Trichoceridae) and to be the sister group to all
other Diptera [5, 20]. Tipulomorpha is thus regarded as the lowest branch of the dipteran
phylogenetic tree. Our data on the sperm axoneme is not compatible with this opinion. Rather, it
seems to us that the family Mycetophilidae is the most primitive dipteran taxon. Only in this
family has the plesiomorphic axonemal pattern - 9+9+2 and accessory tubules with 16
protofilaments - been found. Whether data from molecular biology will confirm that
Mycetophilidae is the most primitive extant dipteran group, or whether data will show the
Tipulimorpha to be so, is a matter that probably will be settled soon.
As mentioned above, some axonemal characteristics are shared between the examined
members of Tipulomorpha and the Brachycera: accessory tubules with 13 protofilaments and
spoke heads that appear double. We believe that these are synapomorphies shared by these groups
and that Brachycera is derived from the tipulomorph group. According to HENNIG [20]
Nematocera contains four large groups: Tipulomorpha, Psychodomorpha, Culicomorpha
(containing, amongst other families, Dixidae, Culicidae, Simulidae and Chironomidae), and
Bibionomorpha (with Bibionidae, Mycetophilidae, Cecidomyidae, Sciaridae). Members of
Psychodoidea have an axoneme of the 9+9+0 type [14], but nothing is known about the number
of protofilaments in the accessory tubules.
The examined members of the culicomorph families Dixidae, Culicidae, and Chironomidae
share an axonemal character: accessory tubules with 15 protofilaments. This number of
protofilaments has been found also in the examined member of Bibionidae and in the mycetophilid
Boletina, but otherwise is a rare feature. Whereas the chironomids have a 9+9+2 axoneme and the
simulid a 9+9+3 one [4], culicid and bibionid flies have a central rod rather than two central
microtubules, a 9+9+' 1' or sometimes a 9+9+0 pattern. This finding indicates that Bibionidae
may be closer related to Culicomorpha than to other families of Bibionomorpha or that
Culicomorpha is derived from Bibionomorpha . The sperm tails of members of Cecidomyiidae
and Sciaridae, finally, have highly aberrant axonemes.
Trichoptera
Based, inter alia, on their free-living larvae, the families Rhyacophilidae, Glossosomatidae,
and Hydroptilidae are considered to be the most primitive trichopterans. The loss of a sperm tail in
Hydroptilidae prevents any comparative study based on those characters that are treated here. The
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309
relatively few modifications from the plesiomorphic insect axoneme in the other two families is
compatible with the opinion that they are primitive.
The division of Trichoptera into two suborders, Integripalpia and Annulipalpia, is based on
the morphology of the palps. If only the sperm axoneme were considered, Trichoptera could also
be divided into two groups, one possessing inner dynein arms and flagellated spermatozoa, the
other without these characteristics. This division would be identical to the classical one and the
two families Rhyacophilidae and Glossosomatidae would be classified as belonging to
Integripalpia. The inclusion of these families (and of Hydroptilidae) into Integripalpia is also
advocated by ROSS [25].
The various species of Integripalpia have axonemal patterns that are characteristic of the
families to which they belong. No significant differences have been found between the three
families Limnephilidae, Goeridae and Odontoceridae. Within Annulipalpia there may be a greater
variability between members of the same family or even the same genus. Since the flagellum is
unable to propagate the spermatozoon, any mutation that changes the pattern from a regular
9+9+2 pattern can be regarded as neutral, in that it will not affect sperm functions. A greater
diversity of the axonemal pattern can hence be expected and has thus been recorded.
ACKNOWLEDGEMENTS
We are grateful to Professor MORETTl for collecting and determining most of the caddisflies and to Professor Hippa
for determining some of the dipteran Hies. The study has been supported by a grant from Murst 60% to R.D.
REFERENCES
1. Afzelius, B. A., Bellon, P. L. & Lanzavecchia, S., 1990. — Microtubules and their protofilaments in the
flagellum of an insect spermatozoon. Journal of Cell Science. 95: 207-217.
2. Afzelius, B. A., Bellon. P. L., Dallai, R., & Lanzavecchia, S.. 1991. — Diversity of microtubular doublets in
insect sperm tails: A computer-aided image analysis. Cell Motility and the Cytoskeleton , 19: 282-289.
3. Afzelius, B. A. & Dallai, R., 1994. — Characteristics of the flagellar axoneme in Neuroptera, Coleoptera, and
Strepsiptera. Journal of Morphology. 219: 15-20.
4. Baccetti, B., Dallai, R., Giusti, F. & Bernini, F., 1974. The ”9+9+3” spermatozoon of simuliid Diptera. Journal of
Ultrastructure Research. 46: 427-440.
5. BlCKEL, D. J., 1982. — Diptera. In: S. P. Parker, Synopsis and Classification of Living Organisms. New York,
McGraw Hill. 2: 563-599.
6. Dallai, R. & Afzelius, B. A., 1990. — Microtubular diversity in insect spermatozoa: Results obtained with a new
fixative. Journal of Structural Biology , 103: 164-179.
7. Dallai, R. & AFZELIUS, B. A., 1991. — Sperm flagellum of Dacus oleae (Gmelin) (Tephritidae) and Drosophila
melanogaster Meigen (Drosophilidae). International Journal of Insect Morphology and Embryology, 20: 215-
222.
8. Dallai, R. & Afzelius, B. A., 1991. — Sperm flagellum of insects belonging to orders Psocoptera, Mallophaga and
Anoplura. Ultrastructural and phylogenetic aspects. Bollettino di Zoologia, 58: 211-216.
9. Dallai, R. & Afzelius, B. A., 1993. — Development of the accessory tubules of insect sperm flagella. Journal of
Submicroscopic Cytology and Embryology, 25: 499-504.
10. Dallai, R. & Afzelius, B. A., 1994. — Sperm structure of Trichoptera. I. Integripalpia: Limnephiloidca.
International Journal of Insect Morphology and Embryology, 23: 197-209.
11. Dallai, R. & Afzelius, B. A., 1994. — The spermatozoon of Trichoptera. II. The aflagellate spermatozoa of
Hydroptila, Orthotrichia , and Stactobia (Hydroptilidae). International Journal of Insect Morphology and
Embryology, 24: 161-170.
12. Dallai, R., Afzelius. B. A., Lanzavecchia, S. & Bellon, P. L., 1991. — Bizarre flagellum of thrips spermatozoa
(Thysanoptera, Insecta), Journal of Morphology, 209: 343-347.
13. Dallai, R., Afzelius, B. A., Lanzavecchia, S. & Bellon, P. L., 1993. — Native microtubules with a variable
number of protofilaments. Cell Motility and the Cytoskeleton, 24: 49-53.
14. Dallai, R., Baccetti, B., Mazzini, M. & Sabatinelli, G., 1984. — The spermatozoon of three species of
Phlebotomus (Phlebotominae) and the acrosomal evolution in nematoceran dipterans. International Journal of
Insect Morphology and Embryology 13: 1-10.
310
R. DALLAI & B. A. AFZELIUS: DIPTERA & TR/CHOPTERA (INSECTA)
15. Dallai, R., Bellon, P. L., Lanzavecchia, S. & Afzelius, B. A., 1994. — The dipteran sperm tail: Ultrastructural
characteristics and phylogenetic considerations. Zoologica Scripta.22 : 193-202.
16. Dallai, R.. Lupetti, P. & Afzelius, B. A., 1994. — Sperm structure of Trichoptera. III. Hydropsychidae,
Polycentropodidae and Philopotamidae (Annulipalpia). International Journal of Insect Morphology and
Embryology, 24: 171-183.
17. Dallai, R., Lupetti, P. & Afzelius, B. A., 1994. — Sperm structure of Trichoptera. IV. Rhyacophilidac and
Glossosomatidae. International Journal of Insect Morphology and Embryology, 24: 185-193.
18. Dallai, R.. Mazzini, M. & Lupetti, P., 1993. — The spermatozoa of Contarinia , Alloc ontarinia, Lestodiplosis and
Myricomyia (Diptera, Cecidomyiidae) with considerations on the systematic relationships within the group.
Dolletino di Zoologia, 60: 7-18.
19. Dallai, R., Xue, L. & Yin, W.. 1992. — Flagellate spermatozoa of Protura (Insecta, Aptcrygota) are motile.
International Journal of Insect Morphology and Embryology. 21: 137-148.
20. HENNIG, W., 1969. — Die Stammesgeschichte der Insekten. Frankfurt am Main, Waldemar Kramer: 1-436.
21. Jamieson, B. G. M., 1987. — The Ultrastructure and Phylogeny of Insect Spermatozoa. Cambridge, Cambridge
University Press: 1-320.
22. Lanzavecchia, S., Dallai, R., Bellon, P.L. & Afzelius, B. A., 1991. — The sperm tail of a gall midge and its
microtubular arrangement studied by two strategies of image analysis (Cecidomyiidae, Diptera, Insecta). Journal
of Structural Biology, 107: 65-75.
23. Mizuhira, V. & Futaesaku, Y., 1972. — New fixation for biological membranes using tannic acids. Acta
Histochemica and Cytochemica, 5: 233-236.
24. Phillips, D. M., 1966. — Substructure of flagellar tubules. Journal of Cell Biology , 31: 635-638.
25. ROSS, H. H., 1967. — The evolution and past dispersal of the Trichoptera. Annual Review of Entomology, 12: 169-
206.
Source : MNHN. Paris
Vertebrates
Source : MNHN. Paris
Source : MNHN, Paris
Comparative Morphology of the Sperm
in Chondrichthyan Fishes
Sho TANAKA *, Hand KUROKAWA * & Masako HARA **
* School of Marine Science and Technology, Tokai University
** Ocean Research Institute, University of Tokyo
ABSTRACT
The external features of the spermatozoa of 35 species belonging to 19 families in 12 orders were observed with light
and scanning electron microscopes. Six characters, i.c., type of sperm aggregate, number of helical gyres, total, head,
midpiece and flagellum length, were used as an operational taxonomic unit for the computation of the resemblance of the
sperm among species in a cluster analysis. The sperm of Orcctolobiformes, Lamniformes, Carcharhiniformes,
Torpediniformes and Rajiformes form sperm aggregates: spermatozeugmata and spermatophores. The sperm of 29 species
are helical from the tip of the head to the midpiece, but Chlamydoselachus anguineus , Dalatias lie ha, Etmopterus spp., and
Squatina japonica do not display a helical form. The sperm length ranges from 93 pm to 224 pm. The sperm of
holocephalans have a shorter head and longer flagellum in proportion to that of elasmobranchs. The results of the cluster
analysis suggest the external features of the sperm show a similarity within the genus and/or family.
RESUME
Morphologie comparee des spermatozoides des poissons Chondrichtyens
La morphologie externe des spermatozoides de 35 espcces appartenant £ 19 families dans 12 ordres a 6t6 observee en
microscopie photonique et & balayage. Six caractfcres, le type d'agregation des spermatozoides. le nombre de tour de
l’helicoide, la longueur du spermatozoide, de la tete, de la piece intermediate, du flagelle. ont ete utilises comme unites
taxonomiques opSrationnelles pour le calcul de la ressemblance des spermatozoides parmi les esp&ces par une methode
agglomerative. Les spermatozoides des Orectolobiformes, Lamniformes, Carcharhiniformes. Torpediniformes et
Rajiformes forment des agregations: spermatozeugmata et spermatophores. Les spermatozoides de 29 especes sont
hSlicoidaux & partir de l’extremite de la tete jusqu'a la piece intermediate, mais Chlamydoselachus anguineus, Dalatias
licha , Etmopterus spp. et Squatina japonica n’ont pas de spermatozoides helicoidaux. La longueur des spermatozoides
varie de 93 pm & 224 pm. Les spermatozoides des Holoc6phales ont une tete plus courte et un flagelle plus long que les
Elasmobranches. Les resultats de 1’ analyse par methode agglomerative suggerent que la morphologie externe des
spermatozoides est similaire & l’intdrieur des genres et/ou des families.
Chondrichthyans are a small group of about 900 species, compared with osteichthyans.
They include two subclasses, 14 orders, and 51 families [1, 9]. Recently, the systematics and
phylogeny of elasmobranchs have been considered using the external, skeletal and muscular
systems of the body, and new schemes have been presented [2, 12]. Female chondrichthyan
Tanaka, S., Kurokawa, H., & Hara. M.. 1995. — Comparative morphology of the sperm in chondrichthyan
fishes. In: Jamieson, B. G. M., AUSIO, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy.
Mem. Mus. natn. Hist, nat., 166 : 313-320. Paris ISBN : 2-85653-225-X.
314
S. TANAKA. H. KUROKAWA & M. HARA : CHONDR1CHTHYES
fishes have diverse and complex reproductive styles, which have also been used as characters tor
consideration of their systematics and phylogeny [3, 10]. In contrast, the reproduction of male
fishes is simple. Claspers, which are copulatory organs, differ in structure among species.
Therefore, the claspers have traditionally been used to provide characters in taxonomy and
systematics [5, 7],
During the last decade, electron microscopy (EM) has been refined. It provides new
information on the fine structure and function of organs, tissues and cells in various animals. The
sperm of many fishes has been observed with EM, and the interrelationships of fishes have been
discussed in relation to the structure of the spermatozoa [6, 8], However, comparative studies on
the sperm of chondrichthyan fishes are few [4], The present paper deals with the comparative
morphology of the sperm in 35 chondrichthyan fishes on the basis of observations with light and
scanning electron microscopes.
MATERIALS AND METHODS
Chondrichthyan fishes, belonging to 12 orders, 19 families and 35 species, were collected on the northeast coast
of the United States, and in Suruga Bay and adjacent waters of central Japan from 1990 to 1994 (Table 1). Spermatozoa,
seminal fluids and tissues were taken from various portions of the reproductive tract of the specimens just after the death.
For observing under light microscopy, they were fixed in 10% neutral formalin. Sperm smears were prepared by smearing
and air-drying seminal fluids on glass slides, and were used for measuring the dimensions of 30 sperm under Nomarski
differential interference microscopy. For scanning electron microscopy (SEM), they were immersed in a cold fixative
containing 2% paraformaldehyde and 2% glutaraldehyde in a 0.1 M cacodylate buffer (pH 7.3) with 10% sucrose added.
They were rinsed in the above buffer, post fixed in 2% osmium tetroxide in the same buffer, dehydrated in graded ethanol,
and displaced with t-butyl alcohol, frozen and dried in a vacuum desiccator. They were sputter-coated with gold, and
observed and photographed using an Akashi ABT-55 scanning electron microscope.
For numerical taxonomy on the sperm of each species, six characters of the sperm: type of sperm aggregate,
number of helical gyres, total length, head length, midpiece length, and flagellum length, were used as an operational
taxonomic unit (OTU). The following five types of sperm aggregate were recognized: 1. a solitary sperm or sperm clump,
2. single-layered spermatozeugma, 3. compound spermatozeugma, 4. “rice-grain" type of spermatophore, 5. atypical rod
shaped spermatophore [11]. The types were scored from 1 to 5, respectively. Data sets containing each character were
standardized; values of each character were calculated as units of standard deviation from the mean value of each character.
The correlation coefficient as a measure of the overall similarity was computed by comparison of each OTU pair. Clusters
of the OTUs were created under the unweighted pair-group method using arithmetic averages (UPGMA).
RESULTS
Observations with light and scanning electron microscopes.
Formation of sperm aggregates. The sperm were separate within secretions in the
epididymis of all species. In Orectolobus japonicus, 3 species of Lamniformes, 13 species of
Carcharhiniformes, Torpedo tokionis and Raja eglanteria, the sperm in the ductus deferens
formed clumps with heads adhering. They formed sperm aggregates in the ampulla ductus
deferentis as reported in [11], The type of sperm aggregate for each species is shown in Table 2.
Single layered spermatozeugmata were found in 10 species of Carcharhiniformes except Prionace
glauca and Sphyrna lewini, T. tokionis, and R. eglanteria (Fig. 1A). O. japonicus formed
compound spermatozeugmata (Fig. IB). The sperm aggregates of P. glauca, S. lewini,
Carcharias taurus, and Isurus oxyrinchus have been described in detail [11]. Alopias pelagicus
possessed rice-grain typed spermatophores. In Heterodontus japonicus and Squatina japonica, the
sperm gathered in clumps in the ductus deferens as in the above species, and retained this
arrangement in the ampulla ductus deferentis. Though Chlamydoselachus anguineus, 9 species of
Squaliformes, Rhinobatos schlegelii, and 2 species of Myliobatiformes made sperm clumps
temporarily, most of the sperm were solitary in the ampulla. The sperm of Chimaeriformes also
formed clumps in the ductus deferens by adhesion of the midpieces, a condition differing from
elasmobranchs. However, the sperm clumps were not found in the seminal fluid from the distal
end of the ampulla.
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
315
Fig. 1. — Sperm aggregate and the head and midpiece of sperm in several chondrichthyan fishes. A: Single layered
spermatozeugma of Galeus nipponensis. B: Compound spermatozeugma of Orectolobus japonicus. C: Sperm of
/surus oxyrinchus. D: Sperm of Mustelus griseus. E: Sperm of Sphyrna lewini. F: Sperm of Dalatias licha .
G: Sperm of Rhinobalos schlegelii. H: Sperm of Chimaera phantasma. Scale indicates 25 pm in A and B, and 5
pm in C to H.
Source : MNHN, Paris
316
S. TANAKA, H. KUROKAWA & M. HARA : CHONDRICHTHYES
Table 1. — Species and sample size of specimens from which sperm were obtained.
Heterodontiformes
Heterodontidae
Heterodontus japonicus 1
Orectolobiformes
Orectolobidae
Orectolobus japonicus 2
Lamni formes
Odontaspididac
Carcharias taurus 1
Alopiidae
Alopias pelagicus 1
Lamnidae
Isurus oxyrinchus 2
Carcharhiniformcs
Scyliorhinidae
Cephaloscy Ilium umbratile 2
Gale us eastmani 5
Galeus nipponensis 3
Triakidae
Hemitriakis japanica 4
Mustelus canis 1
Mustelus griseus 1
Mustelus manazo 2
Carcharhinidae
Carcharhinus plumbeus 1
Galeocerdo cuvier 1
Prionace glauca 3
Sphyrnidae
Sphyrna lewini 3
Hexanchiformes
Chlamydoselachidae
Chlamydoselachus anguineus 1
Squali formes
Squalidae
Centroscymnus owstoni 3
Dalatias licha 2
Deania calcea 2
Deania hisioricosa 5
Etmopterus brachyurus 3
Etmopterus molleri 1
Etmopterus pusillus 2
S qua l us brevirostris 2
Squalus japonicus 3
Squatiniformes
Squatinidae
Squatina japonica 1
Rhinobatiformes
Rhinobatidae
Rhinobatos schlegelii 1
Torpcdiniformes
Torpedinidae
Torpedo tokionis 2
Raj i formes
Rajidae
Raja eglanteria 2
Myliobatiformes
Urolophidae
Urol op bus aurantiacus 2
Myliobatididae
Myiiobatis tobijei 1
Chi maeri formes
Chimaeridae
Chimaera phantasma 1
Hydro lagus mitsukurii 2
Rhinochimaeridae
Rhinochimaera pacifica 1
External features of sperm. The sperm of 35 species consisted of a head including an
acrosome, a midpiece and a slender flagellum. The total length of the sperm ranged from 93 |im in
Galeocerdo cuvier to 224 pm in S. japonica (Table 2). The external features of the head and
midpiece of the sperm varied in each species (Fig. 1C-H). The head in most species was helical,
but in C. anguineus , Dalatias licha, three species of the genus Etmopterus, and S. japonica, the
sperm in the ampulla did not display a clear helical form in the head (Fig. IF). The tip of the head
was bent like a gaff (Fig. 1C, H). The number of gyres of the sperm with the helical form ranged
from 3 to 24 (Table 2). The head length in elasmobranchs was more than 30 pm, while that in
holocephalans was less than 25 pm. The longest sperm head was 93 pm in Centroscymnus
owstoni. The standard deviation of the head length in each species was 1.03 to 2.91 pm. The
proportion of the head to the total length ranged from 7 to 45 % (Table 2).
The midpiece was much shorter than the head in elasmobranchs. In contrast, two species of
Chimaeridae had a long midpiece compared to the head. The width of the midpiece was slightly
thicker than the head (Fig. 1C-H). The midpiece length ranged from 6 to 21 pm, and the
Source . MNHN. Paris
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
317
Table 2. — Summary of the measurement and condition of the sperm in 35 chondrichthyan fishes.
Type of sperm aggregate: 1, Solitary or sperm clumps, 2, Single-layer spermatozeugmata, 3, Compound
spermatozeugmata, 4, Spermatophores (rice-grain type), 5, Spermatophores (atypical rod). Values in parentheses
indicate a ratio to total length.
disparities among species were small. The standard deviation of the midpiece length in each
species was 0.37 to 1.71 pm. The flagellum length ranged from 49 pm in G. cuvier to 143 pm in
S. japonica. The standard deviation of the flagellum length was 1.42 to 3.45 pm. In Isurus
oxyrinchus, C. owstoni and Urolophus aurantiacus, the flagellum was almost the same length as
the head. The proportion of the flagellum in Chimaeriformes was more than 75%.
Source
318
S. TANAKA, H. KUROKAWA & M. HARA : CHONDRICHTHYES
Numerical taxonomic analysis.
The cluster analysis indicated the similarity of external features of the sperm within the
genus and/or family (Fig. 2). The sperm of the two species in Gale us and Squalus were similar,
while the similarity in two species of Deania was low compared to the former genera. In
Etmopterus and Mustelus, the sperm of two of the three species were much alike, but that of each
remaining species was similar to that of other genera. The external features of the sperm in
Squalus spp. were very different from those in the other genera of Squalidae. The similarity of the
families, except Scyliorhinidae, within the Carcharhiniformes was almost equal to that of the
genera, except Squalus, within the Squalidae. The cluster analysis also divided the 35 species into
4 groups (Fig. 2).
-0.4
-0.2
OTU CORRELATIONS
0.2 0.4
0.6
I
0.8
■ r '
1.0
“1
"T
FlG. 2. — Phenogram of 35 species in chondrichthyan fishes, using six characters of sperm morphology in cluster
analysis. Three letter codes at right indicate species name (see Table 2). Cophenctic correlation coefficient: 0.820.
Group I CSU to SLJ. This group included four orders, five families; Scyliorhinidae of the
Carcharhiniformes, Heterodontiformes, two families of Chimaeriformes, and Squaliformes. Only
the Scyliorhinidae was distant from the other families of Carcharhiniformes. The sperm of three
species of Scyliorhinidae were much longer than those of the other species of Carcharhiniformes.
Cephaloscyllium umbratile , in which the sperm had 24 gyres, differed from the other species in
the group. The proportion of the head and flagellum in Squalus spp. was smaller and larger than
that in the other genera of Squaliformes, respectively.
Group II STJ to RBS. This group consisted of five orders, five families; Squatiniformes,
Squaliformes, Hexanchiformes, Urolophidae of Myliobatiformes, and Rhinobatiformes. Most of
the species of Squalidae belonged to the group. Urolophidae was separated from Myliobatididae
of the same order. The group was divided into two subgroups. The number of the gyres of the
Source :
ADVANCES [N SPERMATOZOAL PHYLOGENY AND TAXONOMY
319
sperm in the subgroup from STJ to DTL was less than that in the subgroup from CTO to RBS.
The sperm of C. owstoni, alone in the Squalidae, had a large number of gyres.
Group III RJE. This group contained only one species. Raja eglanteria. The proportion of
the midpiece in this species was large compared to the other species, while the sperm had a small
number of gyres.
Group IV TPT to MBT. This group included five orders and nine families;
Torpediniformes, Orectolobiformes, three families of Lamniformes, three families of
Carcharhiniformes, and the Myliobatididae in the Myliobatiformes. All species of
Carcharhiniformes except Scyliorhinidae belonged to this group. The group was divided into two
subgroups. The sperm in the subgroup from TPT to IRO was longer than that in another
subgroup. Only I. oxyrinchus was separated from the other species of Lamniformes. In the
Triakidae, Hemitriakis japanica was separated from the other species. The sperm of H. japanica
had a larger number of gyres than other species of the same family.
DISCUSSION
The internal morphology of the sperm, especially the structure of the flagellum, displays
differences between elasmobranchs and holocephalans [4, 6, 8]. However, literature which deals
with the relationship between the external features of the sperm and the phylogeny in
chondrichthyan fishes is scanty. The external features of the sperm in chondrichthyan fishes were
recognized to be species specific. They showed a similarity within the genus and/or family. Four
groups based on the cluster analysis also suggested a similarity of sperm morphology within the
order. COMPAGNO [1] and SHIRAI [12] divided elasmobranchs into four and two groups,
respectively. Both authors recognized the four orders Heterodontiformes, Orectolobiformes,
Lamniformes and Carcharhiniformes, as one group. The present study also recognized one group
consisting of three orders excepting the Heterodontiformes. The remaining three groups of
COMPAGNO: 1. Hexanchiformes, Squaliformes and Pristiophoriformes, 2. Batoids (Skates and
Rays), and 3. Squatiniformes, are equal to the group 2 of SHIRAI. COMPAGNO [1] considered
that the three groups are independently derived, while SHIRAI [12] regarded them as one of the
two groups derived from a basal group. Group II in the present study includes five orders and is
close to SHIRAI's grouping. COMPAGNO [2] divided Carcharhiniformes into two suborders;
Scyliorhinoidei and Carcharhinoidei. The Scyliorhinidae of Group I belongs to the former, and
the three families of Group IV belong to the latter.
320
S. TANAKA, H. KUROKAWA & M. HARA : CHONDR1CHTHYES
The families of Group I, except two species of Squalidae, and only one family, Rajidae, of
Group 111 are oviparous [3], The formation of sperm aggregates has been demonstrated in various
species of elasmobranchs [1 1]- In this study, it was found to be of the same type within the order.
This may be related to the similarity of reproductive modes within the order |3J. 1 hus, the
grouping of chondrichthyan fishes based on the external features of the sperm in this account
reflects the systematics and phytogeny derived from consideration of the external, skeletal and
muscular systems of the body and the female reproductive modes.
ACKNOWLEDGEMENTS
We wish to express our thanks to Harold L. Pratt, Jr. and Soichi Hagiwara for kindly supplying samples for this
study, Shozo Sawamoto for permitting us to use the Nomarski differential interference microscopy, and Masahiro OCURA
for computer analysis of numerical taxonomy.
REFERENCES
1 . CompaGNO, L. J. V., 1977. — Phyletic relationships of living sharks and rays. American Zoologist, 17: 303-322.
2. COMPAGNO, L. J. V., 1988. — Sharks of the Order Carcharhiniformes. New Jersey. Princeton University Press: 1-
486.
3. CompaGNO, L. J. V., 1990. — Alternative life-history styles of cartilaginous fishes in time and space.
Environmental Biology of Fishes, 28: 33-75.
4. Hara, M. & Tanaka, S., 1990. — An overview of chondrichthyan seminiferous follicles using electron
microscopy. National Oceanic and Atmospheric Administration Technical Report National Marine Fisheries
Service, 90: 131-142.
5. Ishiyama, R.. 1967. — Fauna Japonica Rajidae (Pisces). Tokyo, Tokyo Electrical Engineering College Press: 1-82.
6. Jamieson, B. G. M., 1991. — Fish Evolution and Systematics: Evidence from Spermatozoa. Cambridge, Cambridge
University Press: 1-319.
7. Leigh-Sharpe, W. H., 1926. — The comparative morphology of the secondary sexual characters of elasmobranch
fishes. Journal of Morphology , 42: 307-358.
8. Mattei, X., 1991. — Spermatozoon ultrastructure and its systematic implications in fishes. Canadian Journal of
Zoology, 69: 3038-3055.
9. NELSON, J. S., 1994. — Fishes of the World, 3rd Edition. New York, John Wiley & Sons: 1-600.
10. Otake, T., 1990. — Classification of reproductive modes in sharks with comments on female reproductive tissues
and structures. National Oceanic and Atmospheric Administration Technical Report National Marine Fisheries
Service, 90: 111-130.
1 1 . Pratt, H. L., Jr., & Tanaka, S., 1994. — Sperm storage in male elasmobranchs: A description and survey. Journal
of Morphology, 219: 297-308.
12. Shirai, S., 1992. — Squalean Phytogeny: A New Framework of “Squaloid” Sharks and Related Taxa. Sapporo,
Hokkaido University Press: 1-151.
Source . MNHN. Paris
Comparative Spermatology of Anurans with
Special References to Phylogeny
Ae Sook KWON & Young Hwan LEE
Department of Biology Education, Taegu University, Kyungsan 713-714, Korea
ABSTRACT
The anurans so far examined can be divided into three groups based on the data of comparative spermatology. The first
group, Ascaphidae and Discoglossidae, is considered to be plesiomorphic because it has most plesiomorphic characters,
endonuclear canal, rod-shaped endonuclear perforatorium and mitochondria adjacent to only the axial rod as in urodeles.
The second group, including Myobatrachidae, Bufonidae, Hylidae and Leptodactylidae is characterized by a conical
acrosome and a conical extranuclear perforatorium. However, Myobatrachidae differ from the other three families, in
mitochondrial position and centriolar arrangement. These characteristics are rather similar to those of ascaphids,
discoglossids and urodeles. Thus Myobatrachidae appears to be the most primitive among this group. The tail of the first
two groups consists of an axoneme, axial rod and undulating membrane. However it contains an axoneme and axial rod in
Hyla , and an axoneme only in Telmatobius. The third group including Pipidae, Ranidae and Rhacophoridae is characterized
by a 140° angle of centrioles and a tail with only the axoneme. These features might represent a synapomorphy for this
group. The main evolutionary pathways observed in anurans arc: 1) a reduction in the length of the endonuclear canal and
the eventual disappearance of this structure; 2) the disappearance of the subacrosomal cone and the perforatorium; 3) the
disappearance of the undulating membrane and the axial rod; and 4) an increase in the angle between the two centrioles.
Phylogenetic conclusions drawn from sperm ultrastructure coincide in many points with previous data based on somatic
features.
RESUME
Spermatologie comparee des Anoures et rapports avec la phylogenie
Les Anoures examines jusqu’ici peuvent etre divises en trois groupes en fonction de la spermatologie comparee. Le
premier groupe, les Ascaphidae et Discoglossidae, est considere comme plesiomorphe parce qu'il possede la plupart des
caracteres plesiomorphes: canal endonucleaire, perforatorium endonucleaire en forme de baguette et mitochondrie
adjacente seulement a la baguette axiale, comme chez les Urodeles. Le second groupe, comprenant les Myobatrachidae,
Bufonidae, Hylidae et Leptodactylidae est caract£ris6 par un acrosome conique et un perforatorium extranucleaire conique.
Toutefois, les Myobatrachidae different des trois autres families par la position de la mitochondrie et la disposition des
centrioles. Ces caracteristiques sont assez proches de celles des Ascaphidae, Discoglossidae et Urodeles. Les
Myobatrachidae semblent done etre les membres les plus primitifs de ce groupe. La queue, dans les deux premiers groupes,
consiste en un axoneme, une baguette axiale et une membrane ondulante. Toutefois. clle conlient seulement un axoneme et
une baguette axiale chez Hyla, et seulement un axoneme chez Telmatobius . Le troisieme groupe. comprenant les Pipidae,
Ranidae et Rhacophoridae, est caracterise par un angle de 140° des centrioles et une queue contenant seulement T axoneme.
Ces caracteristiques pourraient representer une synapomorphie pour ce groupe. Les Stapes evolutives principals
observees chez les Anoures sont 1) une reduction de la longueur du canal endonucleaire et la disparition finale de cette
structure; 2) la disparition du cone subacrosomien et du perforatorium; 3) la disparition de la membrane ondulante; el 4)
l’augmentation de Tangle entre les deux centrioles. Les conclusions phylogenetiques tracees ^ partir de Tultrastructure des
spermatozoides coincident en de nombreux points avec les donnees precedentes basees sur les caracteristiques somatiques.
Kwon, A. S., & Lee, Y. H., 1995. — Comparative spermatology of anurans with special references to phylogeny.
In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem.
Mus. natn. Hist, nat., 166: 321-332. Paris ISBN: 2-85653-225-X.
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A. S. KWON & Y. H. LEE : ANURA ( AMPHIBIA )
Amphibia are divided into the three orders Apoda, Urodela and Anura. The Anura is often
considered as a super-order [30]. Anura can be subdivided into two distinct levels of
organization, with primitive and higher families, on morphological data. However, the phyletic
relationships between and within the two levels are extremely difficult to clarify because anurans
are morphologically quite conservative.
The ultrastructure of the spermatozoon has been the subject of considerable study in the past
twenty years in several animal groups and is recognized as an important indicator of phylogenetic
relationships [6, 15, 21, 28, 31]. Anuran spermatozoa provide a useful suite of taxonomic
characters because they have sufficient variety in the structure of the acrosome, perforatorium,
and tail, and the arrangement of the centrioles. Despite the taxonomic value of sperm structure,
there have been little data available on phylogeny of anuran spermatozoa.
The ultrastructure of the spermatozoon is now known in 3 1 species ( 1 7 genera) of anurans
[12-13, 18, 22-27, 29, 34-39]. This enables us to give a summary of the ultrastructure of the
spermatozoon and its phylogeny.
The purpose of this chapter is to describe the ultrastructure of spermatozoa in six species of
four families of anurans and to compare it to those of other groups of amphibians. The results are
also discussed with regard to the phylogenetic position.
MATERIALS AND METHODS
Spermatozoa from the following anuran species were used in this study (asterisks indicate new data); Ascaphus iruei
(Ascaphidae); Discoglossus pictus, Alytes obstetricians, Bombina variegata , *B. orientalis (Discoglossidae); Adelotus
brevis, Limnodynastes peronii, Mixophyes fasciolatus , Neobatrachus pelobatiodes (Myobalrachidae); Bufo arenarum ,
B. marinus , *B . bufo gargarizans (Bufonidae); Odontophrynus cul tripes, Telmatobius hauthali (Leptodactylidae); Litoria
caerulea, L. fallax, L. gracilenta, L. lesueuri, L. peronii, L. rubella, Pachymedusa dacnicolor , *Hyla japonica ,
H. meridionalis (Hylidae); Xenopus laevis (Pipidae); Rana clamitans , R. pipiens, *R. nigromaculata , *R. dybowskii,
*R. rugosa (Ranidae); Rhacophorus arboreus, R. schlegelii (Rhacophoridae).
Species of anurans were collected in the neighbourhood of Taegu, Korea. Testes were dissected and fixed in 2.5%-
5% glutaraldehyde in 0.1 M sodium cacodylate buffer and post-fixed in 1% osmium tetroxide in the same buffer. They were
then dehydrated in a graded ethanol series and embedded in Epon 812. The samples were sectioned on a Sorvall MT 2-B
ultramicrotome, stained in 4% aqueous uranyl acetate, post-stained with lead citrate and examined with a Hitachi H-600
electron microscope.
OBSERVATIONS
Bombina orientalis
B. orientalis possesses a spermatozoon with a peculiar architecture unlike the classical
sequence of acrosome, nucleus and flagellum. The flagellum is juxtaposed longitudinally along
the nucleus. The nucleus is cone shaped and some nuclear lacunae, irregular in shape, are
scattered within the nucleus. The chromatin is not completely compact but condensed into large
masses (Fig. la).
The acrosome consists of a thin vesicle truncated at the anterior end and its material is
homogenous and moderately electron dense. The subacrosomal space contains the perforatorium
Fig. 1. — Longitudinal sections of head, a: Bombina orientalis, x 30 000; b: Bufo bufo gargarizans, x 35 700;
c : Hyla japonica, x 44 000; d: Rana dybowskii, x 75 000.
Fig. 2. — Transverse sections of head, a: Bombina orientalis, x 30 000; b: Bufo bufo gargarizans, x 56 000; c: Hyla
japonica, x 63 000; d: Rana rugosa, x 52 000.
Abbreviations used in the figures: A. acrosome; AR, axial rod; Ax, axoncme; C, centriole; EC, endonuclear canal; FS,
fibrogranular sheet; M, mitochondria; MF, marginal filament; N, nucleus; P, perforatorium; PM, pericentriolar
material; UM, undulating membrane; V, vesicle.
Source : MNHN. Paris
Source : MNHN, Paris
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A. S. KWON & Y. H. LEE : ANURA ( AMPHIBIA )
which emerges from an endonuclear canal in the tip of the nucleus (Figs 2a, 3a). The
perforatorium consists of bundles of filamentous material arranged parallel to each other.
The neck region is located in the lateral surface of the nucleus which is furrowed by a
longitudinal depression which contains an anterior portion of the axial rod and proximal centriole
(Fig. 3a, b). Two centrioles form an angle of 70°, approximately, to each other and the proximal
centriole is perpendicular to the longitudinal axis.
The tail contains an axoneme, axial rod, undulating membrane and mitochondria. The
axoneme, of the typical 9+2 pattern, is lateral to the main axis which is occupied by the axial rod.
The axial rod extends almost to the tip of the flagellum. The undulating membrane is short in the
proximal region of the tail and becomes longer toward the middle region. Mitochondria are
arranged in a semicircle and positioned only around the axial rod (Fig. 4a).
Bufo bufo gargarizans
The nucleus is cylindrical in shape and the chromatin is completely condensed. The base of
the nucleus holds an implantation fossa in which the proximal centriole lodges.
The acrosome consists of a very thin vesicle containing homogenous material of moderate
electron density as in B. orientalis (Fig. lb). The subacrosomal space contains the perforatorium
consisting of bundles of microfilaments. The perforatorium runs along the inner acrosomal
membrane, unlike B. orientalist Fig. 2b). An endonuclear canal is not present in this species.
In the neck region two centrioles are perpendicular to each other and embedded in
pericentriolar material (Fig. 3c). The pericentriolar material connecting with the anterior end of the
axial rod shows transverse striations.
The tail contains an axoneme, axial rod, undulating membrane and mitochondria. The axial
rod which is associated with axonemal doublet no. 3 extends to the principal piece of the
flagellum (Fig. 5a). The end piece contains only the axoneme. Mitochondria constituting a sheath
are separated from the flagellum by the cytoplasmic canal and surround the axoneme, axial rod
and undulating membrane (Fig. 4b).
Hyla japonica
The spermatozoon of H. japonica has an ultrastructure very similar to that of B. bufo
gargarizans (Figs lc, 3d). Differences between two will be noted here.
The spermatozoa are shorter and more slender than those of B. bufo gargarizans. The
perforatorium consists of microtubule-like arrays instead of the microfilaments in other anuran
spermatozoa (Fig. 2c). No transverse striations appear in the pericentriolar material.
The tail contains an axoneme and axial rod without undulating membrane. The axial rod is
closer to the axonemal doublet no. 3 in the middle and principal pieces (Figs 4c, 5b) but
disappears in the distal region of the flagellum.
Rana nigromaculata, Rana dybowskii, Rana rugosa
The spermatozoa of all three species examined have a very similar ultrastructure except the
pericentriolar material.
The nucleus, with compact chromatin, is cylindrical and slightly tapered at both ends. The
posterior end of the nucleus has an implantation fossa in which the centrioles do not reside.
Intranuclear inclusions are visible within the nucleus.
Fig. 3. — Longitudinal sections of head-tail junction, a, Bombina orientalis, x 23 000; b: Bombina orientalis,
x 30 000; c: Bufo bufo gargarizans , x 34 000; d: Hyla japonica, x 30 000; e: Rana rugosa, x 36 000.
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326 A. S. KWON & Y. H. LEE : ANURA ( AMPHIBIA )
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Fig. 6. — Diagrammatic representation of sections of amphibian spermatozoa, a-i: longitudinal sections; j-r: cross
sections of the principal piece, a, j: Salamandridae, Pleurodeles [32]. b, k: Discoglossidae, Discoglossus [39].
c, 1: Discoglossidae, Bombina [23]. d, m: Myobatrachidae, Limnodynastes [25]. e, n: Leptodactylidae,
Odoniophrynus [12]. Bufonidae, Bufo [24]. Hylidac, Pachy medusa [37], Hylidae, Litoria [26]. f, o: Hylidae, Hylci
[27]. g, p: Leptodactylidae, Telmatobius [34]. h, q: Pipidae, Xenopus [13]. i, r: Ranidae, Rana.
The acrosome is a saclike structure situated at the most anterior portion of the nucleus (Fig.
Id). It is shorter and thicker than in other species mentioned above. There is no perforatorium nor
an endonuclear canal (Figs Id, 2d).
The neck region contains the centrioles, pericentriolar material and mitochondria (Fig. 3e).
Two centrioles form an angle of approximately 140° and the distal centriole is parallel to the main
axis. Two centrioles are situated outside the nuclear fossa. Mitochondria aggregate around the
base of nucleus, the centrioles and the axoneme. A cytoplasmic canal is not observable.
Fig. 4. — Transverse sections of middle piece, a: Bombina orientalis , x 21 000; b: Bufo bufo gargarizans , x 42 000;
c: Hyla japonica , x 40 000; d : Rana dybowskii, x 70 000.
Fig. 5. — Transverse sections of principal piece, a: Bufo bufo gargarizans, x 72 000; b: Hyla japonica. x 10 800;
c: Rana nigromaculata , x 145 000.
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A. S. KWON & Y. H. LEE : ANUKA ( AMPHIBIA )
The tail contains a 9+2 pattern axoneme without axial rod and undulating membrane (Figs
4d, 5c).
The spermatozoa of the three species have some differences in the neck region. In R.
rugosa , the pericentriolar material surrounds the centrioles and contains transverse striations (Fig.
3e). However, in R. dybowskii, it is only located lateral to the centriole and also has transverse
striations. In R. nigromaculata, no transverse striations are observed within the pericentriolar
material surrounding the centrioles.
DISCUSSION
Comparative ultrastructure of spermatozoa in anurans.
The ultrastructure of spermatozoa is described in six species from four genera of four
families (Discoglossidae, Bufonidae, Hylidae and Ranidae) of anurans in this chapter. B.
orientalis, B. bufo gargarizans and H. japonica have spermatozoa that conform to the generalized
amphibian sperm (see JAMIESON, this volume). On the other hand, Rana species have
spermatozoa that differ markedly from the above anurans in their organization.
The acrosome is conical in most anurans, but is saccular in Rana, and is a coiled structure to
one side of the nucleus in Rhacophorus [29]. This character of the saccular and coiled acrosome is
considered to be apomorphic as compared with the conical acrosome.
The subacrosomal cone present in the sperm of Ascaphus [22] is characteristic of urodeles
[32, 33] but is not present in any other anurans. JAMIESON et al. [22] reported that this structure
is a widespread and plesiomorphic feature of amniote sperm.
The endonuclear canal as a main characteristic structure of urodelan sperm has been
observed in the primitive anuran families, Ascaphidae [22] and Discoglossidae [18, 23, 39]. This
character should be considered as plesiomorphic compared to the other anurans which lack the
canal. Discoglossus [39] especially has a long canal running the entire length of the nucleus as in
the urodeles. In Bombina and Alytes [18] the canal penetrates roughly to the middle of the
nucleus. The perforatorium has been observed in many anurans [12, 14, 18, 22-27, 34, 36-39]
and urodeles [8-11, 31-32] but shows differences in its shape and position. The ascaphids [22]
and discoglossids [18, 23, 39] have a rod-shaped endonuclear perforatorium which is also
characteristic of urodeles. This must be considered plesiomorphic, in comparison with the
completely conical extranuclear perforatorium which has been observed in myobatrachids [25],
bufonoids [14, 24, 26], hylids [26-27, 37] and leptodactylids [12, 34]. The higher anuran
families, Pipidae, Ranidae and Rhacophoridae have no perforatorium [13, 29, 35, 38 J. It can thus
be deduced that absence of the perforatorium is an apomorphic character in anurans. The
perforatorium is progressively simplified and finally disappears in several higher arthropods and
vertebrates during evolution of a terrestrial life [6],
The orientation of the centrioles varies depending on the systematic position of the animal
[1]. Anurans show three types of orientation of the centrioles. In the first type the distal and
proximal centrioles lie at an angle of 70° to each other whereas the proximal centriole and the main
axis are perpendicular to each other. This type has been observed in ascaphids [22], discoglossids
[18, 23, 39] and myobatrachids [25], In the second type the two centrioles are perpendicular to
each other. This type is seen in bufonoids [14, 24], hylids [27, 37] and leptodactylids [12, 34].
The third type is characterized by the centrioles forming an angle of approximately 140° to each
other. This type is seen in pipids [13, 38] and ranids [35],
The tail has been used as an important taxonomic character in many animal groups [1-5, 7,
16, 28]. The tail of most amphibians contains an axoneme, axial rod and undulating membrane.
In pipids [13, 38], ranids [35], rhacophorids [29] and the leptodactylid Telmatobius [34] the tail
has an axoneme only. Hyla in the Hylidae [27, 36] has a tail in which the axoneme and axial rod
lack an undulating membrane. This tail seems to be the intermediate type in anuran sperm. We
thus conclude that a tail consisting of undulating membrane and axial rod is plesiomorphic.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
329
The position of the mitochondria varies in anuran sperm. Mitochondria adjacent to only the
axial rod have been observed in ascaphids (termed a paraxonemal rod in this species) [22],
discoglossids [18, 23, 39], myobatrachids [25] and urodeles [8-11, 32-33]. In bufonoids [14,
24, 26], hylids [26-27, 37] and leptodactylids [12, 34], they surround the axoneme and constitute
a sheath separated by the cytoplasmic canal. The mitochondrial sheath without cytoplasmic canal
is observed in pipids [13, 38] and ranids [34], LEE & JAMIESON [25] suggested in myobatrachids
that the location of mitochondria adjacent to only the axial rod is plesiomorphic as compared with
the mitochondria surrounding the axoneme.
Phylogenetic relationships in anurans based on spermatozoal ultrastructure.
Table 1 shows phylogenetic trends within the anurans, based on the data of comparative
spermatology. The main evolutionary tendencies observed in anurans based on ultrastructural
characteristics of spermatozoa are (1) the disappearance of the subacrosomal cone of Ascaphidae
in the other anuran groups, (2) a reduction in the length of the endonuclear canal relative to
Ascaphidae and Discoglossidae and the eventual disappearance of this structure from the other
anuran groups, (3) the disappearance of the perforatorium and the axial rod in Pipidae and
Table 1. — Phylogenetic relationships based on the ultrastructure of anuran spermatozoa
PC, plesiomorphic characters; AC, apomorphic characters; Ur, Urodeles; As, Ascaphidae; Di, Discoglossidae; My,
Myobatrachidae; Bu, Bufonidae; Hy, Hylidae; Le, Leptodactylidae; Pi. Pipidae; Ra, Ranidae; Rh, Rhacophoridae.
*, no data.
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A. S. KWON & Y. H. LEE : ANURA ( AMPHIBIA )
Ranidae, (4) the disappearance of the undulating membrane in Hylidae, Pipidae and Ranidae,
(5) an increase in the angle between the two centrioles: 30°-70° in Ascaphidae, Discoglossidae
and Myobatrachidae, 90° in Leptodactylidae, Bufonidae and Hylidae, 140° in Pipidae and Ranidae
(Fig. 6).
Anurans studied so far can be divided into three groups with regard to phylogenetic
relationships based on the ultrastructure of spermatozoa. The first group includes Ascaphidae and
Discoglossidae. They appear drastically isolated from the other anurans because they are
characterized by an endonuclear canal, a rod-shaped endonuclear perforatorium, the angle of the
centrioles and location of mitochondria adjacent to only the axial rod. The subacrosomal cone
appears in only Ascaphidae. These characters show most of the common feature of urodeles.
Therefore this group should be considered as plesiomorphic and the Ascaphidae as more
plesiomorphic than the Discoglossidae. This interpretation perfectly agrees with the traditional
classification. Based on the ultrastructure of acrosome and incompletely condensed nucleus,
Bombina and Alytes are more closely related than they are to Discoglossus ; Discoglossus [39] has
the ring structure and the endonuclear canal occupying the whole length of the nucleus which has
also been observed in urodeles. With respect to the above characteristics, Discoglossus seems to
be the most primitive genus among Discoglossidae. However, this interpretation disagrees with
the phylogenetic position of three genera from anatomical and karyological data. Bombina is the
most primitive in an osteological classification [40], Alytes is the most primitive from the
karyological point of view in having a karyotype similar to that of primitive urodeles [30].
The second group, including Myobatrachidae, Bufonidae, Hylidae and Leptodactylidae, is
characterized by common features: the conical acrosome and the conical extranuclear
perforatorium. Myobatrachidae are quite different from this group in having the same structure of
the sperm tail as the Ascaphidae and Discoglossidae. These characteristics suggest that
Myobatrachidae may occupy the most primitive position in this group. Myobatrachidae has been
classified as a subfamily of the family Leptodactylidae. However, recent phylogenies using
morphological data [17] or molecular data [20] have recognized myobatrachids as a family
separate from the Leptodactylidae. Ultrastructural data of the spermatozoa are in agreement with
the latter interpretation [25]. Bufonidae, Hylidae and Leptodactylidae are also united by a single
apomorphy, mitochondrial location surrounding the axoneme. This contrasts with the
plesiomorphic location of the mitochondria associated with the axial rod in ascaphids [22],
discoglossids [18, 23, 39] and urodeles [8-1 1, 32-33]. The phyletic affinity between these three
families is recognized by various authors [19-20, 30].
The third group includes Pipidae, Ranidae and Rhacophoridae. They have common
features, such as no perforatorium and a tail with only the axoneme. Although they are different in
the morphology of the acrosome, Pipidae and Ranidae are closely related based on the centriolar
arrangement as well as the above two characters. The conical acrosome of Pipidae appears as a
plesiomorphic character in comparison to the saccular acrosome in Ranidae. This relationship
between two families coincides with the morphological data suggested by GRIFFITHS [19].
However, according to MORESCALCHI [30], Pipidae are closely related to the Discoglossidae
from karyological data. Rhacophoridae are characterized by the coiled acrosome lying un one side
of nucleus.
In anurans the classification based on comparative spermatology is almost similar to
traditional classification. Comparative spermatology thus may be considered as a useful new tool
for the understanding of anuran phylogeny.
ACKNOWLEDGEMENTS
We are much indebted to Mr. Ku Hwan Kim for printing micrographs and to Jae Hoon HONG for drawing the table.
This work has been supported by a grant from Korea Research Foundation to Y. H. Lee.
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331
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28. Mattel X., 1988. — The flagellar apparatus of spermatozoa in fish. Ultrastructure and evolution. Biology of the
Cell 63: 151-158.
29. Mizuhira, V., Futaesaku, Y., Ono, M., Ueno, J.. Yokofujita, J. & Oka, T., 1986. — The fine structure of the
spermatozoa of two species of Rhacophorus (arboreus, schlegelii). I Phase- contrast microscope, scanning
electron microscope, and cytochemical observations of the head piece. Journal of Ultrastructure Research , 96:
41-53.
30. MORESCALCHI, A., 1973. — Amphibia. In: A. B. CHIARELLI & E. Capanna, Cytotaxonomy and Vertebrate
Evolution. New York, Academic Press: 233-347.
31. Nicander, L., 1970. — Comparative studies on the fine structure of vertebrate spermatozoa. In: B. Baccetti,
Comparative Spermalology. New York, Academic Press: 47-56.
32. Picheral, B., 1967. — Structure et organisation du spermatozoide de Pleurodeles waltlii Michah (Amphibien,
Urodele). Archives de Biologie, 78: 193-221.
33. Picheral, B., 1979. — Structural, comparative and functional aspects of spermatozoa in urodeles. In: D. W.
Fawcett & J. M. Bedford, The Spermatozoon. Baltimore & Munich. Urban & Schwarzenberg: 267-287.
34. Pisano, A. & ADLER, R., 1968. — Submicroscopical aspects of Telmatobius hauthali schreiteri spermatids.
Zeitschrift fiir Zellforschung und Mikroskopische Anatomie , 87: 345-349.
35. Poirier, G. R. & Spink, G. C., 1971. — The ultrastructure of testicular spermatozoa in two species of Rana. Journal
of Ultrastructure Research, 36: 455-465.
36. PUGlN-RlOS, E., 1980. — Etude comparative sur la structure du spermatozoide des Amphiens Anoures. Comportement
des gametes lors de la fecondation. Th£se, Universite de Rennes, Rennes, France.
37. Rastogi, R. K., Bagnara, J. T.. Iela, L. & Krasovich, M. A., 1988. — Reproduction in the Mexican leaf frog,
Pachxmedusa dacnicolar. IV. Spermatogenesis ; a light and ultrasonic study. Journal of Morphology, 197: 277-
302.
38. Reed, S. C. & Stanley, H. P., 1972. — Fine structure of spermatogenesis in the South African clawed toad Xenopus
laevis Daudin. Journal of Ultrastructure Research, 41: 277-295.
39. Sandoz, D., 1974. — Development of the neck region and the ring during spermiogenesis of Discoglossus pictus
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237-247.
40. SLABBERT. G. K. & MAREE, W. A., 1945. Annale Universiteit Stellenbosch, 23: 91-97.
Source : MNHN . Paris
Amphibian Sperm:
Phylogeny and Fertilization Environment
Gerhard VAN DER HORST, Brian WILSON & Alan CHANNING
Departments of Physiological Sciences and Biochemistry,
University of the Western Cape, Private Bag XI 7, Bcllville, 7535, South Africa
ABSTRACT
Sperm characteristics such as the structure and position of the acrosome. the design and ultrastructure of the flagellum,
the presence/absence of a neck-piece and several other fine structural details are important diagnostic features which
distinguish the three orders, families and species of lissamphibians. These sperm features are also important in
phylogenetic inferences. It appears that the axoneme-undulating membrane-axial rod represents a plesiomorphic
character. This pattern has been simplified to a single axoneme as a secondary reversal for external fertilization in many
anurans or has developed as a double axoneme as in Chiromantis xerampelina. Sperm head and acrosome length are
predictive in distinguishing between terrestrial and aquatic anuran fertilizers. Sperm motility is species specific among
representatives of seven of the nine South African families and motile sperm which exhibit forward progression are the
rule for aquatic fertilizing anurans. In contrast, the sperm of terrestrial fertilizing anurans are immotile in a wide range of
physiological/culture media with osmotic concentrations varying from 10 to 300 mOsm/kg. The term ect-terrasperm is
suggested as a new terminology for amphibians which exhibit the terrestrial mode of fertilization.
RESUME
Spermatozoides des Aniphibiens: phylogenie et environnement de la fecondation
Les caracteristiques du spermatozoide telles que la structure et la position de V acrosome, la forme et 1* ultrastructure du
flagelle, la presence ou absence d*un cou et plusieurs autres details ultrastructuraux sont des criteres de diagnostic qui
distinguent les trois ordres, les families et les especes de Lissamphibiens. Les caracteristiques du spermatozoide sont aussi
importantes pour la comprehension de la phylogenie. 11 semble que P axoneme avec membrane ondulante et tibre axiale
represcnte un caractere plesiomorphe. Cette structure a etc simplify en un axoneme simple, commc reversion secondaire
pour la fecondation externe, chez de nombreux Anoures, ou s’est ddveloppee en un axondme double comme chez
Chiromantis xerampelina. La longueur de la tete du spermatozoide ou de P acrosome permet de distinguer de maniere
predictive les Anoures h fecondation aquatique ou terrestre. La motilite du spermatozoide est specifique des especes parmi
les representants de sept parmi les neuf families d’Afrique du Sud, et les spermatozoides mobiles qui montrent une
progression vers l’avant sont la fegle pour les Anoures h fecondation aquatique. Par contre, les spermatozoides des Anoures
k fecondation terrestre sont immotiles dans de nombreux milieux physiologiques et de culture avec des concentrations
osmotiques variant de 10 a 300 mOsm/kg. Le terme ect-terraspermatozoide est suggere comme une nouvelle terminologie
pour les Amphibiens qui posscdent le mode terrestre de fecondation.
van DER Horst, G., Wilson B., & Channing, A., 1995. — Amphibian sperm: phylogeny and fertilization
environment. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 16X : 333-342. Paris ISBN : 2-85653-225-X.
334
G. VAN DER HORST, B. WILSON & A. CHANNING : FERTILIZATION (AMPHIBIA)
Studies at both the light microscopic and the ultrastructural level suggest that sperm
morphology relates to the mode of fertilization (internal versus external) [8, 10, 24] and is of
importance in phylogenetic reconstruction [7, 10, 11], Furthermore, sperm morphology assists in
distinguishing between families [25] and even between genera [6] and closely related species [4,
9, 20, 23],
In their work on the Hyla rubra group, FOUQUETTE & DELAHOUSSAYE [7] regarded sperm
structure as important in phylogenetic reconstruction, rather than an adaptation to the fertilization
environment. LEE & JAMIESON [12] used phylogeny as an important basis for their work on the
ultrastructure of myobatrachid frog sperm. In a later work, JAMIESON et al. [10] compared anuran
and urodele sperm to that of Ascaphus. In this study they indicated which ultrastructural features
of sperm could be considered primitive (plesiomorphic), associated with distinct advanced
characteristics (autopomorphies), and derived features which were considered paedomorphic. It
should be realized that sperm of an individual species may exhibit combinations of these features
and that this may complicate interpretation of their phylogenetic position. In addition the latter
authors [10] also indicated the relationship of these features to the fertilization biology of
amphibians and suggested that the general trend towards simplification of sperm in anurans is a
result of secondary reversion to external fertilization.
Among vertebrates, amphibians represent the widest range of “fertilization environments”.
Internal fertilization is evident in most salamanders and in all caecilians. In anurans such as
Ascaphus internal fertilization takes place and their sperm are classified as introsperm by
JAMIESON & Rouse [1 1] and modified sperm by FRANZEN [8J. External fertilization is the rule
for most anurans and their sperm can be classified as ect-aquasperm [1 1] or in the terminology of
FRANZEN [8] as primitive. The terminology “primitive” used by FRANZEN [8] is, however,
confusing in terms of anurans since they are mainly external fertilizers but have modified sperm.
Furthermore, the mode of external fertilization varies among anurans. Direct sperm deposition on
the eggs occurs in Arthroleptella lightfooti and Breviceps gibbosus and sperm only swim through
the mucous/egg jelly surrounding of the egg but are not substantially in contact with external
fluids or fluids that are not largely from a biological origin of the species themselves. Species
exhibiting this pattern will here be referred to as terrestrial fertilizers (TF). In Bufo spp. and
Xenopus laevis, however, sperm are ejaculated in fresh water and sperm actually swim through
an external medium to reach the eggs. Species exhibiting this pattern will be referred to as aquatic
fertilizers (AF) [24], It is therefore evident that amphibians represent an ideal model to investigate
the relationship of sperm structure to their different fertilization environments.
It can furthermore be expected that sperm shape (particularly the head) and flagellar beat will
largely determine the swimming behaviour of sperm. The importance of sperm motility is that it
represents a summation of form, mitochondrial function, membrane integrity, exchange of
important ions such as calcium and unmasking of receptors. It may be possible that specific sperm
motility patterns are associated with mode of fertilization and also with species specific motility
patterns.
Our aims in this investigation are to compare sperm structure in the urodeles, caecilians and
anurans with special reference to seven of the nine South African anuran families. Flere
fertilization biology is contrasted with features that are important in establishing phylogenetic
relationships. Furthermore, we investigate quantitatively sperm motility patterns in anurans as a
function of their fertilization biology. In this part of the investigation we study quantitative sperm
motility in representative examples of frogs exhibiting either the terrestrial mode of fertilization or
the aquatic mode of fertilization. Lastly, we ascertain whether sperm motion is species specific in
anurans.
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335
MATERIALS AND METHODS
General. All comparisons that relate to sperm of the urodeles are extracted from the literature (Table 1). Sperm of
only six caecilians have been described in the literature [16, 17, 18. 22], including only one paper on ultrastructure, that
of Typhlonectes natans [22]. The data on anuran sperm in this paper refer to our original research and representatives of
seven of the nine South African families are included. Table 1 refers to lissamphibian species used for comparisons in this
investigation.
Capture of animals All anurans used in this study were captured at night during the peak of their breeding season.
Animals were transported to the laboratory, were anaesthetized with MS222. dissected and sperm aspirated from the testes
within 48 hours of capturing.
Sperm aspiration Testes were removed and all superficial blood vessels and connective tissue were removed. One
testis of each specimen was put between two glass slides and squashed. Mainly intact sperm and testicular fluid could be
aspirated by means of a micro-pipette. Five pi of this sperm suspension was used for scanning electron microscopy, 5 pi
for motility studies and 5 pi for making sperm smears. The other testis was cut into smaller blocks and prepared for
transmission electron microscopy.
SEM and TEM preparation Sperm suspension obtained as indicated above was processed for scanning electron
microscopy according to the method of van der Horst et al. [21]. Small testes blocks were fixed in Sprenson's phosphate
buffered glutaraldehyde (2,5%) and post-fixed in 1% osmium tetroxide in the same buffer. Routine processing followed and
the material was embedded in Spurr's resin.
Sperm dimensions Sperm were incubated in a nigrosin-eosin solution, sperm smears made and used for
measurement of sperm dimensions (acrosome length, sperm head length, sperm head width) using a Koniron image
analyzer.
Sperm motility Distilled water, pond water and various physiological media were used at different concentrations
(0-300 mOsm/Kg) to establish the ideal medium for activating anuran sperm. Ham's F10 culture medium at 30 mOsm/kg
provided the best medium and also preserved motility in most species for several hours [24]. Five ml of sperm suspension
was placed in a microscopic bath containing 1 ml of Ham's F10 medium and sperm motion was recorded by means of a
video-camera. The images were later replayed and detailed sperm motion characteristics analysed in the fully automated
mode by means of computer aided sperm motility analysis (CASMA) utilizing the Sperm Motility Quantifier (SMQ.
Wirsam Scientific, South Africa). The curvilinear velocity (VCL), the straight line velocity (VSL), the linearity (LIN), the
average path velocity (VAP), amplitude of lateral head displacement (ALH) and dance (DNC = VCL x ALH) were measured.
Fig. 1 diagrammatically depicts a typical sperm swimming trajectory and shows the applicable terminology.
Fig. 1. — Sperm track indicating relevant terminology. VCL, curvilinear velocity; VSL, Straight line velocity; ALH,
Amplitude of lateral head displacement; BCF, Beal cross frequency. LIN = Linearity = VSL/VCL.
336
G. VAN DER HORST. B. WILSON & A. CHANNING : FERTILIZATION (AMPHIBIA)
TABi e i _ List of species used for drawing comparisons among the Lissamphibia. Only South African anurans
represent our original research are listed. (TF) = Terrestrial fertilizers and (AF) = aquatic fertilizers.
Anurans are used for comparison in text.
which
Other
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ADVANCES IN SPERMATOZOA!, PHYLOGENY AND TAXONOMY
337
RESULTS
Structural considerations of South African anurans
Figs. 2 to 8 are scanning and transmission electron micrographs which represent the full
spectrum of sperm morphology in investigated South African anurans and Table 2 represents
detailed measurements of these sperm. Representative examples of anurans which exhibit the
aquatic mode of fertilization will first be described.
The simplest sperm is that of Rana fuscigula which has a straight, short symmetrical head, a
short midpiece and a single flagellum (Fig. 2). The acrosomal cap is short. The sperm of
Semnodactylus wealii have similar features except that the flagellum has in addition an undulating
membrane and axial rod (Fig. 3). Xenopus laevis has a single flagellum but the head is partly
corkscrew shaped with one and a half coils (Fig. 4). Xenopus laevis sperm furthermore differ
from most anurans in that the first few rows of mitochondria surround the posterior part of the
nucleus (Fig. 4, insert). Bufo rangeri sperm have spear-shaped heads, distinct but short
midpieces and flagella with undulating membranes and axial rods (Fig. 5). The head length of
aquatic fertilizers ranges from 10.3 to 20.5 pm.
In contrast the sperm of South African anurans which exhibit the terrestrial mode of
fertilization have very long and slender heads (Hemisus marmoratus, Leptopelis flavomaculatus,
Breviceps gibbosus, Arthroleptella lightfooti ) or long heavily coiled heads ( Chiromantis
xerampelina) varying from 21.4 to 45 pm in length (Figs 6 & 8) (Table 2). Both the head lengths
as well as the acrosome lengths of the terrestrial fertilizers were significantly (p<0.05) longer than
those of the aquatic fertilizers. However, the flagella of the TF represent either a single axoneme
or an axoneme with an undulating membrane and axial rod like the AF. The exception was
Chiromantis xerampelina which has a single flagellar complex containing two axonemes
surrounded by a multitude of microtubules. The term ect-terrasperm is suggested as a new
terminology for amphibians which exhibit the terrestrial mode of fertilization. A clear distinction
between ect-aquasperm and ect-terrasperm is therefore evident.
In Chiromantis the first few rows of mitochondria also surround the posterior part of the
nucleus as in Xenopus. Furthermore, Chiromantis sperm has spherical mitochondria (Fig. 7) in
contrast to Rhacophorus arboreus sperm which has elongate mitochondria.
Urodeles and caecilians
Sperm structure in the urodeles and caecilians (limited information) shows less variation
than the anurans in terms of basic design. The sperm heads in both these groups are long and
slender. While considerable variation exists in the size and form of the flagellum, the basic pattern
of axoneme-undulating membrane-axial rod (AUA) seems to occur in all urodele and caecilian
species. Their acrosomes are similar in that they extend from the anterior part of the nucleus but
do not form a cap that largely overlaps with the nucleus as in anurans. The detailed structure of
the acrosome of urodeles and caecilians are also complex and mostly contain an acrosomal rod
surrounded by less dense but granular material. All urodele sperm furthermore contain a marginal
filament or juxta-axonemal fibre at doublet 8 next to the axoneme, which is absent in caecilians
and anurans.
Sperm motility of South African anurans
Sperm of all aquatic fertilizers exhibited forward progression and the VCL varied from 20 to
31 pm/s, VSL from 8 to 23 pm/s, LIN from 40 to 72%, BCF from 3 to 6Hz and ALH from 4.7
to 7.5pm. In contrast the sperm of two species of terrestrial fertilizers ( Breviceps and
Arthroleptella ) were immotile in a wide range of physiological media and osmotic concentrations
varying from 10 to 300 mOsm/kg. In Chiromantis xerampelina, sperm only exhibited an initial
rapid starspin movement followed by uncoiling of the head and became immotile within a
338
G. VAN DER HORST, B. WILSON & A. CHANNING : FERTILIZATION (AMPHIBIA)
Figs 2-8. — Scanning electron micrographs of amphibian spermatozoa. 2: Rana fuscigula. 3: Semnodactylus wealii.
4: Xenopus laevis (inset: transmission electron micrograph of midpiece). 5: Bufo rangeri. 6: Chiromantis
xerampelina. 7: Chiromantis xerampelina, transmission electron micrograph of midpiece. 8: Breviceps
gibbosus. A, acrosome; AUA, Axoneme-undulating membrane-axial rod; H. Head; m. mitochondria; MP, midpiece;
N, Nucleus. S, single flagellum only with axoneme. Holes in membrane filters (Figs 2, 3, 5 & 8) are 3 fim in
diameter.
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
339
minute. Star symbol plots were used to visually express sperm motility as a pattern of movement
(Fig. 9). Distinct motility patterns could be constructed that were clearly species specific. Sperm
with partly coiled heads such as Xenopus and Strongylopus swim in a corkscrew fashion and the
linearity of the sperm is almost similar (41.3% and 42.4% respectively) whereas Bufo sperm have
straight heads and a LIN of 67.6%. Furthermore, more closely related species (S. grayii and P.
adspersus) had distinct sperm motility patterns but also shared many similarities for several
motion parameters. This can also be seen in Fig. 9, indicating the similarity in star symbol plots.
Fig. 9. — Star Symbol Plots (seven motion parameters used - see abbreviations elsewhere in text - PM = percent motile
sperm) depicting patterns of sperm motion among six anuran species. For any set of stars (above) the smallest
observed value for a parameter is plotted as an arm one-tenth the length of the arm representing the largest
observed value.
Table 2. — Sperm dimensions of aquatic and terrestrial South African anuran fertilizers.
Source : MNHN. Paris
340
G. VAN DER HORST. B. WILSON & A. CHANNING : FERTILIZATION [AMPHIBIA)
DISCUSSION
It has been hypothesized that primitive lissamphibians were internal fertilizing and that
external fertilization was a secondary reversion in the anurans [10]. The 9+2 axoneme-undulating
membrane-axial rod is typically associated with this pattern as well as distinct acrosomal and neck
piece features. In reptiles, birds and mammals, where internal fertilization is the rule, a flagellum
is associated with a 9+9+2 pattern. There, the nine outer coarse fibres seem to function as a
strengthening device for sperm swimming in a viscous environment. The juxta-axonemal fibre as
well as the axial rod in amphibians seem to be homologous to the coarse outer fibres 8 and 3 of
amniotes and insects [14]. One interpretation may be that the AUA of the early internal fertilizing
amphibians represent an intermediate structure from external to internal fertilization and this has
been retained as a primitive feature in reversion to external fertilization in many anurans.
Alternatively, the AUA of early internal fertilizing Lissamphibia may already represent a
simplification of an ancestor that had a 9+9+2 flagellar arrangement. Our data agrees with the
interpretation that this feature (AUA) in anurans reflects a plesiomorphic character within a
particular group or family [7, 10]. A comparison of three species of Heleophryne furthermore
supports this view [23]. The flagellar arrangement of particularly the Anura is therefore one of the
most important features in determining the relative phylogenetic position of a species. Our results
confirm a change from the AUA to a flagelium without an axial rod when examining the more
primitive to the more advanced South African anurans.
Our results furthermore show a distinct relationship between sperm head length and mode
of fertilization among external fertilizing anurans. The sperm heads and acrosomes of aquatic
fertilizing anurans are significantly shorter than those of the TF. It is also known that the egg
coverings of TF are generally thicker than those of AF, presumably to protect the eggs against
dehydration [13]. A longer sperm head and a larger acrosome which may contain more digestive
enzymes may have been an advantage in penetrating these thick egg coats [24], The information
on head and acrosome length may also be of predictive value in other anuran species in
establishing whether they belong to the TF or AF grouping. Three South African Heleophryne
species posses sperm heads with lengths varying from 23 to 28 |im [23]. They are predominantly
TF and further support our results. It is therefore clear from our data on South African anurans
that sperm head and acrosome dimensions reflect on the fertilization biology rather than the
phylogeny and is independent of the type of flagellum (only axoneme or with undulating
membrane and axial rod).
The sperm of the three orders of Lissamphibia can be distinguished on the basis of distinct
sperm features. A diagnostic feature for urodele and caecilian sperm is a complex acrosome that
predominantly extends from the anterior part of the head and does not form a cap surrounding the
anterior tip of the nucleus as in all anurans and an acrosomal cap is therefore a diagnostic feature
for anurans. Here Ascaphus [10] seems to occupy an intermediate position in having a complex
acrosome like the urodeles and caecilians but it also forms a cap around the anterior tip of the
nucleus. A juxta-axonemal fibre at position 8 is a diagnostic feature for urodeles and separates
them from the caecilians and anurans. Anurans on the other hand have developed a minor juxta-
axonemal fibre at position 3 which is not clearly defined in urodeles and apparently absent in
caecilian sperm [22], Glycogen packets have furthermore been observed in two ranid species [15]
and in Xenopus [19] and while the occurrence of these packets is uncommon, they seem to be
specific for anurans. Chiromantis sperm have spherical mitochondria in contrast to Rhacophorus
sperm which have elongate mitochondria. The spherical mitochondrion appears to represent the
primitive condition in anurans [10] and this may suggest a plesiomorphic character for
Chiromantis in relation to Rhacophorus.
Further sperm structural features that seem to reflect on the fertilization biology of urodeles
and caecilians will be briefly discussed. A correlation appears to exist between the length of the
neck-piece and the length of time that the sperm is retained in the female cloaca. In
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
341
Cryptobranchus external fertilization is the rule and the neck-piece is very small whereas in
Diemictylus, where sperm are retained in the cloaca for several months, the neck-piece is long [3].
In two species of viviparous Typhlonectidae the acrosome is barbed or hooked [6, 22], However,
in four oviparous taxa of caecilians the acrosomes are spatulate [16, 17, 18]. It appears that the
shape of the acrosome of caecilians may be predictive in determining oviparity or viviparity of a
given species [22],
Our results on quantitative sperm motility in anurans indicate that sperm from all AF
possess motile sperm that swim progressively forward in a physiological medium of 30
mOsm/kg. In contrast sperm from TF are immotile in a wide range of physiological and culture
media ranging from 10 to 300 mOsm/kg. In AF, sperm swim in a low osmolality environment
even if the male and female cloacae are in close proximity such as in Xenopus. Sperm of
Chiromantis xerampelina (TF) only exhibited a brief spurt (seconds) of hyperactivated motility. In
TF the sperm are deposited directly on the eggs and the need for vigorous and longer term motility
seems less than in the AF group. It should also be conceded that sperm activation in TF may be
dependant on specific substances associated with the egg coat/surface and may explain the
immotile status of TF sperm in our experiments.
Finally sperm motion is highly species specific among the South African anurans. The
pattern of sperm motion appears to be related to the form of the sperm head, the type of tail
(presence or absence of undulating membrane) and the type of flagellar beat. The AF anurans with
only an axoneme seem to exhibit greater values for most motion parameters than those with an
axoneme-undulating membrane-axial rod and the AF accordingly have larger star symbol plots
(Fig. 9).
In summary it appears that several sperm structural features in the Lissamphibia are
diagnostic in separating the three main orders, families (urodeles), and even closely related
species ( Heleophryne ). It furthermore assists in phylogenetic inferences and is predictive in terms
of fertilization biology. Quantitative sperm motility analysis of representative examples of seven
of the nine South African anuran families suggest that sperm motility is species specific and also
relates to fertilization biology.
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18. Seshachar, B. L., 1945. — Spermateleosis in Uraeotyphlus narayani Seshachar and Gegenophis carnosus Beddomc
(Apoda) Proceedings of the National Institute of India, 11: 336-340.
19. VAN DER Horst, G., 1979. — Spermatozoon structure of three anuran species. Proceedings of the Electron
Microscope Society of Southern Africa, 9: 153-154.
20. van der Horst, G.. Curry, P. T., Kitchin, R. M., Burgess, W., Thorne, E. T., Kwiatowski, M., Parker, M. &
Atherton, R. W., 1991. — Quantitative light and scanning electron microscopy of ferret sperm. Molecular
Reproduction and Development, 30: 232-240.
21. van der Horst, G., Kitchin. R. M., Curry, P. T. & Atherton, R. W., 1989. — Use of membrane filters and
osmium tetroxide etching in the preparation of sperm for scanning electron microscopy. Journal of Electron
Microscopic Technique , 12: 65-70.
22. VAN der HORST, G., Visser, J. & van DER Merwe, L., 1991. — The ultrastructure of the spermatozoon of
Typhlonectes natans (Gymnophiona: Typhlonectidae). Journal of Herpetology, 25: 441-447.
23. Visser, J. & VAN DER Horst, G., 1987. — Description of Heleophryne sperm (Amphibia, Leptodactylidae).
Proceedings of the Electron microscopy Society of Southern Africa, 17: 83-84.
24. Wilson, B. A., 1994. — The relationship between fertilization environment and structure and physiology of
selected anuran spermatozoa. Ph. D. thesis. University of the Western Cape, Bell ville. South Africa: 1-147.
25. Wortham, J. W. E., Brandon, R. A. & Marian, J., 1977. — Comparative morphology of some plethodontid
salamander spermatozoa. Copeia, 1977: 666-680.
Source . MNHN. Paris
Evolution of Tetrapod Spermatozoa with
Particular Reference to Amniotes
Barrie G. M. JAMIESON
Zoology Department, University of Queensland
Brisbane, Q 4072, Australia
ABSTRACT
Synapomorphies of tetrapod sperm appear to be: nuclear ‘shoulders’; elongation, relative to dipnoans, of two
longitudinal elements (dense fibres) peripheral to the axoncme adjacent- to doublets 3 and 8; and, questionably,
development of an annulus. A lissamphibian synapomorphy relative to Neoceratodus may have been loss of one
undulating membrane, leaving a single undulating membrane adjacent to the fibre of doublet 3. Amniote synapomorphies
(retained in Chelonia and Sphenodontida) include: elongation of the distal centriole through the entire length of the
moderately elongate midpiece; subspheroidal mitochondria, with concentric cristae; a fibrous sheath; nine peripheral
axonemal fibres; inward projections (longitudinal columns) of the fibrous sheath aligned with fibres 3 and 8; loss or
transformation of the retronuclear body, present in dipnoans and (as the neck structure) urodeles. A possible crocodilian
synapomorphy is a thick dense sheath around the singlets of the axoneme or the distal centriole. Synapomorphies of birds
are loss of the subacrosomal cone and, less certainly derived, adhesion of all nine dense fibres to their axonemal doublets
(also in monotremes). The conical acrosome, fibrous sheath, and elongate centriole of ratites are symplesiomorphies not
proving monophyly. Restriction of the cndonuclear canal to the anterior region of the nucleus in other non-passerines and
passerines may be a synapomorphy of these, homoplasic with crocodiles and derived ratites (emu). Squamate
synapomorphies are: loss of endonuclear canals with restriction of the perforatorial rod to a prenuclear location;
intermitochondrial bodies; forward extension of the fibrous sheath into the midpiece; a paracrystalline subacrosomal
cone; and, homoplasically, shortening of the centriole. Mammal sperm are distinguished by loss of the perforatorium (and
canal), homoplasic with some non-ratite birds, great reduction of the centrioles, and. in therians, (apomorphic?)
detachment of peripheral fibres, except sometimes 3 and 8, from the doublets.
RESUME
Evolution des spermatozoides des Tetrapodes, en particulier des Amniotes
Les synapomorphies des spermatozoides des tetrapodes semblent etre: des “epaules' nucleates; Elongation, en
comparaison des Dipneustes, de deux elements longitudinaux (fibres denses) en peripherie de Eaxon£me et adjacents aux
doublets 3 et 8; et de maniere incertaine, le d£veloppement de I’annulus. Une synapomorphie des Lissamphibiens en
comparaison de Neoceratodus peut etre la perte d'une membrane ondulante, ce qui laisse une seule membrane ondulante
adjacente a la fibre du doublet 3. Les synapomorphies des Amniotes (conserves chez les Chelonia et les Sphenodontia)
comprennent: Elongation du centriole distal sur toute la longueur de la piece intermediaire. qui est moddrement allong^e;
des mitochondries subspheriques, avec des cretes concentriques; une gaine fibreuse; neuf fibres axonemales peripheries;
des projections vers Einterieur (colonnes longitudinales) de la gaine fibreuse alignees avec les fibres 3 et 8; la perte ou la
transformation du corps retronucl^airc, present chez les Dipneustes et (comme structure du cou) chez les Urodeles. Une
synapomorphie possible des Crocodiliens est la gaine dense epaisse entourant les singulets de Eaxoneme ou le centriole
distal. Les synapomorphies des Oiseaux sont la perte du cone subacrosomien et E adhesion des neuf fibres denses h leurs
Jamieson, B. G. M., 1995. — Evolution of tetrapod spermatozoa with particular reference to amniotes. In:
Jamieson, B. G. M.. Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoa! Phylogeny and Taxonomy. Mem. Mus.
natn. Hist, nat., 166 : 343-358. Paris ISBN : 2-85653-225-X.
344
B. G. M. JAMIESON : AMNIOTA (TETRAPODA)
doublets respectifs. Cette derniere structure existe aussi chez les Monotremes et son caract^re evolue est moins assure.
L’acrosome conique, la gaine fibreuse et le centriole allongd des Ratites sont des symplesiomorphies qui ne prouvent pas
leur monophylie. La restriction du canal endonucI6aire & la region anterieure du noyau chez les autres non-passereaux et
chez les Passereaux peut etre une synapomorphie pour ces groupes, homoplasique avec les Crocodiles et les ratites evolues
(Emu). Les synapomorphies des Squamates sont: la perte des canaux endonucleaires avec la restriction de la baguette du
perforatorium a une position pr^-nuclaire; des corps intermitochondriaux; une extension vers Lavanl de la gaine fibreuse
dans la piece intermediate; un cone subacrosomien paracristallin; et de manure homoplasique. le raccourcissement du
centriole. Les spermatozoides des Mammiferes se distinguent par la perte du perforatorium (et du canal) qui est
homoplasique avec certain Oiseaux non-ratites. la grande reduction des centrioles, et, chez les Theriens (apomorphie?), le
detachement des fibres peripheries, exceptees parfois les 3 el 8, des doublets.
This chapter constitutes a brief review, with new material, of the sperm of the amniotes as a
whole and attempts to reconstruct the features of the spermatozoon of the ancestral Amniota and
the course of spermatozoal evolution, as indicated by deduced synapomorphies, in the amniote
classes. This reconstruction is based on intuitive consideration, and also on a cladistic analysis
[22], of spermatozoal ultrastructure in the constituent tetrapod groups. Further reviews of the
ultrastructure of vertebrate sperm may be found in the recent publications of JAMIESON [21] and
HEALY & JAMIESON [16, 22]. Material of dipnoan and urodele sperm is included for comparative
purposes.
MATERIALS AND METHODS
Testes and ducts were dissected from euthanased specimen(s) of Neoceraiodus forsteri (Dipnoi), Taricha granulosa
(Urodela), Emydura kreffti (Chelonia), Sphenodon punctatus (Sphenodontida), Crocodylus johnstoni (Crocodilia),
Lampropholis delicata (Scincidae, Squamata), Geopelia striata (Columbiformes, Aves) and Rhinolophus megaphylla
(Chiroptera, Mammalia). Processing of the tissues was as in [27].
RESULTS AND DISCUSSION
Comparative ultrastructure of amniote spennatozoa
The acrosome, endonuclear canals and perforatoria. All amniote classes (Reptilia, Birds and
Mammals) contain some species, or a majority of species, in which the acrosome* has a
plesiomorphic tripartite structure which, from the evidence of its presence in all three
lissamphibian orders (Urodela, Gymnophiona and Anura) [25], was already present in early
tetrapods ancestral to Lissamphibia and Amniota. In the plesiomorphic tripartite acrosome the
acrosome vesicle forms an elongate, narrow cone symmetrically located on the tip of the nucleus
which narrows within it to a point. The acrosome vesicle encloses a similarly shaped
subacrosomal cone and axially within this there are one to three rodlike perforatoria. The pointed
form of the acrosome, presence of the subacrosomal cone, and tapering of the tip (rostrum) of a
Fig. 1. — Examples of sperm ultrastructure in selected amniote classes and orders. The species depicted in each vertical
column are 1: Emydura kreffti (Chelonia); 2: Sphenodon punctatus (Sphenodontida); 3: Crocodylus johnstoni
(Crocodilia); 4: Lampropholis delicata (Scincidae, Squamata). 5: Geopelia striata (Columbiformes, Aves); and
6: Rhinolophus megaphylla (Chiroptera, Mammalia). The first transverse row, A, E, I, M, Q, and U, are
longitudinal sections through the sperm head (acrosome and anterior nucleus). The second row, B, F, J, N. R, and
V, are longitudinal sections through the midpiece and, in all except F, the posterior region of the nucleus. The third
row, C, G, K, O, S, W, are transverse sections through the midpiece. The fourth row, D, H, L, P, T and X, are
transverse sections through the principal piece. Abbreviations: a, acrosome vesicle; an, annulus; bp, basal plate;
db, dense bodies (mitochondrial transformations); dc, distal centriole; ec, endonuclear canal; et, electron-lucent
epinuclear region; fs, fibrous sheath; lb, lateral body at anterior region of distal centriole; m, mitochondria; n,
nucleus; p, putative perforatorium; pc, proximal centriole; pf, peripheral dense (coarse) fibres; sc, subacrosomal
cone; stc, striated column. Scale bar = 1 pm unless otherwise indicated.
Source MNHN. Paris
345
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
0,5 pm
0.5 um
0,2 pm
0,2
0,5 pm
an
Source : MNHN, Paris
346
B. G. M. JAMIESON : AM N IOTA ( TETRAPODA )
cylindroid nucleus within this, are seen in the Chelonia [13, 16, 19, 22] (Fig. 1A), Crocodilia
( Caiman crocodylus [45], and Crocodylus johnstoni , Fig. II), Sphenodontida ( Sphenodon ) [16,
22] (Fig. IE), Squamata [5, 6, 13, 27] (Fig. 1M), non-passerine birds [24, 37] (Fig. IQ), and
monotremes [7]. All of these plesiomorphic representatives of their classes, with the exception of
mammals, and, in the non-passerines, the columbiforms (Fig. IQ), possess the perforatorial rod
or rods.
In the Squamata the subacrosomal cone has a paracrystalline substructure [5, 6, 13],
recently confirmed for Sphenomorphus and Eugongylus group skinks, the gekkonid Heteronotia
binoei , and in snakes [35], It constitutes a basal synapomorphy of the Squamata [22],
The perforatorial rod in the Squamata is wholly prenuclear (Fig. 1M) but this restriction is
clearly apomorphic. The plesiomorphic condition is seen in basal lissamphibians where a rod
penetrates the nucleus to varying depths, each within an endonuclear canal: viz. urodeles [41] and
the primitive anurans Ascaphus [25], Discoglossus (spermatid only) [47, 48], and bombinids,
including Bombina and Alytes [43]. The number of endonuclear canals and of enclosed
perforatoria is one in basal Lissamphibia, in the caiman (though poorly substantiated by
micrographs), tinamou, rhea and non-passerines (e.g. galliforms), but in the Chelonia and
Crocodylus johnstoni there are two or three canals and there are two in Sphenodon. There are
three endonuclear canals in the sperm of the sturgeon, Acipenser sturio. Although there appears to
be only a single canal in the coelacanth, Latimeria clialumnae, this contains two or three
perforatoria [21] and two to four perforatoria are seen in Neoceratodus (Fig. 3B). It is therefore
probable that the presumed common ancestor of Amphibia and amniotes possessed more than one
perforatorium and endonuclear canal. A single canal appears basic to all amniotes above turtles
and Sphenodon [22], In Acipenser the canals are spiralled around each other as they are in turtles,
Sphenodon and Crocodylus johnstoni. The spiral arrangement or at least the presence of one or
more endonuclear canals may well be a synapomorphy for the Osteichythes, a monophyletic clade
including the Actinopterygii (Ray-finned fish), Sarcopterygii and, within the latter, the Tetrapoda.
That a canal was present even earlier is evidenced by presence of a canal and filamentous
perforatorium in lamprey sperm. The canals are absent (presumed lost) in the highly simplified
sperm of the Chondrostei and Neopterygii [21].
In birds, a conical acrosome vesicle penetrated almost to its tip by a subacrosomal space
which contains a rodlike perforatorium has been demonstrated ultrastructurally in the non¬
passerines turkey, Meleagris gallopavo, chicken, Gallus domesticus, guinea fowl, Nuniida
meleagris (e.g. [55]), the mallard duck, Anas platyrhynchos [20], and the quail Cotumix cotumix
[20], parrots [24] and in the ratites (palaeognaths) tinamou, Eudromia elegans [1], ostrich,
Struthio camelus [3, 51, 52] and emu, Drornaius novaehollandiae [3], Like the sperm of ratites
and other birds, parrot sperm differ from those of reptiles in reduction of the subacrosomal
material (subacrosomal cone, excluding any perforatorium) to a negligible amount [24], In the
columbiforms even a perforatorium is absent although, at least in Geopelia striata (Fig. 1 Q), some
longitudinally orientated subacrosomal material is present and lacunae are present in the nucleus
which may represent a vestigial endonuclear canal.
Not all non-passerines possess a conical acrosome. A small, approximately spherical
acrosome has been described for the white-naped crane, Grus vipio [40], for Jacana jacana [46]
and most Charadriiformes [11], and for the wood pecker Melanerpes carolinus ([18], These latter
avian taxa are considered to be advanced non-passerines, on the basis of DNA hybridization
studies [49, 50].
In the non-passerine and suboscine spermatozoon the acrosome is short relative to the
nucleus, as in reptiles [22, 27, 28], in contrast to the oscine spermatozoon which has an
extremely large acrosomal complex. In passerines the acrosome vesicle becomes an elongate
single-keeled helix, with no evident subacrosomal cone, in, for instance, finches [30, and
Koehler, this volume].
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
347
The endonuclear canal extends almost to the base of the nucleus in Chelonia and Sphenodon [16,
17, 22], and this is considered a plesiomorphic state [22], This condition also occurs in putatively
more primitive ratites (i.e. tinamou [1]), where it is probably also plesiomorphic relative to the
shorter condition in other non-passerines and in advanced ratites, being wholly prenuclear in the
emu [3]. However, there is a possibility that deep penetration in ratites is secondary [22], The
canals extend deeply into the nucleus in Crocodylus johnstoni (Fig. II). A single canal is depicted
as penetrating only the nuclear rostrum in the caiman [45] but the extent requires further
investigation. The endonuclear canal is limited to the anterior 1 to 2 (im of the nucleus in rooster,
guinea fowl, turkey and parrots and the anterior third of the nucleus in the ostrich [3]. The canal is
lost in mammals and, homoplasically, in squamates.
In mammals, as in the bat Rhinolophus megaphylla (Fig. 1U), rodlike perforatoria do not
occur. Although monotremes [7], retain the elongate conical structure of the acrosome and the
subacrosomal cone, this form is greatly modified in the Eutheria (e.g. [44]) and Metatheria [53].
In Eutheria the acrosome is flattened, with the notable exception of the pangolin [33].
The nucleus and nuclear fossa. The nucleus is plesiomorphically elongate in amniotes from
Chelonia through Sphenodon, crocodiles, squamates, birds, monotremes and, in therian
mammals, the pangolin alone [33], as in lissamphibians.
Representation of the basal nuclear fossa, loosely termed the implantation fossa, is variable
in amniotes but some of this variation may be spurious as it is difficult to establish the shape of
the fossa. It appears poorly developed in the sperm of Caiman crocodylus [45] but has a low
dome-shaped form in Crocodylus johnstoni (Fig. 1J). It is small and compact in turtles, tuatara,
rooster, guinea fowl, and squamates excepting some skinks in which it is narrowly funnel-
shaped. In the ratites it has a a triple profile (references in [22]). It is dome-shaped to rounded
conical in eugongyloid skinks [28]. It is a shallow cone in the gekkonid Heteronotia hinoei and
compactly conical in the pygopodid Lialis burtoni [26]. The compact form appears to be
plesiomorphic for amniotes [22].
The annulus. A dense ring, the annulus, at the posterior end of the midpiece is a feature of
many metazoan sperm. It is clearly plesiomorphic for amniotes, occurring in all classes [22] but
absence in Dipnoi possibly indicates apomorphic re-acquisition in tetrapods. It is well developed
in Chelonia, Sphenodon [16, 17, 22], Caiman crocodylus [45], the American Alligator [39] and
Crocodylus johnstoni (Fig. 1J). In squamates, it has been demonstrated for Lacerta vivipara [8],
Cnemidophorus sexlineatus [34], sphenomorph and eugongyloid skinks [27, 28], Heteronotia
binoei, Varanus gouldii flavirufus [35], and, though reduced in some species, in snakes [23, 35].
The annulus is basic to birds, being seen in ratites, rooster, guinea fowl, and columbiforms [2]
but is apomorphically absent in parrots [24], It is weakly developed in monotremes [7]. Two
structural categories of annulus have been recognized in therian mammalian sperm, based on the
profile of the annulus as viewed in longitudinal section: triangular (e.g. bats, dormouse, Chinese
hamster, antelope) and semicircular (e.g. mouse, guinea pig, chinchilla, ram) [12]. However, the
taxonomic and phylogenetic significance of the shape of the annulus is questionable, given that in
the Rodentia both categories are encountered.
The number of mitochondria. The number of mitochondria seen in transverse section of the
midpiece is very variable in amniotes but some of the apparent variation requires confirmation,
particularly as there is variation along the midpiece. Determination of the total number by scanning
electron microscopy would be desirable. It appears that a number, in transverse section, in the
order of 6 to 9 may have been plesiomorphic; 6 has been recorded for the Chelonia, 9 in
Sphenodon, 6 to 8 in Caiman crocodylus [22, 45] and 6 in Crocodylus johnstoni (Fig. IK). In
the remaining amniotes, a trend towards reduction, in transverse section, to 4 in birds and
monotremes has been suggested [22]. It is 4 in ratites (e.g. [51]) and in the turkey [54] and in the
order of 5 in Geopelia striata (Fig. IS). However, in squamates the number remained
plesiomorphically high in lizards or showed apomorphic increase to as many as 14, in snakes.
348
B. G. M. JAMIESON : AMNIOTA (TETRAPODA)
while a reduction to 2 in gekkos was correlated with intrusion of intermitochondrial material of
supposed mitochondrial origin into the transverse section of the midpiece. Further variability in
numbers is now known though it cannot be fully documented here. For instance, large numbers
of small mitochondria occur, it seems apomorphically, in eugongyloid skinks [28] and
approximately 9 have been observed in transverse section of the budgerigar sperm [24], The
mitochondria are subspheroidal in Chelonia, Sphenodon and crocodiles and this may reasonably
be inferred as the plesiomorphic condition. The number of tiers of mitochondria in longitudinal
sequence is in the order of 10 in turtles [16] which is also presumed to be plesiomorphic. In the
spiral midpiece of mammals, the number of gyres varies from 55 to 300 [10] but is not specified
for monotremes [7],
Structure of the mitochondria. In turtles (Fig. IB, C), tuatara (Fig. IF, G), Caiman
crocodylus and Crocodylus johnstoni (Fig. 1J, K), the mitochondria have a form known only in
the sperm of one other amniote, the Woolly opossum, Caluromys philander (see [36]; [9]). The
mitochondrial cristae in these three taxa are concentric and usually surround a large central dense
body. In all other amniotes studied, the cristae have a “conventional” appearance, being linear or
curved, as in Lissamphibia, but never concentric, and do not surround a dense body.
Linear cristae in spermatozoal, as in somatic mitochondria, must be accepted as a
plesiomorphic condition for tetrapods as they are normal for metazoan sperm, including fish. The
concentric arrangement with dense body appears to be an apomorphy acquired early or initially in
amniote evolution and retained paraphyletically in the tuatara, crocodile, and turtle clades [16, 22].
In spermatids of Caiman crocodylus [45] and in at least some mitochondria of spermatids of
Sphenodon [16, 22], the cristae have the linear appearance usual for metazoan sperm and the
concentric arrangement is a late development. Phylogenetic “reversion” of mitochondrial of
concentric cristae to the linear condition seen in other amniotes would need only suppression of
this final transformation [22], Presence of concentric cristae and the intramitochondrial body in
the woolly opossum is construed as homoplasic although the possibility that ancestral mammals
retained this condition from basal amniotes cannot be ruled out [16].
The intermitochondrial rings or dense bodies of squamate sperm are regarded as derivations
of the intramitochondrial dense bodies [6, 16, 22] and as such a “reminiscence” of the occurrence
of concentric cristae in the ancestry of squamates. Origin of intermitochondrial material from
mitochondria has been confirmed ontogenetically in the sperm of some squamates [35]. Extra-
mitochondrial dense bodies are almost limited to squamates but are seen, poorly developed, in
Geopelia striata (Fig. 1R) in which, although appearing homoplasic, they may well indicate
persistence of a genetic basis laid down in early amniotes.
The centrioles. Presence of the proximal centriole can be regarded as plesiomorphic for
tetrapods and is seen in all amniote classes. It persists, well developed, in monotremes [7], but is
absent from mature therian mammals, for instance, the rat [10] and the bat, Rhinolophus
megaphylla (Fig. 1 V).
A distal centriole is at most a vestige in mature mammalian sperm [10] (see also Fig. IV),
but is well developed in sperm of anurans [32], Chelonia (Fig. IB, C), Sphenodon (Fig. IF, G),
crocodiles [16, 22] (Fig. 1 J, K), squamates [13] (Fig. IN), and birds [1, 2] (Fig. 1R). The distal
centriole, forming the basal body of the axoneme, is plesiomorphically short in vertebrates,
including the Lissamphibia and squamates [27]. In contrast, the distal centriole extends the entire
length of the long midpiece in turtles (Fig. IB, C), the tuatara (Fig. IF, G), crocodiles (Fig. 1 J,
K), and ratites, an apparent basal synapomorphy of amniotes. These elongate centrioles differ
from most metazoan basal bodies in being penetrated by two central singlets from the axoneme.
Thus in spermatids of the ratite Rhea , the distal centriole elongates and, late in spermiogenesis,
becomes penetrated by a central pair of tubules from the developing axoneme [38]. The shorter,
though still elongate distal centriole in the rooster and the somewhat shorter centriole in guinea
fowl (0.6 pm) and Geopelia striata (0.5 pm) (Fig. 1R), the short centriole in squamates, and the
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
349
vestigial centriole in monotremes possibly represent secondary reduction in length of the centriole
[16], culminating in almost total reduction in therian mammals.
The distal centriole is embedded in a ring of dense material in all of the amniotes for which it
has been investigated. A cross striated dense body lateral to the proximal centriole appears to be a
basal synapomorphy of amniotes but its homology across the various groups requires
confirmation. It is seen in tuatara, and the caiman where homology with the nine striated columns
of eutherian sperm has been suggested [16] but has yet to be demonstrated in Crocodylus
johstoni. In the skink Nangura it is represented by a bilateral laminated structure [27] and shows
various manifestations in other squamates. It is does not appear to have been reported in birds.
The axoneme and appurtenances. An annulated, helical, dense fibrous sheath (Fig. IB, D,
F, H, J, L, N-P, X) must, clearly, have developed in the earliest amniotes as it is present in all
amniote classes. Homology of this sheath across the different classes is reinforced by the
presence in mammals of two spurlike inwardly directed triangular processes, seen in cross section
of the sperm, in the vicinity of doublets 3 and 8, where they form large ‘longitudinal columns’
[10], though at most weakly developed in monotremes, and in ratites. In the non-passerines,
rooster and guinea fowl, the fibrous sheath has transformed into an amorphous sheath [55] while
in parrots [24] and doves ( Ocyphaps lophotes, pers. obs., and Geopelia striata) it is lost.
In most of the amniotes investigated the fibrous, or amorphous, sheath commences
immediately behind the midpiece. This condition is seen in turtles (Fig. IB), Caiman crocodylus
and Crocodylus johnstoni (Fig. 1 J), ratites, non-passerines (absent in parrots and doves) and
mammals. However, in squamates the fibrous sheath extends anteriorly well into the midpiece, a
clear squamate autapomorphy [16, 22, 27], as in Lampropholis delicata (Fig. IN, O).
An external longitudinal protuberance (rib) on each side of the fibrous sheath is seen in
many amniotes, including eutherians, and is particularly well developed in the tinamou [1]. A
sheath of putative glycogen external to the fibrous sheath was found to be limited to the tinamou
[38] and cannot be ascribed to the plesiomorphic amniote sperm.
Nine longitudinal dense fibres (coarse fibres) peripheral to the nine axonemal doublets, or to
the distal centriole also where this is elongated as in Emydura, Sphenodon and Crocodylus, are a
fundamental feature of amniote sperm, being found in all classes [22, 27], The presence of nine
fibres is an autapomorphy and simultaneous symplesiomorphy of the amniotes, though nine
appear homoplasically in other groups, such as some lampreys and the osteoglossomorph fish
Pantodon [21], and in heterobranch and cephalopod molluscs [14, 15], The dense fibres are small
in turtles (Fig. 1C), Caiman crocodylus and Crocodylus johnstoni (Fig. IK), the tuatara (Fig.
1G), squamates (Fig. 10), birds and monotremes. They have been observed in turkey, rooster
and, though requiring confirmation, in guinea fowl [55], the mallard duck [20], in parrots [24],
and in the anteriormost region of the principal piece of ratite spermatozoa [1, 3, 51, 52] but are
described as “tiny” for the rhea, are absent from the tinamou [1], and are greatly reduced in
columbiforms (Fig. IS). They are present in suboscine and the more apomorphic oscine
passerines, being larger in the latter. They are large and diverse in shape in marsupials above the
didelphids, and in eutherian mammals (Fig. 1W). There thus appear to be trends to enlargement
of the peripheral fibres in passerines and non-monotreme mammals, with diversification in the
latter, and to reduction in ratites, and doves. In Chelonia, Sphenodon, Caiman crocodylus (but
not apparently Crocodylus johnstoni) and in squamates, the fibres at doublets 3 and 8 are enlarged
[16, 22] and are possibly homologous with the axial fibre, at 3, and juxta-axonemal fibre at 8, in
lissamphibian sperm.
Significant synapomorphies are here suggested for amniote groups on the basis of the
configuration of the peripheral axonemal fibres. In reptiles the peripheral fibres at 3 and 8 are
detached from their corresponding doublets while the other seven fibres are attached to their
doublets. In birds and monotremes all of the peripheral fibres are attached to the corresponding
doublets [12], In contrast to both of these assemblages, in therian mammal sperm the peripheral
350
B. G. M. JAMIESON : AM N IOTA ( TETRAPODA )
fibres are detached from their doublets with the exception that fibres 3 and 8 may be close to or
attached to their doublets [12, pers. obs.].
The peripheral fibres are usually situated in the midpiece with some extension into the
principal piece as in turtles [22], the caiman [45], non-passerines [2], tuatara [22], and
monotremes [7]. In the rhea and tinamou dense fibres are present only in the proximal principal
piece. Very small dense fibres are present only in the distal region of the midpiece in the rooster
and mallard; dense fibres in turtle dove sperm disappear before maturation is complete (see review
by Asa & PHILLIPS [2]), though they persist through a short region of the midpiece in Geopelia
striata. In eutherians and marsupials they extend far into the principal piece. However, in
squamates, the only well developed, though small, peripheral fibres at the level of the annulus are
the double fibres at doublets 3 and 8 and by the beginning of the principal piece all nine dense
fibres are already vestigial or absent [27]. The fibres extend through most of the length of the
sperm cell in oscine passerines [38].
In turtles, Sphenodon [16], Crocodylus johnstoni , and in skinks [13, 27, 28], the nine
peripheral dense fibres are partly displaced from the radii of the triplets of the distal centriole into
the gaps between adjacent triplets; the fibres are coradial with the doublets in the axoneme. These
locations have been regarded as plesiomorphic for amniotes in Fig. 1 . Dense material surrounds
the central singlets in Chelonia (Fig. 1C), Sphenodon (Fig. 1G), and in crocodiles (Fig. IK ). In
crocodiles the compact dense sheath appears to be a distinctive synapomorphy.
Snake sperm are characterized, apomorphically, by multilaminar membranes in place of the
normal plasma membrane of the midpiece and axoneme [23, 35]; see also pygopodids [26].
The hypothetical plesiomorphic amniote spermatozoon (Fig. 2)
From the above comparative and cladistic considerations of the anatomy of amniote sperm,
the following features may be attributed to the hypothetical plesiomorphic amniote spermatozoon.
Relative to the tetrapod ground plan, deduced from common features of the amniote and
lissamphibian sperm, amniotes are seen to have few basal synapomorphies. These are indicated,
among plesiomorphic features of the amniote sperm, below.
The spermatozoon was elongate and filiform, with a hollow anterior conical acrosome
vesicle overlying a simple subacrosomal cone. The base of the acrosome invested the tapered
anterior tip (rostrum) of the nucleus and rested on pronounced nuclear ‘shoulders’. The
subacrosomal space within the acrosome contained two or three axial rods (putative perforatoria)
or possibly only one rod. These penetrated the nucleus deeply, almost to its base, in endonuclear
canals. At the base of the nucleus there was a compact fossa (implantation fossa) with which were
associated two triplet centrioles of which the distal formed the basal body of the flagellar
axoneme. The distal centriole was extremely elongate (amniote synapomorphy), traversing the
entire length of the moderately elongate midpiece, the latter being defined by the presence of
mitochondria. The mitochondria were subspheroidal, with concentric cristae and
intramitochondrial dense body (amniote synapomorphies), and formed a circlet of several in cross
sectional view around the distal centriole and several tiers in longitudinal section. The posterior
end of the midpiece was defined by a subplasmalemmal dense annulus possibly homologous with
the ring seen in urodele sperm [42], The axoneme proper, consisting of nine peripheral doublet
and two central singlet microtubles, posterior to the midpiece and constituting the principal piece,
was surrounded by a fibrous sheath (amniote synapomorphy) which plesiomorphically was
annulated. The terminal portion of the axoneme formed a short endpiece defined by the absence of
the sheath. The elongate distal centriole and, internal to the fibrous sheath, a long anterior region
of the axoneme, was surrounded by nine dense peripheral fibres, one to each triplet or doublet
respectively (an amniote synapomorphy relative to fibres at 3 and 8, only, in lissamphibians). In
the principal piece, two fibres, at doublets 3 and 8, were aligned with an inward projection
Source :
ADVANCES IN SPERM ATOZOAL PI I YLOGENY AND TAXONOMY
351
Acrosome vesicle-
Plasma membrane-
Simple subacrosomal cone
Paracrystalline in squamates -
Lost in ratites
Two? endonuclear canals
2 or 3 in Chelonia and Crocodylus. 2
in Sphenodon. 1 in other amniotes or
lost in monotremes and squamates
Endonuclear canals deep
As in Chelonia,
Crocodylus, Sphenodon, and rhea.
Most of length of nucleus in tinamou.
Lost in monotremes and squamates
Anterior only in other amniotes
Perforatorium prenuclear in
squamates
Elongate nucleus
In basal members of all
amniote classes
Basal nuclear fossa compact -
Triple in ratites. Funnel-like in
skinks
Dense body lateral to centriole*
Sphenodon, caiman and snakes
= striated columns in mammals?
Several mitochondria in-
sperm cross section
Mitochondrial cristae concentric
As in Chelonia, Sphenodon. and-
crocodiles (and Wooly opossum).
'Conventional' in other amniotes
WlA
9 dense peripheral axonemal fibres 1 rj
All amniotes excepting tinamou
In mid- and principal piece or, in rhea,
in principal piece only -
Proximal centriole
Distal centriole extending
throughout midpiece
As in Chelonia, Sphenodon, Crocodilia
and ratites. Lost in mammals
Dense intramitochondrial body
As in Chelonia, Sphenodon, and
Crocodilia.
Transformed into intermitochondrial
structures in squamates. Lost in
birds and monotremes
2 central singlets
Annulus -
In all amniotes but reduced or absent in
some squamates and some birds and
reduced in monotremes
No glycogen sheath
Present only in tinamou
Fibrous sheath of axoneme -
Annulate, excepting non-passerines
in which it is amorphous or lost.
Not extending into midpiece
(does so only in squamates)
Fig. 2. — Diagrammatic representation of the hypothetical plesiomorphic amniote spermatozoon, the features of which
are deduced in the text.
352
B. G. M. JAMIESON : AM N I OTA ( TETRAPODA )
(longitudinal column) of the fibrous sheath. It is possible that the two fibres were enlarged and
laterally displaced, and that all fibres in the centriolar region intruded into the inter-triplet radii, as
in ‘lower’ amniotes (Chelonians, Sphenodon and crocodiles). As nine peripheral fibres are seen
in lampreys and Pantodon, it might be considered that nine is the basic tetrapod, rather than
merely amniote, number and that amphibians have lost all but those represented by the fibres at
doublets 3 and 8 but there is no evidence in extant Lissamphibia for such a reduction and the
presence of only two lateral elements in dipnoans and Latimeria suggests that nine fibres were an
amniote synapomorphy, albeit homoplasic with some lish taxa. The retronuclear body seen in
dipnoans and urodeles was lost or possibly transformed into the striated pericentriolar material.
The hypothetical plesiomorphic lissamphibian spermatozoon (Fig. 3)
A survey of the literature, of which only a few references can be given here, together with
personal observations, on the sperm of urodeles [42], Gymnophiona [56], and Anura [25, 31,
32, 43] permits the following generalizations as to the ultrastructure of the sperm of ancestral
lissamphibians (Fig. 3).
The hypothetical plesiomorphic lissamphibian spermatozoon may be attributed an anterior
acrosomal vesicle forming a hollow, cone which overlay a cone of subacrosomal material. This
embraced the tapered anterior end (rostrum) of the elongate nucleus. In Anura, basically, the
acrosome vesicle and underlying cone embrace the nuclear rostrum. Urodeles (at least
salamandrids and plethodontids, pers. obs.) are apomorphic in that the posterior limit of the
acrosome vesicle is anterior to the nucleus, only the subacrosomal cone investing the nuclear
rostrum. At the posterior end of the acrosome (vesicle and subacrosomal cone) the lissamphibian
sperm nucleus plesiomorphically forms characteristic ‘shoulders’ posterior to which its form is
cylindrical. Axially, within the acrosome, there is a rod, the putative perforatorium, which deeply
penetrates the nucleus within an endonuclear canal. An axial rod (perforatorium), though lodged
posteriorly in a much shorter endonucler canal, is also present in gymnophionids. As it is also
present in the primitive frogs Discoglossus [43] and Ascaphus [25] and in the bombinids
Bombina and Alytes [43], it is reasonable to deduce that a long acrosome rod and endonuclear
canal is plesiomorphic for the Lissamphibia. This is endorsed by persistence of this condition in
lower amniotes, the Chelonia, Sphenodontida and at least Crocodylus johnstoni in the Crocodilia.
The base of the nucleus was indented as a basal nuclear fossa which contained the proximal
centriole behind which lay the distal centriole which formed the basal body of the axoneme.
Behind this, retronuclear material may have persisted to form the neckpiece but not as well
developed as that seen in urodeles [42] above the cryptobranchs.
The structure of the sperm tail or flagellum is highly distinctive of the Lissamphibia. The
axoneme has the structure usual for eukaryotes of nine peripheral doublet and two central singlet
microtubules but it has distinctive appurtenances. To the generalized lissamphibian sperm can be
attributed two longitudinal fibres, one on each side of the axoneme, adjacent to doublets 3 and 8.
The fibre at doublet 8 is closely adjacent to the ring of doublets and may be termed the juxta-
axonemal fibre at 8. This fibre is usually, but secondarily, absent in Anura. The fibre associated
with doublet 3 is separated from the axoneme by a thin sheet of cytoplasm, the undulating
membrane (with or without intervention of a juxta-axonemal fibre), and is termed the axial (major)
fibre. The epithet axial refers to reported undulation of the axoneme around this stiff fibre in the
Urodela and some primitive frogs (see KwON & LEE, this volume).
In urodele sperm, the axial fibre, at doublet 3, is connected to the axoneme by the
undulating membrane but, typically, as in Taricha granulosa (Fig. 4C), there is no intervening
juxta-axonemal fibre [4, 42], Plethodon albagula [25, 32] is exceptional, of urodeles studied, in
having a density on the adaxonemal end of the undulating membrane but it is questionable,
because of its connection to dense bodies near the major fibre rather than to the fibre, that this is
Source MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
353
■Proximal centnole
Juxla-axonemal fibre
at 3 (mino« l*>»e>
■Distal centnole
Juxta-axceemai
fibre at 8 >-
Axial fibre
(major fibre)
Axoneme
Juxta-axonemal
fibre
(mator fibre)
UmMating membrane -
Minor (juxta-
axonemal)
membrane
(maior fibre)
Jndutal no
Fig. 3. — Diagrammatic representation of the hypothetical plesiomorphic lissamphibian spermatozoon, the features of
which are deduced in the text.
Source : MNHN, Paris
354
B. G. M. JAMIESON : AMNIOTA ( TETRAPODA )
homologous with the anuran juxta-axonemal fibre at 3. An axial fibre with undulating membrane
is present in caecilians, in which juxta-axonemal fibres are absent [56]. Presence of a longitudinal
fibre, near doublet 3, connecting to the axoneme via an undulating membrane appears, at least on
first analysis, to be a synapomorphy of the Lissamphibia irrespective of whether it is subdivided
into an axial (major) and juxta-axonemal (minor) fibre or is accompanied by a fibre at doublet 8.
The possibility has been mooted [21, 25] (see also next section) that it is the unilateral location of
the undulating membrane and its axial fibre, rather than presence of undulating membranes per se,
which constitutes the synapmorphic condition for the Lissamphibia.
Pre-tetrapod presence of undulating membranes
Two longitudinal elements occur, one at each of doublets 3 and 8, in the dipnoan
Neoceratodus forsteri (Fig. 4A) and the actinistian Latimeria. Sarcopterygian fish, and particularly
dipnoans, appear to be the nearest extant non-tetrapod relatives of amphibians. Two, bilateral,
elements which also occur at doublets 3 and 8 in Chondrichthyes are deduced to have been
convergently acquired [21].
Fig. 4. — A: Neoceratodus forsteri. Transverse section through the anterior region of the tail of the spermatozoon. There
is a large dense rod or fibre on each side of the axoneme, at doublets 3 and 8, and each rod is continuous with a Tin’
which terminates with a smaller, lateral density or fibre. B: Transverse sections of the axonemes more
posteriorly where the fin is sufficiently extensive to be termed an undulating membrane. Three transverse sections
of nucleus and endonuclear canals with contains perforatoria are also seen in the top of the figure. C: Taricha
granulosa. Transverse sections through sperm tails showing urodele features of a longitudinal juxta-axonemal fibre
at doublet 8, a long undulating membrane, and, in salamandroids, as in plethodontids, the Y-shaped transverse
section of the axial (major) fibre. Abbreviations: af, axial (major) fibre; jf3 and jf8, juxta-axonemal fibres at
doublets 3 and 8, respectively; If, lateral fibre; p, perforatoria in the endonuclear canal of the nucleus; um,
undulating membrane. Scale bar = 1 pm.
In Neoceratodus the anterior region of the sperm axoneme (not merely within the
cytoplasmic canal as previously reported [29]) has a large dense rod on each side, at doublets 3
and 8, and each rod is continuous with a ‘fin’ which terminates with a further, smaller density
(Fig. 4A, B). It is tempting to recognize homology between each fin and an amphibian sperm
undulating membrane, between the large dense rods at doublets 3 and 8 and the amphibian juxta-
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
355
axonemal fibres and between the terminal densities (lateral fibres in Fig. 4A, B) and the axial
fibre. This, if valid, would suggest that lissamphibians have retained, from an ancestor shared
with dipnoans (and with other sarcopterygian fish?), only one of two former, bilateral, undulating
membranes, and only one of a former pair of axial rods, but that the two juxta-axonemal fibres of
urodeles are a persistence of the paired ancestral condition, the fibre at doublet 8 normally being
lost in the Anura.
The condition in ray-finned fishes (Actinopterygii) of two lateral fins (also at doublets 3 and
8) [21] could conceivably have been precursory to lissamphibian undulating membrane and to
dipnoan fins but homoplasy cannot be ruled out as lateral axonemal fins occur also in some
echinoderms and protostomes.
Ground plan of the ancestral tetrapod spermatozoon
To the ground plan sperm of the basal tetrapod can be attributed all of the features which
have been ascribed above to the ground plans of the lissamphibian and amniote spermatozoa with
the exclusion of the synapomorphies identified for each of these groups. Synapomorphies of the
tetrapod sperm relative to a presumed common ancestor shared with dipnoans, as exemplified by
Neoceratodus, are deduced to have been few: nuclear ‘shoulders’ were developed as in basal
amniotes and lissamphibians; an annulus (persisting in amniotes and possibly as the ‘ring’ in
urodeles in the lissamphibians) may have developed or, alternatively, may have been retained
from a pre-dipnoan ancestry. There were probably two longitudinal elements (dense fibres)
peripheral to the axoneme adjacent to doublets 3 and 8, as in dipnoans, but these were
apomorphically more extensive or, at least, well developed over a greater length, traversing much
of the length of the axoneme. As noted above, a synapomorphy relative to Neoceratodus may
have been loss of an undulating membrane between the fibre at 8 and the axoneme, leaving an
albeit initially short undulating membrane between the fibre at 3 and the axoneme. The fibre at 8
would have persisted close to the axoneme. Plesiomorphic features deduced for the tetrapod
sperm ground plan are the short distal centriole, and mitochondria, probably few immediately
behind the nucleus, which had linear cristae as in lissamphibians. It appears unlikely that there
were nine dense fibres as in amniotes.
Synapomorphies of higher amniote taxa
Chelonia and Sphenodontida. Turtles and Sphenodon conform to the hypothetical
plesiomorphic amniote sperm and no convincing apomorphy seems demonstrable for the sperm of
Sphenodon relative to the Chelonia.
Crocodilia. The ground plan for the Crocodilia, as exemplified by Crocodylus johnstoni , is
very similar to that of the Chelonia and Sphenodon. All three have two or more endonuclear
canals and concentric cristae with intramitochondrial bodies. Reduction to one perforatorial rod in
Caiman crocodylus and Crocodylus johnstoni and restriction of the endonuclear canal to the
anterior region of the nucleus in Caiman (though requiring confirmation) are apomorphies of the
Crocodilia relative to Chelonia and Sphenodon. However, a possible synapomorphy of
crocodiles, seen in Caiman crocodylus [45] and Crocodylus johnstoni (Fig. IK) is investment of
the two central singlets of the axoneme or of the distal centriole in a thick dense sheath which
differs from the density, resembling a fibre, associated with the singlets in Chelonia, Sphenodon
[16] and (homoplasically?) snakes [23, 35].
Aves. Loss of the subacrosomal cone may be a synapomorphy of birds as a whole. If they
are the sister-group of crocodiles, they have apomorphically lost the concentric mitochondrial
cristae. Ratites, though forming a monophyletic clade, have appeared paraphyletic [22] relative to
non-ratite birds+monotremes but the three synapomorphies unifying the latter clade were
insubstantial and probably artefactual. Monophyly of ratites cannot be considered proven,
356
B. G. M. JAMIESON : AMNIOTA ( TETRAPODA )
however, as features considered to unify them (conical acrosome, fibrous sheath, and elongate
centriole) [3] are all symplesiomorphies. Those in the earlier, cladistic analysis (supposed
reversion to a long endonuclear canal, loss of the subacrosomal cone) [22] were also
unconvincing and were associated with a computed paraphyletic origin of birds, in which ratites
appeared as the sister group of galliforms+monotremes. If, however, the long endonuclear canal
in more primitive ratites is a plesiomorphy carried over from basal amniotes, restriction of the
canal to the anterior region of the nucleus in other non-passerines and passerines may be a
synapomorphy of these, albeit homoplasic with crocodiles and derived ratites (emu). The avian
feature of adhesion of all nine dense fibres to their axonemal doublets is also seen in monotremes;
that it is apomorphic is debatable.
Squamata. Squamates are unified and distinguished by striking spermatozoal
synapomorphies: loss of endonuclear canals and restriction of the perforatorial rod to a prenuclear
location; development of intermitochondrial bodies (apparently from intramitochondrial bodies of
lower amniotes); forward extension of the fibrous sheath into the midpiece; probably the
paracrystalline structure of the subacrosomal cone; and, homplasically with other groups,
shortening of the centriole from the elongate basal amniote condition.
Mammalia. Loss of the perforatorium (and endonuclear canal), homoplasic with some non-
ratite birds, and great reduction of the distal centriole are synapomorphies jointly diagnosing
mammal sperm. Separation, in marsupials and eutherians, of the peripheral dense fibres from
their doublets with the exception that those at 3 and 8 may be attached to their doublets (the
reverse of the situation in ‘reptiles’), may be a therian synapomorphy.
ACKNOWLEDGEMENTS
I am very grateful to Linna Daddow, David SCHHLTINGA and Christopher TUDGE for excellent technical assistance.
For access to material I thank Dr. Anne KEMP ( Neoceratodus ), Dr. Harry Grier ( Taricha ), Dr. Arthur Georges (Emydura), Dr.
Alison CREE (Sphenodon), Tony TUCKER ( Crocodylus ), Simon Oliver ( Lampropholis ) and Professor John D. PETTIGREW
( Rhinolophus ).
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Squamata. II. Agamidae, Varanidae, Colubridae, Elapidae, and Boidae (Reptilia). Herpetologica, (in press).
36. PHILLIPS, D. M., 1970. — Ultrastructure of spermatozoa of the wooly opossum Caluromys philander. Journal of
Ultrastructure Research, 33: 381-397.
37. Phillips, D. M. & Asa, C., 1986. — Ultrastructure of avian spermatozoa. Journal of Cell Biology, 103: 239a.
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38. Phillips, D. M. & Asa, C. S., 1989. — Development of spermatozoa in the Rhea. Anatomical Record , 223: 276-
282.
39. Phillips, D. M. & Asa, C. S., 1993. — Strategies for formation of the midpiece. In: B. BacCETTI, Comparative
Spermatology 20 Years After. New York, Raven Press: 997-1000
40. Phillips, D. M., Asa, C. S. & Stover, J.. 1987. — Ultrastructure of spermatozoa of the white-naped crane. Journal
of Submicroscopic Cytology , 19: 489-494.
41. Picheral, B., 1967. — Structure et organisation du spermatozoide de Pleurodeles waltlii Michah. (Amphibien
Urodele). Archives de Biologie, (Liege), 78: 193-221.
42. Picheral, B., 1979. — Structural, comparative, and functional aspects of spermatozoa in urodeles. In: D. W.
Fawcett & J. M. BEDFORD, The Spermatozoon. Baltimore, Urban & Schwarzenberg: 267-287.
43. PUGIN-RlOS, E., 1980. — Etude comparative sur la structure du spermatozoide des Amphibiens Anoures.
Comportement des gametes tors de la fecondation. These, Universite de Rennes, Rennes, France: 1-114.
44. Rouse, G. W. & Robson, S. K., 1986. — An ultrastructural study of megachiropteran (Mammalia : Chiroptera)
spermatozoa: implications for chiropteran phylogeny. Journal of Submicroscopic Cytology , 18: 137-152.
45. Saita, A., Comazzi, M. & Perrotta, E., 1987. — Electron microscope study of spermiogenesis in Caiman
crocodylus L. Bollettino di Zoologia . 4: 307-318.
46. Saita, A., Longo, O. M. & Tripepe, S., 1983. — Osservazioni comparative sulla spermiogenesi. III. Aspetti
ultrastrutturali della spermiogenesi di Jacana jacana (Caradriformes). Accademia Nazionale dei Lincei
(Rendiconti della Classe di Scienze fisiche, matematiche e naturali), 74: 417-430.
47. Sandoz, D., 1970a. — Etude ultrastructurale et cytochimique de la formation de l’acrosome du discoglosse
(Amphibien Anoure). In: B. Baccetti, Comparative Spermatology. Rome, Accademia Nazionale dei Lincei: 93-
1 13.
48. Sandoz, D., 1970b. — Etude cytochimique des polysaccharides au cours de la spermatogen£se d’un amphibien
anoure: le discoglosse Discoglossus pictus (Otth.). Journal de Microscopie , 9: 243-262.
49. SIBLEY, C. G. & AHLQUIST, J. E., 1990. — Phylogeny and Classification of Birds: A Study in Molecular Evolution.
New Haven, Yale University Press.
50. Sibley, C. G., Ahlquist, J. E. & Monroe, B. L., 1988. — A classification of the living birds of the world based on
DNA-DNA hybridization studies. The Auk , 105: 409-423.
51. Soley, J. T., 1993. — Ultrastructure of ostrich (Struthio camelus) spermatozoa: 1. Transmission electron
microscopy. Onderstepoort Journal of Veterinary Research , 60: 1 19-130.
52. Soley, J. T., 1994. Centriole development and formation of the flagellum during spermiogenesis in the ostrich
( Struthio camelus). Journal of Anatomy, 185: 301-313.
53. Temple-Smith, P., 1987. — Sperm structure and marsupial phylogeny. In: M. Archer, Possums and Opossums:
Studies in Evolution. Sydney, Surrey Beatty & Sons and the Royal Society of New South Wales: 171-193.
54. Thurston, R. J., Hess, R. A., Hughes, B. L. & Froman, D. P. 1982. — Ultrastructure of the guinea fowl ( Numidia
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55. Thurston, R. J. & Hess, R. A., 1987. — Ultrastructure of spermatozoa from domesticated birds: Comparative study
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56. van der Horst, G., Visser, J. & van der Merwe, L., 1991. — The ultrastructure of the spermatozoon of
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Source : MNHN. Paris
The Ultrastructure of Spermatozoa of the Squamata
(Reptilia) with Phylogenetic Considerations
Barrie G. M. JAMIESON
* Zoology Department, University of Queensland,
Brisbane, Q 4072, Australia
ABSTRACT
Comparative ultrastructure of squamate families is reviewed, with new data for the South African chamaeleon,
Bradypodion karroicum. Parsimony analysis is conducted, using Chelonia as the outgroup and branch and bound
searching. Two major spermatozoal autapomorphies for the Squamata are extension of the fibrous sheath into the midpiece
and (not computed) the paracrystalline subacrosomal cone. Further synapomorphies defining the Squamata sensu strictu
are a single perforatorium in place of the two or three of Sphenodontida and Chelonia; loss of the endonuclear canal,
presence of sinuous mitochondria (possibly an artefactual parsimony resolution as a columnar form is intuitively
preferred); intermitochondrial location of dense bodies (mitochondrial transformations); presence of a well developed
epinuclear electron lucent region and, equivocally, arrangement of the dense bodies as periodic rings. A major inference is
polyphyly of the ‘Sauria’, the Scincomorpha and the Scincidae. Sphenomorphus group and egernid skinks show no close
relationship to Eugongylus-group skinks which form the sister-group of the pygopodid Lialis. Snakes are the sister-group
of the Eugongylus+pygopod clade. Gekkonidae appear to be a relatively plesiomorphic group, separated by several
families from the Pygopodidae. The Iguania is not a monophyletic assemblage, iguanids and Pogona occur in the same
clade but Pogona appears to be the sister-taxon of Varanus. Another iguanian, the chamaeleon Bradypodion , has an
unresolved relationship with the gekkonid+snake+pygopodid+Eugongylus-clade. Sphenomorph and egernid skinks form
an unresolved clade with Chalcides and lacertids but linkage of lacertids with the Teiidae in the Lacertoidea is not upheld.
Pending further investigations of a larger number of taxa, these results can only be considered heuristic.
RESUME
L’ultrastructure des spermatozoides des Squamata (Reptilia) et considerations phylogeniques
Ce chapitre comprend une synthase de T ultrastructure comparee des families de Squamata, avec des observations
nouvelles sur le Cameleon d’Afrique du Sud Bradypodion karroicum , et une analyse de parcimonie utilisant les Chelonia
comme outgroup et une recherche par branch and bound. Les deux autapomorphies majeures des spermatozoides pour les
Squamata sont Textension de la gaine fibreuse dans la pi£ce intermediate et (n’intervenant pas dans le calcul) le cone
subacrosomien paracristallin. D’autres synapomorphies d6finissant les Squamata sensu strictu sont un perforatorium
unique & la place de deux ou trois chez les Sphenodontida et les Chelonia; la perte du canal endonucteaire; la presence de
mitochondries sinueuses (ce qui pourrait donner lieu & un artefact de resolution car la forme en colonne est pr6f£r£e de
manure intuitive); la position intermitochondriale des corps denses (transformations mitochondriales); la presence d’une
region 6pinucl6aire claire aux electrons bien developp£e, et, de manure equivoque, la disposition des corps denses en
anneaux periodiques. Une consequence majeure est la polyphylie des "Sauria”, des Scincomorpha et des Scincidae. Le
groupe de Sphenomorphus et les Scincidae ergenides ne montrent pas de relations proches avec les Scincidae du groupe de
Eugongylus, qui forment le groupe frere du pygopodide Lialis. Les Serpents sont le groupe-fr&re du clade
Jamieson, B. G. M., 1995. — The ultrastructure of spermatozoa of the Squamata (Reptilia). with phylogenetic
considerations. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 359-383. Paris ISBN : 2-85653-225-X.
360
B. G. M. JAMIESON : SQUAMATA (REPTILIA)
Eugongylus+pygopodes. Les Gekkonidae semblent etre un groupe relativement pISsiomorphe, s6par6 par plusieurs
families des Pygopodidae. Lcs Iguania ne sont pas un assemblage monophyletique, les iguanides et Pogona se trouvent
dans le meme clade mais Pogona semble etre le groupe-frere de Varanus. Les relations d un autre iguanien, le cameleon
Bradypodion , ne sont pas resolues avec le clade gekkonides+serpents+pygopodides+Eugongylus. Les Sphenomorphes et
les Scincidae egernides forment un clade non resolu avec Chalcides et les lacertides mais la liaison des lacertides avec les
Teiidae dans les Lacertoidea n’est pas soutenue. En attendant d’autres etudes sur un plus grand nombre de taxons, ces
resultats peuvent seulement etre consideres comme heuristiques.
There is no comprehensive well-corroborated phylogeny available for the Squamata [28].
As the utility of spermatozoal ultrastructure as a source of characters for phylogenetic analysis is
well established [26, 27, 31, 32], JAMIESON et al. [37] and OLIVER et al. [45] attempted to shed
light on squamate classification and phylogeny by a comparative study of spermatozoal
ultrastructure which is reviewed and extended here.
The ultrastructure of spermatozoa or spermiogenesis has been studied, though often
cursorily, in the major groups of squamate reptiles. Families studied are: Scincidae [8, 14, 21,
29, 34, 35, 37, 44]; Lacertidae [7, 11, 21]; Teiidae [19, 41, 42]; Iguanidae [22, 48]; Anolidae
[10]; Tropiduridae [12, 13]; Agamidae [2, 9, 15, 16, 17, 18, 45]; Chamaeleonidae [52
(spermiogenesis only), and this account]; Varanidae [45]; Gekkonidae [21, 37, 46]; and
Pygopodidae [25, 37]. A brief account of the kinetic apparatus of the sperm of Amphisbaena
darwinii (Amphisbaenidae) and Liolaemus weigmanii (Iguanidae) by SOTELO & CENOZ [50] is
chiefly of historic interest and will not be reviewed here. The spermatozoa of Serpentes have been
more thoroughly investigated, in terms of taxa examined, than those of other squamates [1, 3, 5,
6, 20, 21, 24, 33, 45, 46, 49],
The present account reviews squamate sperm, provides the first description of mature
chamaeleonid sperm, for the rare South African chamaeleon Bradypodion karroicum , and gives a
preliminary parsimony analysis.
MATERIALS AND METHODS
Testes and ducts were dissected from a euthanased specimen of Bradypodion karroicum (Chamaeleonidae).
Processing of the tissues was as in [34]. A cladistic analysis was performed using the PAUP program of Swofford [51]
(for details see parsimony analysis).
RESULTS AND DISCUSSION
Comparative sperm ultrastructure
Spermatozoal ultrastructure in the Squamata is summarized in Tables 1 and 2.
Scincidae. Descriptions of the male gametes of the Scincidae include a description of the
mature spermatozoon of Chalcides ocellatus tiligugu [21]; an account of spermiogenesis, with
some description of mature, epididymal sperm, in the same subspecies [8]; brief descriptions of
spermiogenesis in C. ocellatus [14, 29]; an account of development of the midpiece in Eumeces
laticeps [44]; and descriptions, with phylogenetic considerations, by JAMIESON & SCHELTINGA,
of the sperm of the sphenomorph skink Nangura spinosa [34] and of Tiliqua scincoides, and
Ctenotus taeniolatus, with brief reference to Anomalopus verreauxii [35]. JAMIESON et al. [37]
Fig. 1. — A generalized spermatozoon (diagrammatic), in longitudinal and corresponding transverse sections, of the
Sphenomorphus group of the Scincidae (Nangura spinosa, Ctenotus taeniolatus, C. robustus, Anomalopus
verreauxii ) and Egemia-group (Tiliqua scincoides scincoides). Scales of various components are only approximate.
Regional zonation of the acrosome vesicle is shown only for the transverse sections. After [34, 37).
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Fig. 2. — A generalized spermatozoon (diagrammatic) of the Eugongylus-group species of the Scincidae. in longitudinal
and corresponding transverse sections. It is drawn from the sperm of Carlia pectoralis but is applicable also to
Cryptoblepharus virgatus (Eugongylus-subgroup), and Lampropholis delicata (with Carlia, in the Lampropholis-
subgroup). Scales of various components are only approximate. Regional zonation of the acrosome vesicle,
though present, is not indicated. After [37].
Source MNHN. Paris
362
B. G. M. JAMIESON : SQUAMATA (REPTIL1A)
have compared the sperm of Ctenotus robustus, a scincid of the Sphenomorphus group and other
sphenomorphs ( Ctenotus taeniolatus, Nangura spinosa), with those of Cryptoblepharus virgatus,
Lampropholis delicata, and Carlia pectoralis, in the Eugongylus-group, in the classification ol
GREER [23].
Spermatozoa of skinks (e.g. Ctenotus robustus, Carlia pectoralis , Cryptoblepharus
virgatus, and Lampropholis delicata) conform with other squamate sperm in the following
respects: the sperm are filiform; the acrosome vesicle is in the form of a hollow, concentrically
zoned cone which basally overlies a paracrystalline subacrosomal cone which invests the tapered
anterior end of the nucleus; the perforatorium is a slender rod extending anteriorly from the
subacrosomal material; the midpiece terminates with an annulus; peripheral dense fibres are
associated with the 9 triplets of the distal centriole and the doublets of the axoneme within the
midpiece; the peripheral fibres adjacent to doublets 3 and 8 are enlarged and each forms a double
structure associated with the annulated fibrous sheath; usually all nine peripheral fibres are absent
from the principal piece though in Lampropholis delicata they remain well developed in its anterior
region and fibres 3 and 8 sometimes extend into the endpiece; the fibrous sheath extends
anteriorly into the midpiece (squamate autapomorphy).
The sperm of species of the Sphenomorphus group (e.g. Ctenotus) and the Egernia-group
( Tiliqua ) (Fig. 1) differ from Eugongylus-group species ( Cryptoblepharus virgatus,
Lampropholis delicata and Carlia pectoralis) (Fig. 2), in the classification of GREER [23], in the
following features: (1) the acrosome is elongate (it is relatively short in Eugongylus-group
species); (2) the acrosome is depressed near its tip; (3) the perforatorium is strongly oblique (it is
very slightly oblique in Eugongylus-group species); (4) a conspicuous laminated structure is
present on each side of the proximal centriole (it is absent, though possibly represented by striated
column(s) in Eugongylus-group species); (5) the midpiece is shorter absolutely and relative to the
nucleus; (6) the midpiece has four dense ring structures in longitudinal succession (in
Eugongylus-group species mitochondrial transformations are scattered irregular dense bodies of
varying sizes); (7) mitochondria between the mitochondrial transformations form columnar
structures in a circle around the fibrous sheath with numerous predominantly longitudinal cristae
(in Eugongylus-group species mitochondria are elongate, tubular structures, with indistinct
cristae, and weave between the intermitochondrial bodies); (8) enlargement of the peripheral fibres
adjacent to doublets 3 and 8 occurs, as in all squamates, but not the gross enlargement which
occurs in the anterior region of the axoneme in Carlia and Lampropholis.
From microcomplement fixation of albumin, BAVERSTOCK & DONNELLAN [4] showed the
Eugongylus-group to be monophyletic as suggested by sperm ultrastructure [35].
The sperm of the European scincid species Chalcides ocellatus tiligugu [21] conforms
closely to the description given above for the Sphenomorphus group, particularly in having a
longitudinal series of four dense rings alternating with columnar mitochondria. An annulus was
not described but absence is doubtful in view of its presence in all squamates which have been
examined by the author. Ch. ocellatus tiligugu differs from the Sphenomorphus group in the
composition of the dense rings which are shown diagrammatically as each being composed of
large juxtaposed granules in single file. CARCUPINO et al. [8] describe the ring structures as “four
rings of electron-dense material” and do not mention a granular composition.
For the scincid Eumeces laticeps, OKIA [44] described a midpiece with nine mitochondrial
columns around the axoneme, a condition reminiscent of that in sphenomorphs. These “columns
lie segmented by partial or complete rings of dense material” but it is not clear whether their
arrangement conforms to the sphenomorph pattern.
A suite of character states which JAMIESON & SCHELTINGA [35] noted for the
Sphenomorphus and Egernia-groups of the Scincidae is also seen in the teiid lizard,
Cnemidophorus sexlineatus [41, 42] (see below): 1. Anterior depression of the acrosome. 2. The
conical nuclear fossa containing dense material projecting from the proximal centriole. 3. The
ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY
363
3
-plasma mentorano
- acrosome vesicle
Pig, 3, — Pogona barbata (Agamidae). A diagrammatic representation of the spermatozoon, in longitudinal and
corresponding transverse sections. After [37].
FlG. 4. — Bradypodion karroicum (Chamaeleonidac). A diagrammatic representation of the spermatozoon, in longitudinal
and corresponding transverse sections. Original.
Source :
364
B. G. M. JAMIESON : SQUAMATA (REPT1LIA)
presence of a laminar structure extending from the pericentriolar apparatus (possibly unilateral in
the teiid, and possibly homologous with the striated column(s) in Eugongylus-group sperm). 4.
Presence of four, or in Cnemidophorus five, intermitochondrial rings alternating with columnar
mitochondria. 5. The absence of sharply defined nuclear shoulders. 6. The apparent wide
separation of the plasma membrane from the fibrous sheath in the anterior region of the principal
piece. However, features 3 and 6 require confirmation for teiids. .
The parsimony analyses (below) which are necessarily preliminary in view ol our lmperlect
knowledge of these and other characters across the spectrum of squamate taxa, strongly suggest
that skinks are not monophyletic and that sphenomorph-egemid and Eugongylus-group skinks are
two distinct groups of squamates, the Eugongylus-group appearing to be the sister-group of
pygopods (Fig. 10). As snakes form the sister-taxon of these two groups, it would appear that the
legless condition has been independently derived in snakes as compared with pygopods, though
as a parallelism from an ancestor shared also with Eugongylus-group skinks.
Lacertidae. Spermatozoa of the Lacertidae have been examined ultrastructurally in Lacerta
sicula campestris, L. lepida lepida , L. laevis, L. viridis and Algyroides alleni [21], L. vivipara
[11] and, with reference to development of the sperm head, Podarcis ( =Lacerta ) taurica [7], They
are morphologically very similar and resemble those of the sphenomorph-egernid Scincidae in
many respects [21]. The head is curved and depressed [7, 11, 21]. The perforatorium (“apical
groove”) is oblique and the subacrosomal cone is paracrystalline [7, 21] as is typical of
squamates. The midpiece is distinctive in having only two sets of dense bodies, the first has the
appearance of two opaque masses on each side immediately posterior to the nucleus [21] and
termed the “nuclear plate” [11]; this is probably correctly regarded by NEWTON and TRAUTH [42]
as the equivalent of the first ring structure in teiid sperm. The second is a large ring (more
certainly equivalent to ring structures of sphenomorph skinks) and questionably regarded as a
“chromatoid body” [11], between the mitochondria in the distal third of the midpiece. In
L. vivipara, there are only three tiers of mitochondria in longitudinal section and the
intermitochondrial ring lies between mitochondrial tiers 2 and 3 [11]. The appearance and size of
the dense bodies varies from species to species but not clearly enough to be used as a defining
characteristic. The internal surface of the posterior ring is indented by numerous niches containing
small masses of moderately opaque material [21]. In the order of 10 mitochondria are illustrated in
transverse section for L. lepida [21] but only five in L. vivipara [11]. Though said to be
filamentous [21], the mitochondria appear compact and only a few times longer than wide, with
linear cristae. Peripheral dense fibres at 3 and 8 are enlarged and fused with or adpressed to the
fibrous sheath [11, 21]. In mature sperm no annulus is indicated diagrammatically but in a
micrograph a very small, weakly developed annulus appears to be present [21], and it is present
in the late spermatid [11]. There are two centrioles and the implantation fossa is compact and
rounded [1 1, 21].
Teiidae. The subacrosomal rod (putative perforatorium) has been shown in Cnemidophorus
lemniscatus lemniscatus to develop from a subacrosomal granule [19]. The spermatozoon of
Fig. 5. — Bradypodion karroicum (Chamaeleonidae). A, B, C: Longitudinal sections (LS) of acrosome and anterior
region of nucleus. D, E: LS of posterior region of nucleus and anterior region of midpiece. F-M: Successive
transverse sections through F-J, the acrosome. K, the nucleus, L, the midpiece and M, the principal piece. N: LS
centriolar region and midpiece. O: LS posterior region of midpiece, including annulus, and anterior region of
principal piece, a = acrosome vesicle; an = annulus; db = dense body (mitochondrial transformation); dc = distal
centriole; et = electron lucent space; fs = fibrous sheath; m = mitochondria; n = nucleus; nf = nuclear fossa; p =
perforatorium; pc = proximal centriole; pf = peripheral dense fibre (coarse fibre); pm = plasma membrane; sc =
subacrosomal cone. Original. Scale bar 1 pm.
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366
B. G. M. JAMIESON : SQUAMATA (REPTILIA)
C. sexlineatus has been the subject of detailed description [41, 42], augmented here by reference
to published micrographs. A suite of characters shared with the Sphenomoiphus and Egernia-
group Scincidae is discussed under that family, above.
In C. sexlineatus , the acrosome caps and invests the anterior 1 |im of the nucleus which
forms a pointed nuclear rostrum. The acrosome is depressed and spatulate, as in Sphenomorphus
and Egemia-group scincids. Although an acrosome vesicle is recognized, no subacrosomal cone
is described. Instead, a large vesicle and a more basal small vesicle are described posterior to the
main part of the acrosome vesicle. From a micrograph there seems some possibility that the large
and small vesicles represent subacrosomal material. It appears that a basal extension of the
acrosome vesicle envelops this material, as in typical squamate and, indeed, tetrapod
spermatozoa. This interpretation is supported by presence of an electron dense connective
between the posterior acrosome and the cell membrane. A well developed rodlike perforatorium,
composed of longitudinal fibres, possibly in helical array, is present. It does not appear to be
oblique but is unusual in being strongly eccentric. A narrow epinuclear electron lucent zone is
visible anterior to the rostrum. The nucleus is curved and strongly condensed and has a low,
conical, basal implantation fossa. The absence of sharply defined nuclear shoulders at the base of
the rostrum is a resemblance to the above-mentioned scincids. The midpiece consists of five tiers,
usually, of shortly columnar mitochondria separated by intermitochondrial dense “ring structures”
and terminating with an annulus, giving the formula rsl/mil, rs2/mi2, rs3/mi3, rs4/mi4, rs5/mi5,
an. Each mitochondrial set consists of 8 to 10 (usually 9) mitochondria arranged around the
axoneme. The axoneme is surrounded by the fibrous sheath from rs3 to the posterior end of the
principal piece, leaving a 0.7 pm endpiece. The A subtubules of the 9+2 axoneme are filled with
dense material. A transverse section of the distal centriole (basal body) shows coarse fibres
enveloping the triplets, extending both peripherally and internally to each triplet, and, as usual,
displaced clockwise into the inter-triplet space. Dense material is associated with the two central
singlets. Coarse fibres are absent from illustrated transverse sections of the principal piece.
Longitudinal extracellular tubules surround the nucleus and flagellum in the ductus deferens. The
suggested presence of a laminar structure extending from the pericentriolar apparatus, equivalent
to the bilateral structure in scincids [35], requires confirmation.
Iguanidae. The account by FURIERI [22], based chiefly on three species of the Iguanidae,
Cupriguanus scapulatus, Phymaturus palluma and Liolaemus austromendocinus , is summarized
here, using the terminology employed in the present account. The general structure of the
spermatozoon is much as described for skinks, with the following details. The acrosome is
circular in cross section; the single, well developed perforatorium appears to have a pointed, not
square, tip and to lack a basal plate but this requires confirmation. An endonuclear canal is absent.
The subacrosomal cone (“inner cap”) is paracrystalline and surrounds a pointed nuclear rostrum
which is preceded by an epinuclear electron lucent region. The nucleus is elongate, with
condensed chromatin, and has a shallow basal fossa which houses the anterior portion of the
proximal centriole; this is at an angle to the distal centriole. There are distinct nuclear shoulders,
apparently intermediate in shape between the sharp and rounded forms. The midpiece is relatively
short, and, with a length of 7 (im, is much shorter than the head. Although the mitochondria,
which are long, thin and numerous, are arranged in a regular circlet around the axoneme and
fibrous sheath, they form helices, with a regular arrangement in C. scapulatus and especially
L. austromendocinus. They have a very fine calibre in L. austromendocinus, thus being
reminiscent of those of snake sperm. The quantity and arrangement of the dense
intermitochondrial bodies are characteristic of each of the three species. In Phymaturus, the
mitochondrial sleeve is subdivided into six long sections, separated by contiguous small masses
of opaque material arranged in a ring which is sometimes not completely closed. A sixth dark ring
is present at the beginning of the mitochondrial sleeve and a seventh, much smaller ring (here
considered to be the annulus) closes the sleeve, giving the formula, in the system of NEWTON and
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
367
TRAUTH [42], of rsl/mil, rs2/mi2, rs3/mi3, rs4/mi4, rs5/mi5, rs6/mi6, an. In Cupriguanus , the
dense material is more scanty and does not seem to form closed rings. In Liolaemus, the dense
bodies form small dark plates which are few in number and are distributed without order.
Development of nine peripheral axonemal fibres is limited to the short region of the midpiece
anterior to the fibrous sheath. Within the fibrous sheath there are only two, enlarged fibres and
these are joined to the sheath. These are said to lie at doublets 3 and 7 but the micrographs and
diagram indicate that they are in the usual position adjacent to doublets 3 and 8.
Anolidae. The account of spermiogenesis in the so-called American chamaeleon, Anolis
carolinensis (Anolidae) [10] reveals no characters which can conclusively be ascribed to the
mature spermatozoon. However, the spermatid has the usual acrosome vesicle and subacrosomal
material investing the nuclear rostrum; a condensed, curved nucleus; a low, dome shaped but
apically pointed implantation fossa; and a proximal centriole tilted relative to the distal centriole.
The fibrous sheath is posterior to the annulus but whether it later extends into the midpiece is not
known, though likely.
Tropiduridae. Accounts of spermiogenesis in Tropidurus torquatus [12, 13] yield little
information with regard to the mature spermatozoon. The following account is derived from text
and micrographs. As usual for squamates, the acrosome vesicle and underlying subacrosomal
cone ensheath a tapered anterior extension of the nucleus (nuclear rostrum). Smooth nuclear
shoulders support the posterior end of the acrosome. There. is a large lacuna within the nuclear
rostrum, near it tip. The sperm head does not appear to be depressed. The very short midpiece has
only three gyres of mitochondria. The statement that the fibrous sheath is found only in the
principal piece, except for a few dense spots around the annulus (unlike any other known
squamate sperm) is to be doubted as the observation is not derived from a mature spermatozoon.
Peripheral dense fibres are limited to a short segment near the short neck cylinder which lies
between the proximal centriole and the base of the nucleus. Multiple cytoplasmic membranes
around the acrosome tend to disappear by maturity whereas those in snakes and pygopods appear
to persist. No evidence of dense bodies or ring structures is provided [13].
Agamidae. In the Agamidae, differentiation of the sperm head has been described for
Agama stellio [2], Uromastyx philbyi [15], Stenodactylus selvini [16], for A. adramitana [18]
and, for the nuclear manchette, A. agama [9]. Differentiation of the sperm tail has been described
in Uromastyx philbyi [17]. The entire sperm has been described for Pogona barbata [45] (Fig. 3).
The mature spermatozoon of Agama stellio illustrated by AL-HAJJ et al. [2] conforms
closely in ultrastructure to that of the cofamilial Pogona barbata [45]. Flattening of the acrosome is
again seen [2], as in A. adramitana [18], but supposed flattening of the nucleus is not supported
by micrographs. A well. defined narrow acrosomal cortex is present (see also [8, 18]). From
published accounts [37], division of the acrosome into cortex and medulla can be claimed not only
for the Agamidae but also for Lacertidae, Teiidae, Chamaeleonidae, Varanidae and all families of
the Serpentes. This subdivision is not apparent in Gekkonidae but it is possible that layering
described for sphenomorph skinks [34] and the anterior saccular enclave seen in the Eugongylus-
group skink Lampropholis delicata and in the pygopod Lialis burtonis [37] is equivalent. The
wide zone between the acrosomal cortex and the perforatorium [2, 1 8] is clearly the subacrosomal
cone. Late spermiogenic stages of A. agama [9] confirm the circular nuclear section but flattened
acrosome and wide, distinct nuclear shoulders which, as in Pogona barbata , are intermediate
between the concave, angular shoulders of, for instance, Eugongylus-group scincids, and the
rounded shoulders of sphenomorphs.
With regard to affinities of the sperm of Pogona barbata, it shares a larger number of
character states with the sperm of Varanus gouldii than with other non-agamid squamate taxa
which have been studied. Those also common to the sphenomorph-teiid-varanid assemblage have
been indicated above.
368
B. G. M. JAMIESON : SQUAMATA ( REPTILIA )
A varanid-agamid relationship does not appear to have been suggested on the basis of
somatic morphology (see review by RlEPPEL [47]). Of the three noteworthy spermatozoal
similarities of depressed acrosome, alternating, intermitochondrial rings and the not entirely
similar basal plates, only the knob-like form of the basal plate appears to be an agamid-varanid
synapomorphy and it is homoplasic with Eugongylus-groups skinks. Nevertheless, the validity of
recognizing a varanid-agamid relationship deserves further consideration.
"a number of features shared between Pogona barbata and sphenomorph skinks, Tiliqua
scincoides (Egernia-group), Cnemidophorus sexlineatus (Teiidae) and Varanus appear, on
intuitive consideration and in parsimony analysis, to be symplesiomorphic for these taxa: the
single, pointed perforatorium and linear cristae (plesiomorphic for squamates); the epinuclear
electron lucent zone, the elongate, cylindrical nucleus (plesiomorphic for tetrapods); absence of
multilaminar membranes around the midpiece (presence has recently been recognized as a
similarity and possible synapomorphy of snakes and pygopods [37, and this account]; and
extension of the fibrous sheath into the midpiece (autapomorphy of squamates).
Chamaeleonidae. A preliminary study of the spermatozoon of the rare South African
chamaeleon, Bradypodion karroicum (Figs 4, 5), reveals close similarity, on intuitive
consideration, to the spermatozoa of the Agamidae (see above). The acrosome is sharply
attenuated but is depressed in one plane, The acrosome vesicle is divisible into cortex and
medulla. Within the acrosome medulla the subacrosomal space encloses a perforatorium in the
form of a narrow cylinder which tapers to a blunt point anteriorly and is cross striated. A basal
plate, questionably present in Pogona barbata, is absent. The subacrosomal cone, occupying the
basal two thirds of the length of the acrosome, is conspicuously paracrystalline. The cylindrical,
electron dense nucleus has slight, smoothly rounded shoulders and a long, slightly curved nuclear
point (rostrum) which projects almost to the tip of the subacrosomal cone and is preceded by an
electron lucent epinuclear region. There is a broad basal nuclear fossa which, in being pointed
anteriorly, resembles that of sphenomorph skinks more closely than the shallow rounded fossa of
Pogona. The proximal centriole, consisting of nine triplets, lies at right angles to the long axis of
the sperm with its anterior region within the nuclear fossa (implantation fossa); a narrow density
anterolateral to this centriole appears to continue to the summit of the fossa. As in agamids, a
distinct lamellar structure is not discernible. As in Pogona, the distal centriole, forming the basal
body of the axoneme, is elongate but similarly does not extend into the fibrous sheath. In
transverse section of the midpiece through the fibrous sheath approximately 10 circular
mitochondrial profiles form a single circlet around the sheath. In longitudinal sections, the profiles
are again mostly circular but may show elongation consistent with a spiral arrangement and this is
particularly true of the mitochondria in the short region of the midpiece anterior to the fibrous
sheath. Small intermitochondrial dense bodies sporadically interrupt the mitochondria in
transverse and longitudinal sections. Dense rings of the type seen in sphenomorph skinks are not
present. A large dense granule is visible in some mitochondria and there is evidence of
transformation of mitochondria into intermitochondrial dense bodies. The midpiece is moderately
elongate and terminates at a distinct if small annulus. In the midpiece the axoneme has ninr coarse
fibres; those at 3 and 8 are enlarged, appear double, and are adpressed to the fibrous sheath. No
coarse fibres are visible in the principal piece, posterior to the mitochondrial midpiece. For a
considerable distance posterior to the annulus a wide band of cytoplasm surrounds the fibrous
sheath as in Pogona barbata and Varanus gouldii [45] but also in Eugongylus, sphenomorph, and
egemid group skinks [35] and in the gekkonid Heteronotia binoei [37],
In the parsimony analysis (Fig. 10) chamaeleonids have an unresolved position, with
gekkonids, at the base of the Eugongylus-group-pygopodid-snake clade. Their usual iguanian
relationship or placement in the Agamidae is not upheld.
Varanidae. In view of the fact that only one varanid has been examined for spermatozoal
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
369
"Him
7
nuclear fossa
mitochondria
proximal centriole
pericentnolar.
matrix
distal centriole.
■pencentriolar-
collar
mitochondrion
peripheral fibre
fibrous sheath
litochondrion
•peripheral dense-^
fibre at 8
- intermitochondrial
projection
(dense body or
mitochondrial
transformation)
plasma membrane
acrosome vesicle
- cortex
— perforatorium^
medulla
basal plate
acrosome vesicle*
nuclear rostrum-
subacrosomal com
flange of the
,subacrosomal
cone
nucleus-
nuclear fossa
annulus
pnncipal piece
1.0 pm
sndpiece
Fig. 6. — Varanus gouldii flavirufus (Varanidae). A diagrammatic representation of the spermatozoon, in longitudinal and
corresponding transverse sections. After (45].
Fig. 7. — Heteronotia binoei. (Gekkonidae). A diagrammatic representation of the spermatozoon, in longitudinal and
corresponding transverse sections. After (37].
Source . MNHN, Paris
370
B. G. M. JAMIESON : SQUAMATA (REPTILIA)
ultrastructure (Fig. 6), the following comparison with the sperm of other squamates can be
considered only a most preliminary indication of affinities of the family. A suite of character states
has been described for the sperm of Varanus gouldii flavirufus [45] which is known elsewhere in
squamates in the Sphenomorphus and Egernia ( Tiliqua ) groups of the Scincidae [37] and in the
teiid lizard, Cnemidophorus sexlineatus [42] although some are also seen in agamids (e.g.
Pogona barbata [45]). This suite includes the following states. 1 . Anterior depression of the
acrosome (also in agamids). 2. The conical nuclear fossa containing dense material projecting
from the proximal centriole. 3. The presence of a laminar structure extending from the
pericentriolar apparatus (possibly unilateral only in the teiid) although this is less definitely
laminated in V. gouldii. 4. Presence of four intermitochondrial rings alternating with columnar
mitochondria. The rings differ in V. gouldii in being composed of many large granules in
contrast with the solid condensed structure in the sphenomorph-Egernia-group and in
Cnemidophorus. The difference is lessened, however, by supposed constitution of the rings in
the scincid Chalcides ocellatus from a single circlet of large granules [21]. (An alternation of
mitochondria and albeit incomplete rings occurs in also in P. barbata). 5. The rounded rather than
angular nuclear shoulders are apomorphic relative to the “sharp” or angular shoulders of
Sphenodon [32] and primitive frogs [36]; a feature occurring only in the Eugongylus-group
skinks [37]. (P. barbata shows an intermediate condition.) 6. The wide separation of the plasma
membrane from the fibrous sheath in the anterior region of the principal piece (also in
P. barbata ), although this has not been demonstrated with certainty for Cnemidophorus.
In the parsimony analysis (Fig. 10) the alternation of ring structures with mitochondrial
columns computes as an ambiguous basic synapomorphy of the squamates as a whole, rather than
merely of the iguanid through sphenomorph assemblage. Flattening of the acrosome computes as
the sole basal synapomorphy of this assemblage, independently developed in Brady podion.
Consideration may be given, however, to the possibility that flattening is basic to squamates and
has been lost in the snake+pygopodid+Eugongylus-group clade.
Gekkonidae. In the Gekkonidae, FURIERI [2 1 ] briefly described the sperm of Lygodactylus
picturatus and made reference to Hemidactylus frenatus, H. mabouia, and Tarentola mauritanica
mauritanica-, PHILLIPS & ASA [46] described formation of the midpiece in Sphaerodactylus
cinereus and JAMIESON et al. [37] described the sperm of Heteronotia binoei.
In the spermatozoa of H. binoei (Fig. 7) no epinuclear electron-lucent region has been
observed (computing as a loss); nuclear shoulders are smooth, as in sphenomorph skinks;
mitochondria are large and discrete, arranged in a circle around the fibrous sheath, with
intervening mitochondrial transformations, and extend longitudinally as slender columns. A
feature not known in skinks is indentation of the median surfaces of the mitochondria at intervals
by triangular dense bodies which are perhaps always longitudinally interconnected.
The sperm of Lygodactylus picturatus , Tarentola mauritanica and Hemidactylus frenatus
[21] are generally similar to those of H. binoei, but the amount of intermitochondrial material
(dense bodies or putative mitochondrial transformations of the present work) is greater in
Lygodactylus picturatus and is present in decreasing amounts in Tarentola mauritanica and
Hemidactylus frenatus respectively. The stellate arrangement of dense bodies, seen in cross
section of the midpiece, in T. mauritanica closely resembles that in H. binoei. A stopper like
electron dense perforatorial plate, seen in H. binoei, is illustrated for T. mauritanica and, less
clearly, L. picturatus. A micrograph of the midpiece of Sphaerodactylus cinereus [46] shows a
longitudinal series of four columnar mitochondria on each side (said to total 20 mitochondria for
the midpiece) alternating with dense bodies, of comparable length, and the small annulus. In the
absence of transverse sections it is difficult to compare this arrangement with that of other
gekkonids but the columnar form of the mitochondria is reminiscent of that in Heteronotia.
Pygopodidae. The spermatozoa of Lialis burtonis (Fig. 8) are again like those of scincids in
Source :
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
371
FIG.
Q.5nm
. — Lialis burtonis (Pygopodidae). A diagrammatic representation of the spermatozoon, in longitudinal and
corresponding transverse sections. After [37].
Source : MNHN , Paris
372
B. G. M. JAMIESON : SQUAMATA (REPTILIA)
their chief features, as was noted by HARDING et al. [25] in their preliminary report for the
pygopods Aprasia repens , Delma tincta, L. burtonis and Pygopus lepidopus. However, in
L. burtonis the acrosome is fore-shortened and apically domed and the perforatorium extends
virtually to its tip; nuclear shoulders are absent; the mitochondria are small subspheroidal
structures, four or five in a transverse section, and appear very numerous in longitudinal single
file, alternating singly or in groups with one or more dense bodies, with evidence that they are
sinuous; dense bodies also form an interrupted collar around the distal centriole [37]. The
L. burtonis sperm shares a suite of apparently apomorphic character states of varying
distinctiveness with Eugongylus-group skinks ( Cryptoblepharus virgatus, Carlia pectoralis and
Lampropholis delicata). They are as follows [37], The perforatorium is square-ended rather than
pointed. The midpiece is elongate, that in the Eugongylus-group species being less elongate but
strikingly longer than that of sphenomorphs or other investigated squamates with the exception of
the very long mitochondrial sheath of snake sperm [33, 45], However, the mitochondria in
L. burtonis, though forming a single layer as in snake sperm, are not as narrow. They are not
irregularly interspersed with the dense bodies as they are in the Eugongylus-group skinks and are
not as elongate as the mitochondria of the Eugongylus-group and snakes. Nevertheless, an
elongated zigzag configuration much as in snake sperm is illustrated in a superficial longitudinal
section of the midpiece of Aprasia repens by HARDING et al. [25] and these authors have noted a
further striking similarity to snakes in the existence of a multilaminar membrane around the
midpiece in their material of L. burtonis, and around the flagellum in Delma tincta. These
multilaminar membranes had previously been considered unique to snake sperm [21, 33], The co¬
occurrence of elongate, tubular, zigzagged mitochondria and an, albeit transient, multilaminar cell
membrane in the sperm of pygopodids and snakes is here considered to warrant serious
consideration that these two groups of legless squamates share a common origin despite lack of a
direct sister-group relationship in the cladistic analysis (Figs 10, 11).
Serpentes. AUSTIN [3] gave a detailed account of the fine structure of the sperm tail in
Lampropeltis getulus, Coluber constrictor, Drymarchon corais, Crotalus adamanteus, Micrurus
fulvius and Constrictor sp., BOISSON & MATTEI [5, 6] described spermiogenesis in Python
sebae. HAMILTON & FAWCETT [24] gave details of the neck and midpiece in Lampropeltis
getulus and Constrictor constrictor, SAITA et al. [49] described spermiogenesis in Coluber
viridiflavius, PHILLIPS & ASA [46] described the formation of the midpiece with reference to the
behaviour of the annulus in Masticophis flagellum flagellum and AFZELIUS [1] described
occlusion of microtubules in Liophis miliaris. Nevertheless, only FURIERI [20, 21] had given an
account of the ultrastructure of the entire spermatozoon (giving in the latter work a general account
for four species of Colubridae, Coluber viridiflavius viridiflavius, Natrix tesselata tesselata,
N. natrix, and Coronella austriaca, and one species of Viperidae, Vipera aspis aspis) until the
spermatozoon of Nerodia sipedon was described by JAMIESON & KOEHLER [33], OLIVER et al.
[45] describe the sperm of Boiga irregularis, Stegonotus cucullatus (Colubridae),
O. microlepidotus (Elapidae), and Aspidites melanocephalus (Boidae).
Snake sperm (Fig. 9) present features [3, 20, 21, 24, 33, 45, 46] common to squamate
sperm: they are filiform; the acrosome vesicle is in the form of a hollow, concentrically zoned
cone which basally overlies a subacrosomal cone which invests the tapered anterior end of the
nucleus; the perforatorium is a slender rod extending anteriorly from the subacrosomal material;
the midpiece contains dense bodies (mitochondrial transformations) in addition to the
mitochondria; the fibrous sheath surrounding the axoneme extends anteriorly into the midpiece
(squamate autapomorphy); nine peripheral dense fibres accompany the triplets of the distal
centriole and the doublets of the axoneme in the midpiece; the fibres adjacent to doublets 3 and 8
are enlarged, each as a double structure associated with the fibrous sheath; the endpiece lacks
peripheral fibres and the fibrous sheath. A poorly developed “stopper-like” perforatorial base plate
Source :
Fig.
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
373
►principal piece
-endpiece -
iviiiira
w
9. — A diagrammatic representation of the spermatozoon of the Serpentes, in longitudinal and corresponding
transverse sections. After [45].
Source : MNHN. Paris
374
B. G. M. JAMIESON : SQUAMATA ( REPTILIA )
in the colubrid Nerodia sipedon, unknown in other snakes, is presumably homoplasic with that of
gekkonids. An electron-lucent space caps the nuclear point in the snakes Boiga irregularis and
Stegonotus cucullatus as in some other squamate orders [33, 45] but is poorly developed.
Less widespread is a suite of apparently apomorphic characters states of snake sperm [33,
45] shared with Eugongylus-group skinks ( Cryptoblepharus virgatus , Carlia pectoralis and
Lampropholis delicata) and pygopodids. The shared states are as follows. The midpiece is greatly
elongated in snakes (mitochondrial sheath of FURIERI [21]), that in the Eugongylus-group
species, and pygopodids but also in gekkonids, being less elongate but strikingly longer than that
of sphenomorph skinks or other investigated squamates. The mitochondria in at least some
pygopodids [25] form zigzagged tubes as in snakes. They are not irregularly interspersed with the
dense bodies as they are in the Eugongylus-group skinks. A multilaminar membrane is seen in
some pygopods as in snakes (see above). JAMIESON et al. [37] stated that these similarities of
snake, pygopodid and Eugongylus-group sperm warranted further investigation with a view to
determining the degree of homoplasy as against synapomorphy. Individually these characters
appear in some other squamates. Thus we have seen that extracellular tubules occur in immature
teiid sperm and that multilaminar membranes invest the acrosome in immature tropidurid sperm.
Possibly both types of structure are a normal feature of developing squamate sperm. Two
apomorphies of snakes and pygopodids, the multilaminar membrane and the tubular zigzagged
mitochondria are striking, and intuitively were considered synapomorphic resemblances.
However, multilaminar membranes computed as basal not only to these two groups but also to
Eugongylus-group skinks, in which they were computed as lost, and sinuous mitochondria
parsimoniously (but not, perhaps, plausibly) computed as a basic squamate feature.
The evidence, albeit uncertain, for transformation of extracellular tubules into multilaminar
membranes in Boiga irregularis possibly represents a stage in production of the membranes.
Conversion of microtubules into membrane-like laminate appendages of testicular sperm is known
in the Lepidoptera (see review in Jamieson [30]). It has been suggested that the multiple layers of
membranes provide a source of endogenous phospholipid that could be utilized as a source of
energy for motility [24],
The dense collar (termed the neck cylinder by AUSTIN [3]) is considered to be homologous
with the intermitochondrial dense bodies [24, 45] which in turn have been shown in skinks (e.g.
Cryptoblepharus virgatus) to be derived from mitochondria [37]. The dense element in the central
axis of the proximal centriole, seen in Aspidites , is also illustrated and described for Lampropeltis
getulus [24] and is stated to be regularly found in mammalian sperm. They [24] also observe the
presence, unknown in other vertebrates, of extracellular microtubules, observed here in at least
Aspidites melanocephalus and Boiga irregularis. With regard to the axoneme, they remind us that
enlargement and prolongation of the peripheral fibres 3 and 8 in snakes (as, we note, in all
squamates) is the reverse of the situation in the mammalian sperm tail where these are the smallest
fibres and terminate first. The density associated with one of the two central singlets [3, 24],
thought to be unique to snake sperm, has since been demonstrated for the skinks Nangura spinosa
[34], Tiliqua scincoides and less certainly Carlia pectoralis [35], the gekko Heteronotia binoei
[37] and the teiid Cnemidophorus sexlineatus [42] and may well be more widely demonstrated in
squamates when favourable sections of the centriole are obtained. Dense material filling the A
subtubules in the posterior portion of the axoneme has been demonstrated for doublets 1, 2, 5, 6
or 1, 2, 5, 6 and 7 in Liophis miliaris [1].
Parsimony analysis
A posteriori to intuitive consideration of comparative spermatozoal ultrastructure, with some
inferred relationships, which has been detailed above, preliminary parsimony analyses have been
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
375
Table 1. — Ultrastructural characters of squamate sperm used in parsimony analysis. Character states numbered in
parentheses are not transformation series as polarity was determined by use of an outgroup. The first state is
nevertheless considered plesiomorphic.
performed using the PAUP program. The characters employed are those listed in Table 1 and the
states of these characters are given in Table 2 which provides a summary of ultrastructure
discussed in this account.
A branch and bound search was performed for the total data matrix (Table 2). The following
options were applied: addition sequence: furthest; 1 tree held at each step during stepwise
addition; MULPARS option in effect; steepest descent option not in effect; initial upper bound:
unknown (compute via stepwise); branches having maximum length zero collapsed to yield
polytomies; no topological constraints; trees unrooted; multi-state taxa interpreted as
polymorphism; character-state optimization accelerated transformation (ACCTRAN). All
characters were treated as unordered.
The branch and bound analysis produced 240 most parsimonious trees from which a strict
(Fig. 11) and a 50% majority rule (Fig. 10) consensus tree was computed. These had the
following characteristics in both analyses: tree length = 43; consistency index = 0.767; homoplasy
index = 0.302; retention index = 0.839; rescaled consistency index = 0.644. Character state
changes are included in Fig. 10.
If the search was made with multi-state taxa scored as uncertainty (to allow for the equivocal
condition of the mitochondrial derivatives, character 9, in Pogona) an identical number of trees
and topology were obtained but the tree was three steps shorter, with consistency index = 0.750;
homoplasy index = 0.250; retention index = 0.839; rescaled consistency index = 0.629.
It must be stressed that the strict consensus tree (Fig. 1 1), agreed closely with the majority
rule tree, differing only in placing Cnemidophorus on the same level as the remainder of its clade
and Lialis on the same level as the Carlia and snake clades. These two polytomies correspond
with the only percentages below 100% on the majority rule tree.
376
B. G. M. JAMIESON : SQUAMATA (REPTILIA)
Table 2. — Comparative ultrastructure of spermatozoa of Squamata, Sphenodon punctatus and Chelonia. (Input data
matrix).
Taxon Characters Reference
11111111
12345678901234567
Chelonia
Sphenodon punctatus
Ctenotus robust us
Chalcides ocellatus
Lacertidae
Cnemidophorus sexlineatus
Tiliqua scincoides
Carlia pectoralis
Lampropholis delicata
Heteronotia binoei
Lygodactylus picturatus
Lialis burtonis
Pogona barbata
Varanus gouldii
Colubridae
Elapidae (Oxyuranus)
Boidae (Aspidites)
Iguanidae
Bradypodion karroicum
00000000000000000
00000000000000000
10011001111101001
100 1700113 1101001
10011001501101001
10012001111101001
10010001111101001
01112112201011101
01112112201011101
02010101401101001
02012101401101001
07112112301211011
11012023111101001
3
11012001121101001
00011212301101011
00017212301101011
00017212301101011
00012002111001001
2 1
10012102201101001
[26, 27]
[26, 27, 32]
[37]
[21]
[7, 11, 21]
[41, 42]
[35]
[35]
[37]
[37]
[21]
[25, 37]
[45]
[45]
Serpentes:
[3, 20, 21, 24. 33, 45, 46]
[22, 48]
This study
Discussion of the phylo grams
The majority rule phylogram (Fig. 10) indicates those similarities of spermatozoal
ultrastructure within the Squamata, and relative to Sphenodon and the Chelonia, which are
synapomorphic for the respective nodes. These similarities, as in all spermiocladistic studies,
must reflect similar fertilization biology but also phylogenetic, i.e. genetic, constraints.
Spermatozoal morphology is here considered sufficiently conservative to reflect at least close
phylogenetic relationship as it can be expected to be subject to stabilizing selection [22], The
evidence of many other studies (see, for instance, [26, 27, 31, 32] and references in [34]) shows
that, although caution is required in their interpretation, such spermiocladistic phylograms hold
significant phylogenetic information. Bearing in mind the necessity for caution and also the
pressing need for inclusion of reliable data from a much larger sample of taxa, and confirmation
of some data from the present sample, many of the following conclusions drawn from the
phylogram can be regarded only as heuristic. Findings which both intuitively and by computation
are unequivocal are stressed. Because of limitations of space, references to the literature will
chiefly be confined to the excellent review of squamate classification by RlEPPEL [47].
Squamata. The Squamata, which are monophyletic in all most parsimonious trees in the
analysis, are apomorphic relative to the Sphenodontida and Chelonia in the following features
which are synapomorphies defining the Squamata s. strict. Sphenodon and the Chelonia are
equally plesiomorphic and indistinguishable on the characters employed and therefore reference
will chiefly be made to Sphenodon. It should be stressed, however, that Sphenodon is not here
Source :
ADVANCES IN SPERMATOZOAL PH YLOGENY AND TAXONOMY
377
CHELONIA
Sphenodon punctatus
Ctenotus robustus SCINCIDAE
Chalcides ocellatus SC1NCIDAE
LACERTIDAE
•10. 1==>3
■ 9. 1 ==>5 10. 1==>0
- 5. 1==>0
■7. 0 ==>2 8. 1 ==> 3
4.
5.
8.
9.
■ll. 0==>1
12. 0==>1
14. 0==>1
17. 0==>1
6. 0==>1
9. 1 — >2
Tiliqua scincoides SCINCIDAE
Cnemidophorus sexlineatus TEIIDAE
Pogona barbata AGAMIDAE
Varanus gouldii VARANIDAE
10. 1==>2
9. 3-->2 15. 0==>1 16. 1~>0
| Carlia pectoralis SCINCIDAE
I Lampropholis delicata SCINCIDAE
i Lialis burtonis PYGOPODIDAE
COLUBRIDAE 12' °"">2
2 - Oxyuranus microlepidotus ELAPIDAE
6. i==>2 1 Aspidites melanocephalus BOIDAE
Heteronotia binoei GEKKONIDAE
Lygodactylus picturatus GEKKONIDAE
i Bradypodion karroicum CHAMAELEONIDAE
1. 0==>1
' 5. 2==>0
■2. 0==>2
8. 2 ==>1
9. 2 — >4
Fig. 10. — Branch and Bound 50 % majority rule consensus phylogram for 240 equally most parsimonious trees, for
character states of chelonian, sphenodontid and squamate sperm ultrastructure listed in Tables 1 and 2. Settings as
in text. Tree length = 43; consistency index = 0.767; homoplasy index = 0.302; retention index = 0.839; rescaled
consistency index = 0.644. Numbers in circles are the percentage of trees supporting the node to which they
attach. All other nodes were supported in 100 % of the trees.
regarded as the immediate sister-group of the Squamata sensu strictu but as a very basal amniote
in a sphenodontidan lineage which possible predates emergence of the Crocodilia, Aves, and, it
appears, the Mammalia [26, 32],
The squamate synapomorphies follow: 1. Possession of a single perforatorium in place of
the two or three of Sphenodontida; 2. Loss of the endonuclear canal. The wholly epinuclear rather
than partly endonuclear perforatorium is a notable difference of the Squamata from Chelonia,
Sphenodontida, Crocodilia, non-passerine birds, and also basal lissamphibians (urodeles and
Ascaphus). 3. Presence of the (well developed) epinuclear electron-lucent region. 4. Presence of
sinuous mitochondria (possibly an artefactual parsimony resolution as a columnar form is
intuitively preferred). 5. linear cristae, not the subspherical mitochondria, with concentric cristae
of Sphenodon. 6. Dense bodies (mitochondrial transformations) intermitochondrial, in contrast
with the intramitochondrial dense body in Sphenodon, and (equivocally) forming regular rings.
Although the intermitochondrial position may well be basic to all squamates, one might query that
the arrangement in regular rings is in their ground plan. 7. The fibrous sheath extends into the
midpiece. This is a most significant and convincing synapomorphy and autapomorphy of the
378
B. G. M. JAMIESON : SQUAMATA ( REPTILIA )
CHELONIA
Sphenodon punctatus
Ctenotus robustus
Chalcides ocellatus
LACERTIDAE
Cnemidcphorus sexlineatus
™ Tiliqua scincoides
Pogona barbata
Varanus gouldi
IGUANIDAE
_ | Carlia pectoralis
■ Lampropholis delicata
Lialis burtoni
ICOLUBRIDAE
Oxyuranus microlepidotus
Aspidites melanocephalus
Heteronotia bionei
Lygodactylus picturatus
Bradypodion karroicum
Fig. 11. — Branch and Bound 50 % strict consensus phylogram for 240 equally most parsimonious trees, for character
states of chelonian, sphenodontid and squamate sperm ultrastructure listed in Tables 1 and 2. Settings, length and
indices as in Fig. 10.
Squamata and is not seen in the Sphenodontida [26, 32]. 8. Development of rounded nuclear
shoulders is basic to squamates. In addition, though not included in the parsimony analysis,
squamates are diagnosed by the paracrystalline substructure of the subacrosomal cone.
These squamate spermatozoal autapomorphies constitute a striking endorsement of the
“highly corroborated'’ monophyly of the Squamata for which evidence is reviewed by RlEPPEL
[47].
‘Sauria’. The Sauria is the third group of the Squamata, the other two being the
Amphisbaenia and the Serpentes. Whereas the latter two groups appear to be monophyletic,
RlEPPEL [47] stated that monophyly of the Sauria could not be established and that the name
should be dropped or, in effect, broadened to equate with Squamata. If the evidence of the
spermatozoal phylograms (Figs 10, 1 1) be accepted, the view that Sauria (in the strict sense of
‘lizards’ rather than squamates in general) are not monophyletic is emphatically endorsed as some
saunans (invariably Eugongylus-group skinks and pygopodids, and, less closely, gekkonids and
the chamaeleonid) appear more closely related to snakes than they are to other ‘lizards’. Thus,
Sauria is only monophyletic when it is, indeed, equated with Squamata and it includes lizards and
snakes. The position of the amphisbaenians remains to be investigated when spermatozoal
samples become available.
Scincomorpha. Monophyly of the Scincomorpha, though well supported by somatic
morphology, has been considered to deserve further critical study [47]. The group is generally
held to include the Xantusioidea, Lacertoidea (Lacertidae, Teiidae and Gymnophthalmidae) and
the Scincoidea (Scincidae and Cordylidae). Spermatologically (Figs 10, 1 1) the Scincomorpha is
diphyletic or possibly polyphyletic. This is due to the fact that the Scincoidea, represented in the
parsimony analysis by the Scincidae only, are themselves at least diphyletic. notably because the
Eugongylus-group skinks associate with the Serpentes, Pygopodidae, Chamaeleonidae and
uekkomdae
Source :
ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY
379
RlEPPEL (e.g. [47]) has repeatedly questioned the monophyly of the Scincidae and his
doubts are underlined spermatologically. Thus the Eugongylus-group skinks ( Carlia and
Lampropholis) constitute the sister-group of the Pygopodidae; and the pygopodid-Eugongylus-
clade is in turn the sister-group of the Serpentes in the majority rule tree (Fig. 10). However, the
pygopodid forms a trichotomy with the Eugongylus-group and Serpentes clades in the strict
consensus tree (Fig. 1 1).
The apparent synapomorphies joining the Carlia through Serpentes clades are weak. They
are loss of the regular mitochondrial circlet, and, equivocally, development of linear dense bodies
and of multilaminar membranes. Of these, the last two conditions are not seen in Carlia and
Lampropholis. These two genera are linked to the pygopodid by four apomorphies. Two of these,
the square-ended perforatorium and alteration of the nucleus to a stout form appear strong
synapomorphies, while two are equivocal, acquisition of a knob-like basal plate (homoplasic with
Pogona and Varanus) and development of sharp nuclear shoulders.
The severance of the Eugongylus-group from other skinks merits further investigation using
non-spermatozoal characters. The spermatozoal synapomorphies defining the Eugongylus-group
are striking. They are the characteristic development of scattered dense bodies, forming more than
one layer around the axoneme; the fact that coarse fibres 3 and 8 are grossly enlarged anteriorly in
addition to the usual enlargement in squamates; and, equivocally, reversion to no multilaminar cell
membranes.
The position of the Lacertidae and Teiidae is poorly resolved (Fig. 10), and further data on
these families are required. Linkage of the Lacertidae and Teiidae in the Lacertoidea [47] is not
upheld. A teiid relationship to the Iguanidae (references in [47]) is also not supported. In the strict
consensus tree (Fig. 1 1), lacertids, the teiid Cnemidophorus, and the skinks Ctenotus, Chalcides,
and Tiliqua form an unresolved polytomy with the Pogona-Varanus clade. However, in the 50%
majority rule tree (Fig. 10), Cnemidophorus is less close to the other scincomorphs than they are
to each other. Intuitive considerations had placed teiids near sphenomorph ( Ctenotus ) and egemid
(Tiliqua) skinks [37] but an especially close relationship is not upheld in this analysis.
Lacertid sperm have not been examined by the author but the composite data for the family,
while placing them with the other, non-Eugongylus-scincomorphs, do not at present ally them
more closely to any scincid or with the teiid. Their synapomorphies are arrangement of dense
bodies into two groups and, of uncertain validity, reversal to a poorly developed epinuclear
electron-lucent region. As recorded in the comparative section above, the arrangement of dense
bodies in lacertids appears to be unusually diverse but is never strongly similar to the Ctenotus
condition.
Iguania. Traditionally these include the Iguanidae, Anolidae, Tropiduridae, Agamidae and
Chamaeleonidae. The Iguania does not emerge as a monophyletic group in the present analysis
(Figs 10, 11). Pogona groups not with an iguanian but with Varanus in a clade which otherwise
consists of non-Eugongylus scincomorphs while iguanids form the plesiomorph sister-group of
this clade. The plesiomorphic status of the Iguanidae relative to agamids (references in RlEPPEL
[47]) is endorsed but here in a paraphyletic relationship.
The chamaeleon Bradypodion is severed from the Iguanidae and is basal in the gekkonid-
snake-pygopodid-Eugongylus-group clade in the majority rule (Fig. 10) and strict (Fig. 11)
consensus trees in the present analysis. It may be noted, however, that in terms of patristic
distance, the nearest taxon to the chamaeleon is, nevertheless, the Iguanidae. On intuitive
consideration (see comparative account above) the sperm of the chamaeleon appear closely similar
to that of the agamid in many respects but the sinuous tubular mitochondria are an important
difference from agamids and, although a resemblance to iguanids, are also seen in the Cariia-
pygopodid-snake clade. Much further investigation of spermatozoa, and comparison with
morphological characters, is necessary if iguanian relationships are to be elucidated.
The knob-like basal plate appears to be the sole synapomorphy of Varanus and Pogona but
is homoplasic with that in Carlia and Lampropholis.
380
B. G. M. JAMIESON : SQUAMATA ( REPTILIA )
Anguimorpha. Anguimorphs are conventionally divided into the Anguioidea and the
Varanoidea [47], Anguioidea have yet to be examined for sperm ultrastructure. In the comparative
study above, the similarity of the Varanus sperm to that of Pogona has been described. From the
comparative study, varanid sperm resemble those of agamids, sphenomorph and egernid skinks,
and teiids in the depressed acrosome and intermitochondrial rings; they further resemble agamids
and also (homoplasically) Eugongylus-group skinks in possessing a knob-like perforatorial base
plate. Intuitively, as in the analysis (Fig. 10), the varanid is part of a sphenomorph-egernid-
agamid assemblage. It is noteworthy that although a close relationships of varanids with scincids
does not appear to have been previously suggested, brain data have been considered to indicate
that the Teiidae are most closely related to the Varanidae [43],
A very close relationship between varanids and snakes which has formerly been postulated
(see references in [45, 47]) is not supported by spermatozoal ultrastructure. Varanus sperm differ
from those of snakes in the short midpiece, columnar mitochondria in a regular circlet, dense
intermitochondrial bodies forming regular rings and absence of multilaminar membranes. It is
expected that varanid relationships will come nearer to resolution when more than one species of
each family is represented. A unique varanid apomorphy is replacement of the solid condition of
the intermitochondrial rings, also seen in Ctenotus, Cnemidophorus, Tiliqua, Pogona and
iguanids, with a granular condition.
Thus, the relationships of varanids remain enigmatic despite the parsimony analyses but a
close relationship to snakes is not upheld.
Gekkota. Monophyly of the Gekkota, comprising the two extant families Gekkonidae and
Pygopodidae has been considered well corroborated (references in RlEPPEL [47]). However, the
majority rule consensus tree (Fig. 10) represents the Pygopodidae, exemplified by Lialis burtonis,
as the sister-taxon of the Eugongylus-group skinks (for synapomorphies see Scincomorpha
above). In the strict consensus tree (Fig. 1 1), Lialis forms a trichotomy with these skinks and the
snakes. In the parsimony analysis (Figs 10, 1 1) the gekkonids form a monophyletic clade which,
however, has an unresolved relationship with the chamaeleon, at the base of the
snake+pygopodid+Eugongylus-group clade. The only unequivocal synapomorphy for the
composite clade, is development of a moderately long midpiece which becomes very long in
snakes. No unique synapomorphies are apparent between the sperm of gekkonids and pygopods
which would support the special relationship between these two families suggested by KLUGE
[38, 39, 40] who from a cogent cladistic study of general morphology has concluded [39] that the
Pygopodidae should be placed within the Gekkonidae. This departure from somatic
morphological evidence requires further investigation from sperm of larger numbers of taxa.
A relatively primitive position of the Gekkonidae in the ‘Sauna’ proposed by some workers
[47], as opposed to the Pygopodidae, is supported by both consensus phylograms. Gekkos have
somewhat generalized scincid sperm but a stellate arrangement of dense bodies in the midpiece
distinguishes at least some of them. Heteronotia and Lygodactylus are sister-taxa in 1 00% of the
trees. Computed synapomorphies of gekkonids ( Heteronotia and Lygodactylus) are the stopper¬
like perforatorial base, the columnar condition of the mitochondria, supposedly derived
secondarily from a sinuous condition; and, ambiguously, the stellate mitochondrial derivatives
which compute as arising from a scattered condition such as is seen in Eugongylus-group skinks.
Serpentes. The origin of snakes, on the grounds of somatic morphology, continues to be an
enigma [47], However, the spermiocladistic analysis indicates origin of snakes from an ancestor,
which must have been skink-like, shared with Eugongylus-group skinks (Carlia and
Lampropholis) and with pygopodids. The pygopodid has a sister-group relationship with the
Eugongylus-group skinks in the majority rule tree (Fig. 10) but forms an unresolved trichotomy
with these and snakes in the strict consensus tree (Fig. 1 1). At present this origin of snakes must
remain merely an heuristic finding but spermatozoa do provide a serpent apomorphy (see also
[33, 45]) shared only with the pygopodid, presence of multilaminar sperm cell-membranes. These
Source :
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
381
membranes appear much better developed in snake sperm which differ further, and uniquely, in
the immense elongation of the midpiece as a mitochondrial sheath around most of the length of the
axoneme. That the multilaminar membranes have been lost in Eugongylus-group skinks, as
computed, may be questioned. If not, presence would be a snake-pygopod synapomorphy.
Further snake apomorphies appear to be reduction of the epinuclear electron-lucent region and
loss of a perforatorial base plate. Snake sperm also have a greater development of extracellular
tubules (not coded) than is known in any other squamate.
With regard to groups not included in the analyses, tropidurid sperm are insufficiently
known for determination of phylogenetic affinities but the three gyres of mitochondria are also a
lacertid characteristic; supposed failure of the fibrous sheath to penetrate the midpiece (the
squamate autapomorphy) requires confirmation.
Investigations of sperm of additional taxa are required if suggested relationships throughout
the Squamata are to be adequately tested.
ACKNOWLEDGEMENTS
I am grateful to Mrs Lina Daddow, David Scheltinga, Chris Tudge, and Simon Oliver for excellent assistance and
to Chris Tudge and Tom Gorringe for printing the micrographs. Professor Alan Hodgson and Dr. Rick Bernard kindly
supplied a resin block of Bradypodion sperm. This work w'as partly supported by an Australian Research Council grant.
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Ultrastructure of Spermatozoa of Australian Blindsnakes,
Ramphotyphlops spp. (Typhlopidae, Squamata):
First Observations on the Mature Spermatozoon
of Scolecophidian Snakes
H. Ronnie HARDING * ***, Ken P. APLIN ** & Maria MAZUR *
* Institute of Environmental Studies,
University of New South Wales, Sydney, 2052, NSW, Australia
** Western Australian Museum, Francis Street, Perth, 6000. Western Australia, Australia
*** Honorary Research Associate at the Department of Anatomy affd Histology, University of Sydney
ABSTRACT
The structure of mature, epididymidal spermatozoa of three species of Australian blindsnakes. Ramphotyphlops spp.
(Typhlopidae, Squamata) was investigated by a combination of scanning and transmission electron microscopy. Special
attention was paid to features of potential phylogenetic significance, in the hope that aspects of sperm morphology
might shed light on the evolutionary affinities of this highly specialized and enigmatic group of reptiles. Spermatozoa of
Ramphotyphlops spp. share many features in common with other squamates (lizards and snakes), but display some
striking features not known in any other taxa. Special derived features shared by Ramphotyphlops spp. and other
Squamata include: a paracrystalline subacrosomal cone; dense intermitochondrial “rings" or “plaques"; a relatively
inconspicuous annulus; an anteriorly extensive fibrous sheath which penetrates the midpiece; and the absence of an
endonuclear canal. Additional derived features which may be shared exclusively by Ramphotyphlops spp. and other snakes
include: the great elongation of the midpiece; the forward prolongation of the fibrous sheath to the level of the neck; the
presence of a distinct “neck cylinder"; the extremely elongate form of the mitochondria; and the presence of a multi¬
layered midpiece plasma membrane. These cladistic interpretations lend support to the current taxonomic placement of
Typhlopidae within Squamata, and specifically within Serpentes. Several highly distinctive features of Ramphotyphlops
spermatozoa are identified, the most striking being the extreme length and regular zig-zagging arrangement of the
mitochondria.
RESUME
Ultrastructure des spermatozoides des serpents aveugles australiens, Ramphotyphlops spp.
(Typhlopidae, Squamata): premieres observations sur le spermatozoide mur des Serpents
Scolecophidiens.
La structure des spermatozoides murs epididymaires de trois especes de serpents aveugles australiens, Ramphotyphlops
spp. (Typhlopidae; Squamata) a ete etudiee en microscopic electronique h balayage et & transmission. Une attention
particuliere a ete portee aux details d’ importance phylogenique potentielle, dans l’espoir que des caracteristiques de la
morphologie du spermatozoide puissent eclairer les affinites evolutives de ce groupe de Reptiles, tres evolues et
Harding, H. R., Aplin, K. P., & Mazur, M.. 1995. — Ultrastructure of spermatozoa of australian blindsnakes,
Ramphotyphlops spp. (Typhlopidae, Squamata): first observations on the mature spermatozoon of scolecophidian
snakes. In: Jamieson, B. G. M., AusiO, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy.
Mem. Mus. natn. Hist, nat., 166 : 385-396. Paris ISBN : 2-85653-225-X.
386 H. R. HARDING, K. P. APLIN & M. MAZUR : RAMPHOTYPHLOPS ( TYPHLOPIDAE . SQUAMATA)
6nigmatiques. Les spermatozoides de Ramphotyphlops spp. poss£dent de nombreux caracteres en commun avec les autres
Squamata (lizards et serpenis). mais montrent quelques aspects originaux inconnus dans les autres taxons. Les caracteres
deriv6s sp6ciaux partag^s par Ramphotyphlops spp. et les autres Squamata comprennent: un cone subacrosomien
paracristallin, des "anneaux" ou “plaques*’ denscs intermitochondriaux, un annulus relativemcnt peu apparent, une gaine
fibreuse s’etendant vers l’avant et qui pendtre la piece intermediaire et 1’absence de canal endonuclSaire. Les caracteres
derives supplemental qui pourraient etre communs seulement a Ramphotyphlops spp. et aux autres Serpents
comprennent: le grand allongement de la piece intermediaire, le prolongement vers l’avant de la gaine fibreuse jusqu'au
niveau du cou, la presence d’un “cylindre du cou" distinct, la forme extremement allong6e de la mitochondrie et la presence
d’une membrane plasmique & plusieurs couches dans la piece intermediaire. Ces interpretations cladistiques soutiennent le
placement actuellement admis des Typhlopidae dans les Squamata, et specifiquement dans les Serpentes. Quelques
caracteres tres originaux du spermatozoide de Ramphotyphlops sont identifies, dont le plus frappant est la longueur
extreme et la disposition regulierement zigzaguante des mitochondries.
Despite the pioneering efforts of FURIERI [6] to promote interest in the ultrastructure of
reptilian spermatozoa, many significant gaps in taxonomic coverage still mar our understanding of
spermatozoal structure and evolution in this large and important group of vertebrates. This is
particularly marked within the taxonomically diverse Order Squamata (collectively, the lizards and
snakes), for which information is available for less than one third of the extant families.
In this paper we report the first observations on spermatozoa from members of the Family
Typhlopidae, a group commonly known as blindsnakes. These highly specialized, fossorial
snakes are represented on all continents, with major radiations in Australia, Africa and Central
America. Together with members of at least two other families, Leptotyphlopidae and
Anomelepidae, blindsnakes are generally included in the higher taxon Scolecophidia, as one of the
two major groups of living snakes [4, 13]. However, some authors have suggested a possible
closer relationship with certain groups of lizards, thereby challenging the monophyly of both
Scolecophidia and Serpentes [9, 14]. Even more remarkably, one worker has questioned the
placement of Typhlopidae within Squamata, based on peculiar features of the male reproductive
tract [17].
Material examined in this study comes from three species of the Australasian blindsnake
genus Ramphotyphlops, which includes approximately 50 species spread between Malaysia and
southeastern Australia. This study forms one part of a wider comparative survey of spermatozoal
morphology among Australian and Indonesian squamates, from which we hope not only to fill
many of the major gaps in taxonomic coverage, but also to further knowledge of squamate
phylogeny and classification.
MATERIAL AND METHODS
Spermatozoal samples were collected from three Ramphotyphlops species. The associated voucher specimens are
lodged in the collection of the Western Australian Museum as follows: Ramphotyphlops waitii 114891 Waggrakine Pass,
W. A. and 119239 Walyunga National Park, W. A.; R. endoterus 115000 38 km ENE Laverton; R. australis 115125 11
km ESE North Dandalup. Samples from all three species were processed for TEM and for R. waitii also for SEM.
Animals were euthanased with sodium pentabarbitol (Nembutal). Immediately following death, samples of testis
and epididymides were removed, cut into small pieces and fixed for 30-60 minutes in Kamovsky's fixative with picric acid
added. They were washed and stored in 0.1 M cacodylate buffer pending further treatment.
Samples for TEM were postfixed in 1% osmium tetroxide in cacodylate buffer, stained en bloc in 4% alcoholic
uranium acetate, dehydrated in alcohol and acetone, and embedded in Spurr resin. Sections were stained in 4% alcoholic
uranium acetate and lead citrate, and examined using a JEOL 100 TEM.
For SEM a drop of sperm released from the tissue was placed on a cover slip coated with platinum and then with
poly-L-lysine. Samples were postfixed in osmium tetroxide in cacodylate buffer, dehydrated, critical point dried in CC>2,
and examined using field emission, high resolution JEOL JSM-6000, at the accelerating voltage of 3 kV.
Measurements, which are of R. waitii , were taken from micrographs and using the 40/0.7 and 100/1.3 objective
lenses of a Leitz Diaplan microscope with a video camera attached and connected to a MD30 Automated Image Analysis
System (Leading Edge, Australia). However, owing to the extreme length of the spermatozoon and the difficulty of
distinguishing the midpiece and principal piece junction in whole-sperm light microscopy, the measurements given in
this paper should be regarded as approximate only.
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387
OBSERVATIONS
The mature spermatozoon in all three species is clearly very elongate, with lengths up to
179 pm recorded for R. waitii under the image analyser.
Head
The sperm head is elongate and essentially cylindrical in form, but with a slight flexion just
behind the base of the acrosomal cap. Measurements taken from micrographs indicate a total head
length of 15 pm and a basal diameter of 0.6 pm. The nucleus consists of a basal cylindrical
portion measuring 10 pm in length, and a tapered apical portion which projects 2.1 pm into the
acrosomal cap (Fig. la). A distinct “nuclear shoulder” marks the transition from basal to apical
regions (Fig. la, h). The nuclear fossa is broad and relatively shallow except in the very centre
above the basal plate where it is more deeply excavated (Fig. 2c). The nucleus is evenly electron-
dense and is not penetrated by an endonuclear canal.
The acrosomal complex forms a complete cap which sits atop the tapered portion of the
nucleus (Fig. la); it measures 3. 8-4.0 pm from nuclear shoulder to acrosomal tip. In common
with other reptiles, the acrosome consists of three main components: an inner subacrosomal cone;
an outer acrosomal sleeve; and a central, rod-like perforatorium (Fig. la). The perforatorium
projects from the tip of the subacrosomal cone as a slender transversely striated rod (Fig. la, f-g).
In R. waitii it measures 1 pm. As shown by comparison of Figs If and lg the perforatorium
varies in length between the species, being a much longer structure in R. waitii than in R.
endoterus. Basally it is embedded within the subacrosomal cone (Fig. lc, f-g). A narrow
electron-lucent zone surrounds much of the striated perforatorium. In R. waitii and R. australis
this vacuity is irregularly subdivided by a series of vertical folds which arise from the inner
surface of the acrosomal sleeve (Fig. la, f). In R. endoterus the acrosomal vacuity is more
prominent and is regularly partitioned by four vertical “vanes” of the acrosomal sleeve (Fig. lb,
g). The subacrosomal cone has a paracrystalline structure (Fig. la, e, g). It is surrounded by the
acrosomal sleeve which is complex in form. Basally, where the sleeve overlies the subacrosomal
cone, it consists of a single amorphous layer (Fig. la, d-e). Proximally, it is composed of two
distinct units separated by a narrow, electron-dense band (Fig. la-b. f-g). A well-defined “trigger
area” [5] covers the proximal tip of the sperm head (Fig. la, f-g).
The plasma membrane covering the acrosomal portion of the sperm head appears as a rather
heavy and relatively “loose” membrane over most of the acrosome but it is attached to the nuclear
membrane proximally around the margin of the “trigger area” and basally at the “post-acrosomal
ring”, an annulus-like structure which rests upon the nuclear shoulder (Fig. la, h). In common
with various other groups of snakes and lizards [1, 7, 15], the plasma membrane of the sperm
head is surrounded by a pallisade of closely-applied, longitudinally aligned microtubules (Fig. la,
d-e, g-h).
Neck region
The neck region in Ramphotyphlops is exceptionally complex, comprising both proximal
and distal centrioles; a basal plate which lines the nuclear fossa; a series of longitudinal columns
which extend anteriorly from the dense outer fibres (peripheral fibres) of the midpiece; an electron
dense body (referred to here as the “capitulum” in view of its similarity to the structure of that
name in mammalian spermatozoa) which conforms to the shape of the basal plate anteriorly,
surrounds the proximal centriole and joins to the longitudinal columns; and a “neck cylinder”
which encloses the remaining neck elements and extends distally to surround the anterior
extremity of the fibrous sheath.
The short proximal centriole (Fig. 2b, j) is set at an angle of approximately 80 degrees to the
distal centriole and axoneme. It conforms to the usual pattern of nine sets of peripheral triplets and
no central tubules. The central space is penetrated by a body of diffuse, electron dense material
388 H. R. HARDING, K. P, APLIN & M. MAZUR : RAMPHOTYPHLOPS (TYPHLOPIDAE, SQUAMATA)
representing a “centriolar pedicle” of the neck cylinder (Fig. 2b). This unusual structure is
prominent in R. endoterus, in which the neck cylinder and its various processes are lelatively
electron dense (Fig. 2c, f-g). In contrast the neck cylinder in R. waitii and R. australis appears
relatively electron lucent and granular (Fig. 2a-b, d-e). The proximal centriole as a whole is
embedded within an irregular-shaped, electron dense body (the “capitulum ’) which closely
parallels the form of the nuclear fossa (Fig. 2a-c, i). This structure forms the “ball' of a mortise
and tenon joint between the sperm head and flagellum, and is therefore at least analogous with the
capitulum that forms the anterior region of the “connecting piece” of mammalian sperm.
The basal plate forms the functional interface between the nuclear and flagellar portions of
the spermatozoon. It appears to be of composite origin, including both nuclear membranes and
membranes which envelope the midpiece and neck structures (Fig. 2 a-c). The space between the
basal plate and the capitulum is occupied by an unbounded body of granular material, with a
suggestion of radial structure in some micrographs (Fig. 2 a-b).
The distal centriole extends posteriorly from the proximal centriole (but parallel to the long
axis of the flagellum) to approximately the region of the flagellum marked by the anterior
extremity of the fibrous sheath (Fig. 2a, c, d-f), where it meets the axoneme. It possesses the
usual peripheral triplets as well as a pair of central singlets (Fig. 2d). The dense outer fibres
associated with each of the nine axonemal doublets continue forward into the neck where they
meet the longitudinal columns. These nine columns diverge progressively from the centriolar
triplets as they extend in the direction of the sperm head and at their anterior extremity they join
the capitulum, forming a support for the proximal centriole and capitulum (Fig. 2a, c, h-j).
Transverse sections through the distal centriole show a supernumerary dense column lying
between one of the pair of central singlets and one of the centriolar triplets which, on the basis of
presently incomplete information, is judged to be triplet 3 (Fig. 2 d-f). One of the longitudinal
columns is very poorly developed relative to the others and generally is found close to its adjacent
Fig. 1. — a: Longitudinal section through the acrosomal region of the nucleus in a Ramphotyphlops waitii
spermatozoon. The arrow head marks the attachment of the plasma membrane and nuclear membranes at the trigger
area and the post-acrosomal ring marks the basal attachment. The numbered lines indicate the approximate
position of the transverse sections in Fig. lb-e. b: Transverse section through the head of a R. endoterus
spermatozoon in the position marked 1-1 in Fig. la. Note the regular arrangement of the vertical folds in the
acrosomal vacuity, c: Transverse section through the head of a R. endoterus spermatozoon in the position marked
2-2 in Fig. la. A very slight variation in the appearance of the centre of the subacrosomal cone marks the basal
portion of the perforatorium, d: Transverse section through the head of a R. waitii spermatozoon in the position
marked 3-3 in Fig. la. Note the thin layer of subacrosomal material surrounding the nucleus, e: Transverse section
through the head of a R. waitii spermatozoon in the position marked 4-4 in Fig. la. The paracrystalline structure
of the subacrosomal cone is very evident, f: Longitudinal section through the anterior region of the acrosome in a
R. waitii spermatozoon. Compare the length of the perforatorium with that of R. endoterus in Fig. lg printed at
approximately the same magnification, g: Transverse section through the anterior region of the acrosome in a R.
endoterus spermatozoon. Note that the vertical folds enclosed within the acrosomal vacuity are more prominent
and regular than in R. waitii. h: Longitudinal section through the head of a R. waitii spermatozoon in the region of
the post-acrosomal ring, a-h, scale bar = 0.1 pm.
Abbreviations for all figures: a. acrosomal sleeve; an, annulus; b, basal plate; be, basal portion of perforatorium embedded
in the subacrosomal cone; c, subacrosomal cone; ca, capitulum; cy, neck cylinder; d, distal centriole; df, dense
outer fibres; e, electron dense band separating inner and outer layers of acrosomal sleeve; f, fibrous sheath; g,
granular material surrounding fibrous sheath; i, intermitochondrial plaque; lc, longitudinal columns of the neck; m,
mitochondria; n, nucleus; ns, nuclear shoulder; p, striated rod of the perforatorium; pd, centriolar pedicle; pm,
plasma membrane; px, proximal centriole; r, post-acrosomal ring; s, supernumerary longitudinal column; t,
trigger area; tr, centriolar triplets; tu, palisade of longitudinally orientated microtubules surrounding the
spermatozoon; v, vertical folds within acrosomal vacuity; x, axoneme.
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390 H. R. HARDING, K. P. APLIN & M. MAZUR : RAMPHOTYPHLOPS (TYPHLOPIDAE, SQUAMATA)
triplet (Fig. 2 d-e). The spatial relationship between this column and the supernumerary column is
constant in the various transverse sections (Fig. 2 d-e) and, again on the basis of as yet
incomplete information, this poorly developed column appears to be adjacent to triplet number 9.
The fibrous sheath, normally confined to the principal piece in tetrapod sperm, extends in
Ramphotyphlops all the way forward to the level of the axoneme-distal centriole junction. In
longitudinal sections the columns are seen to diverge radially from this point moving anteriorly
(Fig. 2a, c, h-i).
It is not clear precisely where the columns join the dense outer fibres. In a region which
must be just anterior to the commencement of the fibrous sheath (since the sheath is absent but the
columns/fibres are not radially displaced from the axoneme doublets) the supernumerary fibre is
still seen between the central pair and outer doublet 3 (?). Dense outer fibres 3 and 8 are
particularly prominent and the fibres associated with all the doublets appear not only adjacent to
the outer periphery of the circle of doublets, but also extend inwards, between and partially
enclosing each doublet (Fig. 2f).
The neck cylinder in R. waitii and R. australis consists of a diffuse and somewhat uneven
aggregation of granular material, bounded by a narrow membrane. Anteriorly, it surrounds the
capitulum and the centriolar complex, contacting the nucleus around the periphery of the nuclear
fossa, where it appears to be continuous with the basal plate (Fig. 2 a-b). Posteriorly, it extends
well beyond the point of the distal centriole-axoneme union to enclose the anterior portion of the
fibrous sheath (Fig. 2 a). In R. endoterus the neck cylinder is a more solid structure, being
equivalent in electron density to the “capitulum” and the dense outer fibres (Fig. 2 c). However,
its basic form and relations are identical to those described for R. waitii.
Midpiece
The midpiece is extremely elongate, with one complete section measured from a micrograph
reaching 108 (im. Typical cross-sections are circular in shape, have a diameter of 0.5 pm and
show nine mitochondrial sections; eight of which are round and one very elongate (Fig. 3 c).
Fig, 2. — a: Longitudinal section through the neck and anterior midpiece of a Ramphotyphlops waitii spermatozoon.
The longitudinal columns diverge as they extend from the anteriormost portion of the fibrous sheath to the
capitulum, which surrounds the proximal centriole. The numbered lines indicate the approximate position of the
transverse sections in Fig. 2 d-e. b: Oblique longitudinal section through the nuclear fossa of a R. waitii
spermatozoon. Note the radial striations in the granular material (arrowed) separating the basal plate and the
capitulum and the centriolar pedicle of the neck cylinder, c: Longitudinal section through the neck and anterior
midpiece of a R. endoterus spermatozoon. The numbered lines indicate the approximate positions of the transverse
sections in Fig. 2 f-g. d: Transverse section through the distal centriole of a R. waitii spermatozoon in the
approximate region marked 1-1 in Fig. 2a. Note the multiple plasma membranes, the poorly developed
longitudinal column (arrowed), the supernumerary column, and the central pair surrounded by the centriolar triplets,
e: Transverse section through the distal centriole of a R. waitii spermatozoon in the approximate region marked
2-2 in Fig. 2a. Note that the longitudinal columns are not as radially divergent as in Fig. 2d. The poorly developed
column is arrowed, f: Transverse section through the neck region of a R. endoterus spermatozoon in the
approximate region marked 1-1 in Fig. 2c. Note the difference in electron density of the neck cylinder compared
with that in R. waitii (Fig. 2e). Note that the fibres extend between the doublets. Fibres 3 (labelled) and 8 arc
considerably larger than the rest but all 9 fibres are well developed, g: Transverse section through the neck region
of a R. endoterus spermatozoon in the approximate region marked 2-2 in Fig. 2c. Note the supernumerary column
is no longer visible and dense outer fibre (3?) (marked) is particulalry large, h: SEM view of the neck and anterior
midpiece of a R. waitii spermatozoon. The plasma membrane and neck cylinder have been lost to reveal the
underlying structures, i: SEM view of the neck of a R. waitii spermatozoon. The head and flagellum are separated
to reveal the capitulum supported by the longitudinal columns. There appears to be a junction (arrowed) just
anterior to the start of the fibrous sheath, which may be the junction between the longitudinal columns and the
dense outer fibres, j: SEM view of the nucleus-neck junction in a R. waitii spermatozoon. The plasma membrane
and neck cylinder have been lost to reveal the proximal centriole. a-j, scale bar = 0.1 pm.
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392 H. R. HARDING, K. P. APLIN & M. MAZUR : RAM PHOT YPHLOPS (TYPHLOPIDAE, SQUAMATA)
Fig. 3. — a: Grazing longitudinal section of the midpiece of a Ramphotyphlops waitii spermatozoon to show the ordered
and unusual arrangement of the mitochondria. Note the multiple plasma membranes, b: Grazing longitudinal
section of the fibrous sheath in the principal piece of a R. waitii spermatozoon. Note the single plasma membrane
and the granular material surrounding the fibrous sheath, c: Three transverse sections of R. waitii spermatozoa to
show the midpiece (labelled 1), the principal piece (2) and the endpiece (3). Note that in the midpiece the dense
outer fibres 3 (numbered) and 8 are considerable larger than the others and are associated with the inner layer of the
fibrous sheath, and that the fibres are absent in the principal piece section. Note also the multiple plasma
membrane layers in the midpiece and the single layer in the principal and end pieces, d: Longitudinal section of
part of the midpiece of a R. endoterus spermatozoon to show the intermitochondrial plaque, e: Longitudinal
section of part of the midpiece of a R. australis spermatozoon to show the poorly developed intermitochondrial
plaque, f: Longitudinal section of the annulus region of a R. endoterus spermatozoon. Note the multiple plasma
membranes in the midpiece and single in the principal piece and the granular material in the principal piece, a-f,
scale bar = 0.2 pm
Anteriorly, the mitochondrial sheath commences at the posterior extremity of the neck cylinder.
As shown in Figs 2 a, 2 c and 2 h, it surrounds the fibrous sheath, which extends further
anteriorly than the mitochondria.
Sections derived from anterior portions of the midpiece show 9 dense outer fibres, each
closely applied to a peripheral axonemal doublet. As is common among non-mammalian amniote
spermatozoa, fibres 3 and 8 are much larger than the others and make contact with the encircling
fibrous sheath (Fig. 3 c). More distal sections show a progressive reduction, eventually leaving
only fibres 3 and 8 as longitudinal “ribs” of the fibrous sheath.
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
393
The fibrous sheath as revealed in grazing sections has a predominantly transverse
orientation but with irregular bifurcations and anastomoses (Fig. 3 b).
The mitochondria show a uniquely regular and complex arrangement which immediately
distinguishes Ramphotyphlops spermatozoa from those of all other reptiles. As shown clearly in
both grazing TEM sections (Fig. 3 a) and in SEM images (Fig. 2 h), the overall pattern consists
of a series of stacked and alternating chevrons. SEM images of damaged sperm suggest that
individual mitochondria are extremely elongate and narrow, with a diameter of around 0.1 |im
and that they spiral helically around the midpiece for one complete revolution before turning
sharply to repeat the pattern but with the opposite helical polarity (i.e. if clockwise, then
anticlockwise). The exact length attained by any individual mitochondrion, as well as the number
of flexion points per mitochondrion are presently unknown. Order is apparently maintained in this
potential chaos by a very precise alignment of the points of flexion around a common
circumferential point, thereby maintaining the pattern of regular, alternating chevrons. In some
grazing sections of the midpiece the mitochondria show a more chaotic, intertwining and
overlapping arrangement. It is not clear at this stage whether these sections are artefactual,
resulting from cut spermatozoa, or whether they are a natural variant, possibly associated with
posterior sections of the midpiece.
Transverse and longitudinal setions consistently show a broad translucent zone between the
mitochondria and the underlying fibrous sheath (Fig. 3c, f). This apparent lack of contact between
the two flagellar sheaths is surprising in view of the regular alignment of the mitochondria.
Internally, the mitochondria usually show a single, circular crista enclosing granular material of
variable density (Figs 2 a, 3 d).
Most transverse and longitudinal sections through the midpiece show that it is surrounded
by multiple plasma membranes (generally between five and seven). The longitudinally orientated
microtubules which so conspicuously surround the spermatozoal head are also evident in most
sections of the midpiece (Fig. 3 a, c).
Intermitochondrial “rings” or “plaques” are clearly present in R. endoterus, where blocks of
electron dense material are commonly observed in both tranverse and longitudinal sections,
interspersed among otherwise typical mitochondria (Fig. 3 d). Available material suggests that
both complete rings and discrete blocks are represented. Less compelling evidence for the
presence of intermitochondrial rings is available for R. australis and R. waitii. In each species just
one longitudinal section has shown apparently discrete, membrane-bound plaques of granular
material interposed between typical mitochondria (Fig. 3 e). Notably, although the plaques are
very definite structures in R. endoterus, they are not numerous in comparison to the pattern seen
in sperm of many other reptile species [6].
Principal piece
The boundary between mid-piece and principal piece is marked by a small but distinct
annulus. There is only a minor decrease in flagellar diameter between the midpiece and principal
piece (Fig. 3f). The lack of abrupt narrowing of the flagellum posterior to the mitochondrial
sheath is due to the presence of a diffuse granular material both external to, and intercalated
between, the elements of the fibrous sheath of the principal piece (Fig. 3 f). Further posteriorly,
this granular layer is absent, and the principal piece is correspondingly smaller in diameter.
The longitudinal fibres associated with the fibrous sheath, adjacent to axoneme doublets 3
and 8, extend an unknown distance into the principal piece. The absence of these fibres from most
transverse sections of the principal piece (Fig. 3 c) suggests that they must terminate fairly close
to the annulus.
A single plasma membrane is present throughout the length of the principal piece, as are the
associated microtubules (Fig. 3 c). In sections of R. endoterus the multilayered membranes of the
394 H. R. HARDING. K. P. APLIN & M. MAZUR : RAMPHOTYPHLOPS (TYPHLOPIDAE, SQUAMATA)
midpiece are clearly seen to terminate at the annulus, posterior to which a single, heavier
membrane is present (Fig. 3f).
The length of the principal piece could not be measured from available material. However, it
must be at least 55 |im in R. waitii, based on lengths of incomplete spermatozoa and of the other
major components.
Endpiece
Fig. 3 c indicates that the axoneme extends posteriorly beyond the termination of the
fibrous sheath where, surrounded simply by the plasma membrane, it forms the endpiece. It is
notable that the palisade of microtubules extends to the endpiece.
DISCUSSION
As noted in the Introduction, the phylogenetic affinities of Typhlopidae are poorly
understood, with some uncertainty over both their current association with other snakes in the
taxon Serpentes [9, 14], and even their current inclusion within Squamata [17], Recent cladistic
analyses of spermatozoal evolution among higher vertebrates [8, 10, 11] provide a useful
framework against which to assess the broad scale phylogenetic affinities of the Typhlopidae,
based on a previously unexamined aspect of their morphology.
Compared with the various major groups of living amniotes (turtles, crocodiles, birds,
tuatara, mammals, squamates) spermatozoa of Ramphotyphlops species are, overall, most similar
to those of other squamates as described by FURIERI [6] and more recent workers [11, 12, 15].
Some of the features shared by Ramphotyphlops spp. (e.g. the presence of an elongate and
relatively straight sperm head) are undoubtedly plesiomorphic for amniotes as a whole. However,
other features appear to represent derived or apomorphic characters of possible phylogenetic
significance. These apomorphic, squamatan characteristics include: the paracrystalline nature of
the subacrosomal cone; the absence of an endonuclear canal; the presence of electron dense
intermitochondrial “rings” or “plaques”; the extension of the fibrous sheath into the midpiece ([8,
10], see also JAMIESON, Squamates, this volume); and reduction or loss of the annulus. The
occurrence of these features in the spermatozoon of Ramphotyphlops species provides strong
independent confirmation that Typhlopidae are correctly placed within Squamata.
Analysis of phylogenetic relationships within Squamata based on spermatozoal characters is
hindered by the many gaps in current taxonomic coverage. Among the snakes for example,
comparable observations are available only for representatives of three other families, Colubridae
[1, 6, 7, 16, 18, 19], Viperidae [6] and Boidae [2,7], and no data are available for more than a
dozen other families. Similarly, little or no information is available for many important groups of
lizards, including some which have been putatively implicated in the evolution of snakes (e.g.
varanoids [14], dibamids [4]).
Among the various squamate groups for which spermatozoal data are available,
Ramphotyphlops spermatozoa appear particularly close to those of snakes. In all, five features
appear to be uniquely shared among the various families of snakes, namely: (1) the extreme
forward prolongation of the fibrous sheath to the level of the neck; (2) the presence of a distinct
neck cylinder; (3) the multi-layered nature of the midpiece plasma membrane; (4) the extremely
elongate nature of the individual mitochondria; and (5) the extreme elongation of the midpiece.
Each of these features appears to represent a derived or apomorphic character relative to a basal
squamate condition [8, 10] and they thus collectively support the current placement of
Typhlopidae within Serpentes.
Ramphotyphlops spermatozoa differ from those of other snakes in the arrangement of
mitochondria in the midpiece. For other species of snakes, the mitochondria are variably
described as “very slender and rather convoluted” [6] and as “highly contorted and variable in
their orientation ... (but with) .. a predominant direction .. in a loose spiral around the flagellum”
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[7]. These observations are confirmed by our own unpublished findings for various species of
boids, elapids and a viperid. Clearly they are in striking contrast with the highly regular
arrangement described for Ramphotyphlops. Nevertheless, JAMIESON [Squamates, this volume]
has identified zigzagged mitochondria as a characteristics of snakes, also seen in pygopodids.
The mitochondria of lizards and other groups of reptiles (turtles, crocodiles, tuatara etc.) are
even less similar to those of Ramphotyphlops. In these groups, typically the mitochondria are
small, rounded or brick-shaped bodies, arranged either in regular rows intercalated between
complete intermitochondrial rings, or, in irregular fashion with scattered intermitochondrial bodies
[6, 11, 12, 15]. Exactly how the mitochondria in Ramphotyphlops come to be so elongate and
furthermore, how they come to be deployed in such a precise and unusual manner, are clearly
issues of general interest which should be further investigated through examination of the
spermiogenetic process.
Another difference between spermatozoa of Ramphotyphlops and those of other snakes
concerns the granular layer external to the principal piece fibrous sheath. This material appears to
be absent from the spermatozoa of both boid and colubroid snakes, among which the flagellum
shows a more pronounced narrowing at the annulus. Among other squamates, a granular layer is
present in the principal piece of at least some skinks [11, 12] and varanids [unpublished data].
Hence this may represent a plesiomorphic feature within Squamata.
Significant differences in spermatozoal structure were observed between each of the three
species of Ramphotyphlops examined in this study. Of these, the most striking differences
concern the presence and electron density of the intermitochondrial bodies of the midpieces:
conspicuous, but relatively few, electron dense bodies in the midpiece in R. endoterus and
granular structures of low electron density in R. waitii and R. australis. These appear to be even
less common in the midpiece than they are in R. endoterus. Interestingly, these differences in the
relative electron density correlate with that observed in the neck cylinder; dense in R. endoterus
and of low electron density in the other species. The possibility that these characteristics are
related is further strengthened by HAMILTON & Fawcett’s [7] finding that the neck cylinder and
the intermitochondrial plaques both arise from a common body of granular material which lies in
close topographical relationship to the ends of mitochondria as they assemble around the distal
centriole and base of the axoneme during late spermiogenesis. However, because this observation
is at odds with recent claims that derive the intermitochondrial bodies directly from mitochondria
[3, 8], we defer any further speculation pending analysis of spermiogenesis in Ramphotyphlops.
ACKNOWLEDGEMENTS
All the electron microscopy for this project has been carried out at the Department of Anatomy and Histology,
University of Sydney, and we thank A/Prof. Cedric Shorey, Head of Department, for enabling use of facilities and for the
technical support from the Department. This project has been supported by an ARC (Australian Research Council) Small
Grant to H.R. Harding, through the University of New South Wales.
REFERENCES
1 . AUSTIN, C. R., 1965. — Fine structure of the snake sperm tail. Journal of Ultrastructure Research , 12: 452-462.
2. Boisson, C. & Mattei, X., 1966. — La spermiogen£se de Python sebae, Gmelin, observee au microscope
electronique. Annales des Sciences Naturelles, Zoologie, 8: 363-390.
3. Carcupino, M., Corso, G. & Pala, M., 1989. — Spermiogenesis in Chalcides ocellatus tiligugu (Gmelin)
(Squamata, Scincidae): an electron microscope study. Bollettino di Zoologia, 56: 119-124.
4. CUNDALL, D., WallaCH, V. & Rossman, D. A., 1993. — The systematic relationships of the snake genus
Anomochilus. Zoological Journal of the Linnean Society , 109: 275-299.
5. Dan, J. C., Hashimoto, S., Kubo, M. & Yonehara, K., 1975. — The fine structure of the acrosomal trigger. In: B.
Afzelius, The Functional Anatomy of the Spermatozoon. Oxford, Pergamon Press: 39-45.
6. Furieri, P., 1970. — Sperm morphology in some reptiles: Squamata and Chelonia. In: B. BACCETTI, Comparative
Spermatology. Rome, Accademia Nazionale dei Lincei & New York, Academic Press: 115-131.
396 H. R. HARDING, K. P. APLIN & M. MAZUR : RAMPHOTYPHLOPS (TYPHLOPIDAE. SQUAMATA)
7. Hamilton, D. W. & Fawcett, D. W., 1968. — Unusual features of the neck and middle-piece of snake spermatozoa.
Journal of Ultrastructure Research. 23: 81-97.
8. Healy, J. M. & Jamieson, B. G. M„ 1992. — Ultrastructure of the spermatozoon of the tuatara ( Sphenodon
punctatus) and its relevance to the relationships of the Sphenodontida. Philosophical Transactions of the Royal
Society of London. B. 335: 193-205.
9. HEYDER, G., 1973. — Das Blutgefassystem von Typhlops vermicularis Merrem (1820). Morphologisches Jahrbuch ,
119: 492-513.
10. Jamieson, B. G. M. & Healy. J. M., 1992. — The phylogenetic position of the tuatara Sphenodon (Sphenodontida,
Amniota), as indicated by cladistic analysis of the ultrastructure of spermatozoa. Philosophical Transactions of
the Royal Society t of London, B , 335: 207-219.
1 1. Jamieson, B. G. M. & Scheltinga, D. M., 1993. — The ultrastructure of spermatozoa of Nangura spinosa
(Scincidae, Reptilia). Memoirs of the Queensland Museum, 34: 169-179.
1 2. McDowell, S. B., 1987. — Systematics. In: R. A. SEIGEL, J. T. Collins & S. S. Novak, Snakes: Ecology and
Evolutionary Biology. New York, Macmillan: 3-50.
13. McDowell, S. B. & Bogert, C. M., 1954. — The systematic position of Lanthanotus and the affinities of
anguiomorphan lizards. Bulletin of the American Museum of Natural History , 105: 1-142.
14. Newton, W. D. & Trauth, S. E., 1992. — Ultrastructure of the spermatozoon of the lizard Cnemidophorus
sexlineatus (Sauria: Teiidae). Herpetologica, 48: 330-343.
15. Phillips, D. M. & Asa, C. S.. 1991. — Strategies for formation of the midpiece. In: B. Baccetti, Comparative
Spermatology 20 Years After. New York, Raven Press: 997-1000.
16. Robb. J.. 1960. — The internal anatomy of Typhlops Schneider (Reptilia). Australian Journal of Zoology. 8: 181-
216.
17. Saita. A., Comazzi, M. & Perrotta, E., 1988. — Ulteriori osservazione al M.E. sulla spermiogenesi di un
serpente: Coluber viridiflavus (Lacepede) in riferimento ad elementi comparativi nella spermiogenesi dei rettili.
Atti Accademia Nazionale dei Lincei Rendiconti Classe di Scienze Fisiche, Matematiche e Naturali (Ser. VIII)
82: 137-143.
18. SUD, B. N. & Meek, G. A., 1981. — Ultrastructure of the spermatozoon of the green snake, Natrix natrix.
Anatomical Record , 199: 248A.
Source : MNHN. Paris
Ultrastructural and Light Microscopic Observations
of Mature Epididymal Spermatozoa
and Sperm Maturation of the Greater Bilby,
Macrotis lagotis (Metatheria, Mammalia)
Stephen JOHNSTON *, Lina DADDOW ** & Frank CARRICK **
* Department of Farm Animal Medicine and Production,
University of Queensland Veterinary Science Farm, Pinjarra Hills, Q 4069, Australia
** Zoology Department, The University of Queensland, Brisbane, Q 4072, Australia
ABSTRACT
Light microscopic and ultrastructural observations were made on mature Macrotis lagotis cauda epididymal spermatozoa.
Sperm nuclear length measures 13.2 pm, midpiece length 16.2 pm and total sperm length 149.4 pm. The sperm head is
simple and without lateral concavities typical of the Peramelidae. Parachromatin-like material is present as a thin layer on
the dorsal nuclear surface and the lateral margins of the sperm head are uncondenscd and pitted. The acrosomc covers 2/5 o!
the dorso-rostral nuclear surface, extending caudally. The mitochondrial sheath is essentially round but slightly flattened
in transverse section and sculptured on its inner surface to accommodate dense outer fibres. These iibres are widely
separated in the caudal region of the midpiece and connecting lamellae doubled. The annulus is typically perameloid in
structure. Unique cauda epididymal sperm characteristics include: lattice substructural material about the neck region and on
the caudo-ventral inner surface of the nucleus; arcs of double thickened membranes underlying the plasma membrane near
the caudal midpiece and the neck region of axoneme bifurcate about the proximal centriole. Evidence of sperm maturation
during epididymal transit includes: dislocation and cranial migration of the neck insertion from a primary implantation
fossa to a secondary insertion at the inner tip of the nucleus; slight acrosomal compaction; slight rotation of the sperm
head parallel to the longitudinal axis of the sperm and the shedding of a cytoplasmic droplet. Although no cladistic
analysis was attempted, the unique characteristics of M. lagotis spermatozoa support the present taxonomic status ol the
Thylacomyidae as a distinct family. In addition, shared spermatological characteristics between that of the
Thylacomyidae, Dasyuridae, Peramelidae and Tarsipes rostratus are also evidence of a close phylogenetic relationship.
However, the precise phylogeny of the Thylacomyidae requires further investigation.
RESUME
Observations en microscopie photonique et electronique sur le spermatozoide mur de 1 epididyme
et sa maturation chez le grand Bilby Macrotis lagotis (Metatheria, Mammalia).
Des observations en microscopie photonique el electronique ont ete faites sur les spermatozoides de la queue dc
Pepididyme de Macrotis lagotis (Bilby ou Grand Lievre Marsupial). Les longueurs du noyau, de la piece interm6diaire et du
spermatozoide sont de 13.2, 16.2 et 149.4 pm. La tete du spermatozoide est simple et sans les cavites laterales typiques
des Peramelidae. Un materiel de type parachromatinien est present sous forme d'une couche fine sur la surface dorsale du
noyau et les marges laterales de la tete ne sont pas condensees et sont alveolees. L’acrosome couvre les deux cinquiemes de
Johnston , S., Daddow, L., & Carrick, F., 1995. — Ultrastructural and light microscopic observations of mature
epididymal spermatozoa and sperm maturation of the Greater Bilby, Macrotis lagotis (Metatheria, Mammalia). In:
Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mas.
natn. Hist . nat., 166 : 397-407. Paris ISBN : 2-85653-225-X.
398 S. JOHNSTON, L. DADDOW & F. CARRICK : MACROTIS LAGOTIS (METATHERIA, MAMMALIA)
la surface nucleaire dorso-rostrale et s’etend vers la queue. La gaine mitochondriale est essentiellemeni ronde mais
legerement aplatie en coupe transversale et sculptee sur sa surface interne pour laisser la place aux fibres dcnses extemes.
Ces fibres sont largement scparees dans la region caudale de la pi£ce intermediate et les lamelles de connexion sont
doublees. L'annulus a une structure perameloi'de typique. Les caracteristiques originales du spermatozoi'de epididymaire
sont: un materiel a structure de treillis autour de la region du cou et sur la surface intcrieure caudo-ventrale du noyau; des arcs
de membrane k double epaisscur soulignant la membrane plasmique pr£s de la partie caudale de la piece intermediaire; une
bifurcation de la region du cou de 1’axoneme pr&s du centriole proximal. Les preuves de la maturation du spermatozoi'de
pendant le transit epididymaire sont: la dislocation et la migration vers la tete de 1’insertion du cou k partir d’une fossette
primaire jusqu*& une insertion secondaire k Lextremite interne du noyau; une legere compactage de Lacrosome; une legcre
rotation de la tete du spermatozoi'de parallelement k l’axe longitudinal du spermatozoi'de; la perte d’une goutte
cytoplasmique. Bien qu’une analyse cladistique n’ait pas ete tentee, les caracteristiques originales du spermatozoi'de de M.
lagotis soutiennent la position actuellement reconnue des Thylacomyidae comme une famille distincte. De plus, les
caracteristiques spermatologiques partagees des Thylacomyidae, Dasyuridae, Peramelidae and T. rostratus sont la preuve de
relations phylogeniques proches. Toutefois. la phylogdnie precise des Thylacomyidae demande d’autres Eludes.
While spermiogenesis of Perameles nasuta has been thoroughly documented, [19-23],
observations of perameloid sperm maturation and mature spermatozoa are limited [14], HARDING
et al. [14] noted that most descriptions of epididymal sperm structure in the Peramelidae were
incomplete or were included as scattered adjuncts in comparative studies [2, 3, 5, 6, 9-12, 16].
Most studies have examined spermatozoa from P. nasuta and lsoodon macrourus, with only
preliminary observations reported for P. gunni, I. obesulus and Echymipera rufescens
spermatozoa.
HARDING et al. [14] previously described the ultrastructure and epididymal maturation of
P. nasuta and I. macrourus spermatozoa. These authors noted that mature spermatozoa from both
species were similar but also displayed a number of distinct apomorphies. They concluded that
ultrastructural changes during sperm maturation were not so obvious when compared to other
marsupials, except for the dislocation and marked relocation of the neck from its original
implantation fossa to a position close to the rostrum of the nucleus.
Preliminary observations made on Perorcytes longicauda and Echymipera kalubae sperm
have indicated that these species conform to the distinctive perameloid pattern with only minor
differences in size and shape [14], The flagellar structure of M. lagotis sperm has also been
described by HARDING et al. [14] who noted that it conformed to the basic perameloid pattern.
However, poor fixation of testicular tissue from one animal, and the occurrence of degenerate
spermatozoa from another old male, meant that detailed observations of head structure and to a
lesser extent midpiece structure were not possible.
Sperm structure is regarded as a conservative taxonomic character, reflecting evolutionary
affinities in the Marsupialia [24]. A detailed evaluation of the ultrastructure and epididymal
maturation of M. lagotis should, therefore, provide valuable insights into the controversial
phylogenetic position of the genus Macrotis, which to date has been based on skull and dental
characters [8], chromosomal [4] and serological evidence [1],
The present paper describes for the first time the ultrastructure of the mature Macrotis lagotis
spermatozoon as well as morphological changes during sperm maturation. Light microscopic
observations of sperm dimensions are recorded.
MATERIALS AND METHODS
A six year old sexually mature captive male Macrotis lagotis located at Western Plains Zoo, Dubbo (32°15'S.,
148°37'E.), Australia, was euthanased because of a prolonged chronic staphylococcal infection of the hind limbs. The
animal was in poor condition and considered surplus breeding stock.
Under general gaseous anaesthesia (Halothane), the caput and cauda epididymides of one testis were removed and
diced into 2mm x 2mm blocks and immersed into cold 3 % glutaraldehyde in 0.1 M phosphate buffer with 6% sucrose for
approximately 1 h. Samples were then transferred into 0.1 M sucrose in phosphate buffer and transported back to the
laboratory (The University of Queensland, St. Lucia) within 24 hours. Samples for electron microscopy were rinsed in
phosphate buffer, followed by post-fixation for 80 min in 1% osmium tetroxide in 0.1 M phosphate buffer with 6%
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
399
sucrose. Specimens were then washed in buffer, dehydrated through an ascending ethanol series, infiltrated and embedded
in Spurr's low viscosity epoxy resin. Ultrathin sections, 60 to 80 nm, were cut on an LKB 2128 UM IV ultramicrotome.
Sections were collected on carbon stabilised colloidion coated 200 mesh and single slot grids and stained with Reynold s
lead citrate [18], 6% uranyl acetate and further lead citrate [7]. All micrographs were taken on a Hitachi 11-300 electron
microscope at 75KV.
The epididymidis of the remaining testis was then dissected into warm saline and gently leased to release motile
spermatozoa. Nigrosin - eosin stained spermatozoa were used to measure the midpiece and total sperm length via bright-
field microscopy at 400 x. As the marsupial sperm nucleus decondenses on air drying [6], measurements of head length
and width were made on formalin fixed spermatozoa, using Nomarski differential interference microscopy at 1000 x. All
measurements were made using a calibrated eyepiece micrometer.
RESULTS
Light microscope studies: sperm dimensions
Table 1 shows the sperm dimensions recorded for 100 Macrotis lagotis spermatozoa.
Table 1. — Dimensions of mature cpididymal spermatozoa of Macrotis lagotis
Ultrastructure of mature epididymal spermatozoa
The sperm head of Macrotis lagotis is cuneiform-shaped but slightly dorsoventrally flattened
(Fig. lb-c). A deep longitudinal groove runs caudally along the ventral nuclear surface from
where the neck is inserted. The nuclear groove is narrow cranially but widens caudally to
accommodate the midpiece (Fig. la). The majority of the dorsal aspect of the sperm head oveilies
the anterior 3/5 of the midpiece. Serial transverse sections through the sperm head indicate the
presence of a dorsal nuclear ridge which extends from the tip of nucleus to its caudal extremity,
but it is not prominent cranially (Fig. ld-h).
The nucleoplasm is primarily condensed. However, some transverse sections indicate
nuclear indentations along the periphery of the ventral flanges of the nucleus (Fig. If). A thin
layer of parachromatin-like material extends caudally from the rostrum for approximately two
fifths the length of the nucleus, and consists of material which is distinct from that of the
acrosome, but is less electron dense than the nucleus (Fig. 2f-g). Poor preservation of membrane
ultrastructure makes it difficult to ascertain whether the parachromatin-like material is within or
outside the nuclear membrane.
There is an original primary and a distinct secondary implantation lossa (Fig. 21-g). Ihe
original implantation fossa is located deep within the sperm head and lined by a thick layer of
material which appears to form a basal plate. In the vicinity of the second implantation lossa and
surrounding the neck region, granular material which has an ordered substructure is present (Fig.
If). Some micrographs also indicate the presence of electron dense material which is confined to
the caudo-ventral surface of the sperm head in association with the midpiece, (Fig. la, f),
however, this material is particularly prominent in immature spermatozoa (Fig. 3b). It appears to
have a distinct “honeycomb" lattice substructure.
400 S. JOHNSTON. L. DADDOW & F. CARRICK : MACROTIS LAGOTIS (METATHERIA, MAMMALIA)
Fig. 1. — Electron micrographs of longitudinal sections (LS) and transverse sections (TS) of the nuclear and midpiece
region of Macrons lagotis cauda epididymal spermatozoa, a: LS of head and midpiece (x 1 1 200). b-i: TS
through head to midpiece regions of M. lagotis spermatozoa as indicated in Fig. la. a-e: nuclear acrosomal
region; b; x 21 000; c, x 21 000; d. x 21 000; e x 28 000; f, g: neck region; f. x 22 000; g, x 16 500.
h, i: midpiece region; h, x 22 000; i. x 22 000.
Source . MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
401
The majority of the acrosomal matrix of the mature acrosome forms a rudimentary cap over
the cranial dorsal nuclear surface (Figs la, 2f-g). However, it also extends dorso-caudally for two
fifths the length of the nucleus in close association with the parachromatin-like material. Serial
transverse sections through the caudal region of the acrosome indicate that the distribution of the
acrosome conforms to the underlying profile of the nucleus (Fig. lb-f). The cranial margin of the
acrosome forms a lip at the cranial extremity of the nucleus (Figs la, 2f-g, 3a-b).
Longitudinal sections reveal that the axoneme terminates in a bifurcated structure, with the
proximal centriole situated within the fork of the bifurcation (Fig. 2f). The dorsal arm of the
bifurcation extends cranially past the proximal centriole and terminates surrounded by electron-
lucent material. The ventral arm of the bifurcation is also surrounded by electron-lucent material
but extends only adjacent to the proximal centriole.
The midpiece and underlying mitochondrial sheath are slightly flattened in transverse section
(Fig. 2b). The ratio of the cross sectional diameters perpendicular and parallel to the centrally
located axonemal fibres is 1.08 ±0.03 (n= 1 6). Mitochondria in transverse section are sculptured
on their inner surfaces to accommodate the displaced outer fibres (Fig. 2b). Dense outer fibres 3
and 8 have an evenly circular contour, while the rest of the fibres has a semi-circular contour in
cross section (Fig. 2b). The dense outer fibres of the cranial extremity of the axoneme, in the
vicinity of the midpiece, are positioned close to the underlying axoneme doublet. However, in all
transverse sections of the axoneme caudal to the nucleus, the dense outer fibres become widely
separated. The separation is greatest between dense outer fibres 1, 5 and 6 and least between 3
and 8. Connecting lamellae in M. lagotis are double in nature except for fibres 3 and 8 (Fig. 2b).
There is no diffuse granular material surrounding the outside of the mitochondrial sheath.
However, transverse sections of the caudal midpiece region reveal an arc of doubled thicked
membrane structures underlying the plasma membrane in the region of dense outer fibres 2, 3 and
4 and 7, 8 and 9 (Fig. 2b). In longitudinal sections these structures are located from the terminal
caudal portion of the midpiece to a position approximately 10 mitochondrial whorls cranially. The
annulus forms a fibrous ring below the most caudal mitochondria, and is joined to the fibrous
sheath of the principal piece by an array of fine fibres (Fig. 2a).
Serial sections of the cranial region of the principal piece of M. lagotis spermatozoa are
slightly flattened in cross section (Fig. 2c). The ratio of the cross sectional diameters
perpendicular and parallel to centrally located axonemal fibres is 1.04 ± 0.02 (n=47). The fibrous
sheath had only one fenestration (vacuole) in each rib on either side of the longitudinal columns
(Fig. 2c). There is no typical endpiece, as the axoneme terminates before that of the fibrous sheath
(Fig. 2d). Surrounding the fibrous sheath and underlying the plasmalemma there is also a layer of
fine material; the accessory sheath (Fig. 2e).
Epididymal sperm maturation
Light microscopic observations of spermatozoa in the lumen of the seminiferous tubule at
spermiation and the caput epididymides indicate that the long axis of the nucleus ranges from
perpendicular (Fig. 3a) to almost completely rotated parallel to the long axis of the axoneme (Fig.
3b). However, by the time spermatozoon reaches the cauda epididymidis the nucleus has fully
rotated and is streamlined with the tail. The most notable change during epididymal transit is the
dislocation and migration of the neck region from the original primary implantation fossa to a
secondary implantation fossa located cranially (Fig. 3b). The dorso-cranial portion of the
acrosome of caput epididymal spermatozoa appears button-like (Fig. 3c). However, during
epididymal maturation this region appears to undergo compaction; becoming slightly flattened.
The cytoplasmic droplet of the caput epididymal spermatozoon is eccentrically placed to one side
402
S. JOHNSTON, L. DADDOW & F. CARRICK : MACROTIS LAGOTIS (METATHERIA, MAMMALIA)
Table 2. — Ultrastructural characteristics of M. lagoiis cauda epididymal spermatozoa and sperm maturation as compared
with the Pcramelidae and Dasyuridae. Bold type = M. lagoiis unique character. Italics = M. lagotis shared character
Fig. 2. Electron micrographs of Macrotis lagoiis cauda epididymal spermatozoa, a: LS of midpiece and principal piece
region illustrating microtubular structures at the caudal extremity of the midpiece and the annulus (x 22 500).
b: TS (hrough caudal extremity of midpiece (x 39 000). c: TS through annular region (x 36 000). d: LS of
endptece (x 28 000). e: TS through principal and endpiece regions (x 14 000). f: LS of the nuclear-flagellar
connection, illustrating bifurcated neck-piece (x 24 000). g: LS of the nuclear-flagellar connection, illustrating
primary implantation fossa (x 24 000).
Source . MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
403
Source : MNHN. Paris
404 S. JOHNSTON. L. DADDOW & F. CARR1CK : MACROTIS LAGOTIS t METATHER1A . MAMMALIA)
b
Fig. 3. — Electron micrographs of Macrotis lagotis immature caput epididymal spermatozoa, a, b: LS illustrating neck
insertion into primary implantation fossa, button-like acrosome and cytoplasmic droplet. Also note bifurcated
neck in a and honey-comb granular material in the caudal extremity of the nucleus in b. c: LS of nuclear-flagellar
connection highlighting eccentric position of the cytoplasmic droplet, d: TS low power electron micrograph of
immature spermatozoa, a, x 9 800; b, x 9 100; c, x 5 500; d, x 2 800.
Abbreviations for all figures: Ac, acrosome; An, annulus; As, accessory sheath; Ax, axoneme; Bi. bifurcated neck; Cd,
cytoplasmic droplet; Del, double connecting lamellae; EP. endpiece; F, fibres of annulus attached to fibrous
sheath; Fs, fibrous sheath; G. granular material about neck region; Mg, honey comb granular material; Lc,
longitudinal columns; Mi, mitochondria; Mt, double thickened membrane; Ni, nuclear indentations; Nr, nuclear
ridge; Nu, nucleus; Pa. parachromatin-like material; Pc, proximal centriole; Pif, primary implantation fossa; R,
ribs of fibrous sheath; Sc, sculptured mitochondria; Sif, secondary implantation fossa; Sf, single fenestration of
the fibrous sheath; Vf, ventral flange of nucleus; Vg, ventral groove ; 1-9, relative position of dense outer fibres.
of the midpiece and sperm head (Fig. 3a-d). The droplet appears to consist of two types of
electron dense material. The first is easily recognised as composed of cytoplasmic membrane
remnants, while the second is of less dense consistency (Fig. 3c).
Source : MNHN, Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
405
DISCUSSION
Macrotis lagotis is the only extant member of the Thylacomyidae. Its spermatozoa are large
compared to those of other mammals, but smaller than those of the Dasyuridae, Peramelidae and
Tarsipes rostratus. Interestingly, while the total length of the M. lagotis spermatozoon is shorter
than that of the other perameloids, its head length is up to twice as long.
Spermatozoa of M. lagotis display numerous unique ultrastructural characteristics which
differentiate them both from the Peramelidae, (traditionally regarded as sharing a close
phylogenetic relative), and the Dasyuridae. There are also numerous shared sperm characteristics
between M. lagotis and the Dasyuridae and Peramelidae (See Table 2).
The head shape of M. lagotis is simple in profile, characteristically marsupialian [14] and
without the complicated lateral concavities of typical Peramelidae or the hooked profile of the
Vombatidae and Phascolarctidae. Like the Peramelidae, the ventral groove of sperm of M. lagotis
is extensive, reaching almost to the tip of the nucleus. Similarly, both Isoodon macrourus and M.
lagotis , have a nuclear ridge or keel on the dorsal surface of the sperm head, although the ridge of
I. macrourus appears somewhat more flattened [14], It is possible that such a structure could
provide hydrodynamic stability during sperm motility.
The sperm nucleus of the Peramelidae is characterised by two chromatin components; an
electron-dense and an electron-lucent component (or parachromatin). The nuclei of sperm of M.
lagotis also appeared to contain parachromatin-like material;- however, it is confined to the dorsal
surface of the sperm head. It is still equivocal whether this material is part of the nucleoplasm.
The occurrence of indentations along the periphery of the ventral flanges of the sperm nucleus
appears similar to that described in the Peramelidae [14] but they are not as marked as those found
in the Dasyuridae [15]. The anterior extremity of the ventral groove is also surrounded by material
which is less electron dense than that of the parachromatin-like material. Unique to M. lagotis, so
far as is known, this material had a lattice substructure.
During sperm maturation the neck of the axoneme dislocates from the primary implantation
fossa to become located deep within the cranial extremity of the ventral groove. The secondary
implantation fossa, however, does not possess a basal plate, and like other perameloid
marsupials, connecting structures are not evident in association with the neck [14], HARDING et
al. [14] commented that lack of connecting structures might have meant that the head-flagella
connection of the mature spermatozoon is not secure and that the stream-lined alignment of head
and flagellum probably aided in preventing separation. Interestingly, this study also reveals
electron dense material with a honey-comb lattice substructure on the interior surface of the caudo-
dorsal sperm nucleus. This material is also apparent in nigrosin-eosin stained spermatozoa. Given
that the sperm head of M. lagotis is found to be twice as long as that of other perameloid
marsupials, it is possible that this material cements the caudo-dorsal extremity of the nucleus to
the midpiece region, thus adding further strength to the head-flagella connection. One of the most
striking features of the M. lagotis spermatozoon is the bifurcated morphology of the connecting
piece, with the proximal centriole located within the fork of the bifurcation. This unusual structure
appears to be unique to M. lagotis and a functional significance is elusive. Perhaps such an
arrangement also strengthens the head-flagellum connection.
Acrosomal morphology of M. lagotis appears most similar to that of T. rostratus
(Tarsipedidae). HARDING et al. [13] noted that the acrosome of T. rostratus covered the dorsal
surface of approximately the anterior 2/3 of the nucleus. This observation compares with 2/5 ol
the nucleus of M. lagotis reported in this study. Secondly, the acrosome of both T. rostratus and
M. lagotis forms a small lip at the cranial tip of the nucleus (HARDING, pers. comm.). The caudal
portion of the acrosome of spermatozoa in the caput epididymis appears button like, but during
epididymal transit the acrosome in this region flattens. Although acrosomal maturation of M.
lagotis is only comparatively minor, perameloid and dasyuroid acrosomes appear fully compacted
prior to epididymal transit.
406 S. JOHNSTON, L. DADDOW & F. CARR1CK : MACROTIS LAGOTIS (METATHERIA, MAMMALIA )
HARDING et al. [14] had previously described the sperm flagellar ultrastructure of M.
lagotis, noting the wide separation of dense outer fibres from the axoneme, presence of paired
connecting lamellae, the single fenestration on either side of the longitudinal columns and the lack
of a diamond shaped transverse flagellar section with a concave profile. However, owing to poor
fixation and consequent quality of the material examined, midpiece ultrastructure was not
described. . .
As for other perameloid marsupials [14], the dense outer fibres of M. lagotis in the cranial
portion of the midpiece lie close to the underlying axoneme (Fig. lh), but in caudal regions of the
midpiece the dense outer fibres become radially displaced. However, the extent of the
displacement in M. lagotis and the Peramelidae is not as marked as in the Dasyuridae and T.
rostratus [13, 15]. As in the other perameloid marsupials [14] the shape of the dense outer fibres
of M. lagotis sperm (except 3 and 8) is semi-circular in transverse section.
The shape of the caudal midpiece of M. lagotis in transverse section is slightly flattened,
unlike that of the other perameloid marsupials which are essentially circular [14]. Also in common
with the perameloids, dasyuroids and Tarsipes rostratus, the mitochondria are sculptured on their
inner surface to accommodate the displaced dense outer fibres [13, 14, 15]. Spermatozoa of M.
lagotis lack the granular layer surrounding the mitochondrial sheath, which is typical of the
perameloid midpiece. However, there is an arc of double thickened membrane material underlying
the plasma membrane in the region of dense outer fibres 2, 3 and 4 and 7, 8 and 9. These
structures appear to be unique to M. lagotis . Observations from the midpiece region of M. lagotis
sperm failed to detect the presence of an electron dense plate surrounding the midpiece or an
underlying granular wedge; both apomorphies which characterize other perameloid spermatozoa
[14].
The annulus of M. lagotis spermatozoa is “perameloid-like” in forming a fibrous ring caudal
to the most posterior mitochondria and is joined to the fibrous sheath of the principal piece by an
array of fine fibres [14]. Also similar to the sperm of other perameloids, dasyuroids and T.
rostratus, the plasma membrane in the region of the annulus is not indented.
Compared to the other perameloids, dasyuroids and T. rostratus [13-15], transverse
sections of the cranial principal piece are markedly less flattened: a feature unique to M. lagotis.
However, unlike the other perameloids, but similar to the dasyuroids, the fibrous sheath of M.
lagotis has only one fenestration in the rib of either of the longitudinal columns. In common with
the Peramelidae, M. lagotis also has an accessory sheath surrounding the fibrous sheath of the
principal piece, and the axoneme terminates before the fibrous sheath [14].
In conclusion, Macrotis lagotis spermatozoa display a number of unique characters that
appear to separate them from the Peramelidae, Dasyuridae and Tarsipes rostratus. While a detailed
cladistic analysis is currently in preparation, ultrastructural spermatozoal evidence on M. lagotis
reported in this study, appears to support the present taxonomic status of the Family
Thylacomyidae and confirm the close phylogenetic affinities (intermediate) with the Peramelidae,
Dasyuridae and T. rostratus.
ACKNOWLEDGEMENTS
The authors thank Dr. David Blyde of Western Plains Zoo for allowing access to Bilby testicular material and Mr.
Tom Gorringe, Department of Zoology, University of Queensland for printing the electron micrographs.
REFERENCES
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3. Cleland, K. W. & Rothschild, Lord, 1959. — The bandicoot spermatozoon: an electron microscopic study of the
tail. Proceedings of the Royal Society, 150: 24-42.
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4. Close, R. L., Murray, J. D. & Briscoe, D. A., 1990. — Electrophoretic and chromosome surveys of the taxa of
short-nosed bandicoots within the genus Isoodon. In: J. H. Seebeck, P. R. Brown, R. L. Wallis & C. M.
Kemper, Bandicoots and Bilbies. Sydney, Surrey Beatty & Sons: 19-27.
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macrourus. Proceedings of the Annual Meeting of the Australian Society • of Reproductive Biology (Perth): 84.
6. Cummins, J. M., 1980. — Decondensation of sperm nuclei of Australian marsupials: effects of air drying and of
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7. DaddOW, L., 1986. — An abbreviated method of the double lead stain technique. Journal of Submicroscopic
Cytology 18: 221-4.
8. Groves, C. P. & Flannery, T., 1990. — Revision of the families and genera of bandicoots. In: J. H. Seebeck, P. R.
Brown, R. L. Wallis & C. M. Kemper, Bandicoots and Bilbies. Sydney, Surrey Beatty & Sons: 1-11.
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development and structure. Ph. D. Thesis. University of New South Wales, Sydney, Australia.
1 0. Harding, H. R., Carrick, F. N. & Shorey, C. D., 1977. — Spermatozoa of Australian marsupials: ultrastructure and
epididymal development. In: J. H. Calaby & C. H. TYNDALE-BlSCOE, Reproduction and Evolution; Fourth
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151-152.
1 1. Harding, H. R., Carrick, F. N. & Shorey, C. D., 1979. — Special features of sperm structure and function in
marsupials. In: D. W. Fawcett & J. M. Bedford. The spermatozoon. Baltimore-Munich, Urban &
Schwarzenberg: 289-303.
12. Harding, H. R.. Carrick, F. N. & Shorey, C. D., 1983. — AcrosoYhe development during spermiogenesis and
epididymal sperm maturation in Australian marsupials. In: J. Andr£, The Sperm Cell. The Hague, Martinus
Nijhoff: 411-414.
13. Harding, H. R., Carrick, F. N. & Shorey, C. D., 1984. — Sperm ultrastructure and development in the Honey
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Society: 451-461.
14. Harding, H. R., Shorey, C. D. & Cleland, K. W., 1990. — Ultrastructure of spermatozoa and epididymal sperm
maturation in some perameloids. In: J. H. Seebeck, P. R. Brow'N, R. L. Wallis & C. M. Kemper, Bandicoots
and Bilbies. Sydney, Surrey Beatty & Sons: 235-250.
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and epididymal sperm maturation in dasyurid marsupials: phylogenetic implications. In: M. Archer,
Carnivorous Marsupials. Sydney, Royal Zoological Society of New South Wales: 659-673.
1 6. Hughes, R. L., 1965. — Comparative morphology of spermatozoa from Five marsupial families. Australian Journal
of Zoology , 13: 533-543.
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M. Archer, Carnivorous Marsupials. Sydney, Royal Zoological Society of New South Wales: 49-63.
18. Reynolds, E. S. 1963. — The use of lead citrate at high pH and electron opaque stain in electron microscopy.
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19. Sapsford, C. S. & Rae, C. A., 1969. — Ultrastructural studies on Sertoli cells and spermatids in the bandicoot and
ram during movement of mature spermatids into the lumen of the seminiferous tubule. Australian Journal of
Zoology, 17: 415-445.
20. Sapsford, C. S., Rae, C. A. & Cleland, K. W., 1967. — Ultrastructural studies on spermatids and Sertoli cells
during early spermiogenesis in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Australian Journal of
Zoology, IS: 881-909.
21. Sapsford, C. S., Rae, C. A. & Cleland, K. W., 1969a. — Ultrastructural studies on maturing spermatids and on
Sertoli cells in the bandicoot Perameles nasuta Geoffroy (Marsupialia) Australian Journal of Zoology, 17: 195-
292.
22. Sapsford, C. S., Rae, C. A. & Cleland, K. W., 1969b. — The fate of residual bodies and degenerating germ cells
and the lipid cycle in Sertoli cells in the bandicoot Perameles nasuta Geoffroy (Marsupialia). Australian Journal
of Zoology , 17: 729-753.
23. Sapsford, C. S., Rae, C. A. & Cleland, K. W., 1970. — Ultrastructural studies on the development and form of the
principal piece sheath of the bandicoot Perameles nasuta Geoffroy (Marsupialia). Australian Journal of
Zoology, 18: 21-48.
24. TEMPLE-SMITH, F. D., 1987. — Sperm structure and marsupial phylogeny. In: M. ARCHER, Possums and Opossums:
Studies in Evolution. Sydney, Surrey Beatty and Sons & The Royal Zoological Society of New South Wales:
171-193.
Source MNHN, Paris
Source : MNHN. Paris
Variation in Sperm Head Morphology of Muroid
Rodents of Africa: Phylogenetic Implications
William G. BREED
Department of Anatomy and Histology, University of Adelaide. South Australia 5005. Australia
ABSTRACT
Sperm morphology from individuals of the following subfamilies of muroid rodents of southern Alrica is determined:
Cricetomyinae {Saccostomus). Gerbillinae (CerbiUurus and Taiera). Dendromurinae (Dendromus. Malacothnx. Pnonomys.
Sieatomvs, and Deomys ), and Otomyinae (Otomys), Mystromys (subfamily Mystromyinae) is used as an outgroup, the
sperm head of most species including that of Mystromys is falciform in shape but differences in internal organisation
occur. In Mystromys it is long and thin and there is a very large apical acrosomal segment. The falciform sperm heads ol
Saccostomus and Malacotlirix are broader basally but have a similar organisation apically. whereas in Deomys the sperm
head is very small, bilaterally flattened, with no apical hook. Gerbillurus sperm head terminates in a sharp pointed apex,
whereas in Totem it is round apically and a deep invagination occurs in the caudal nuclear region. In Olomys the sperm
head is falciform and the organisation of the perforatorium and acrosome is similar to that of most murine rodents apart
from Acomys and Uranomys. This study suggests that a falciform sperm type is probably the ancestral condition for the
dendromurine-cricetomyine-otomyine-murine clade. Sperm of both Deomys and Totem are highly divergent, and those o
the otomyines and murines are very different from sperm of species in other subfamilies; since a very similar or identical
morphology occurs in most species of the Otomyinae and Murinae it suggests that these two subfamilies are sister groups
to the exclusion of the other subfamilies and of Acomys and Uranomys.
RESUME
Les variations morphologiques de la tete des spermatozoides des Rongeurs Muroides d'Afrique:
implications phylogenetiques
La morphologie du spermatozoide a ete determince chez des specimens appartenant aux families suivantes de Rongeurs
Muroides d’Afrique du Sud : Cricetomyinae ( Saccostomus ). Gerbillinae ( Gerbillurus et Totem). Dendromurinae ( Dendromus .
Malacotlirix. Prionomys, Stealomys ct Deomys). et Otomyinae (Otomys). Mystromys (sous-lamille Mystromyinae) a ete
utilise comme outgroup. La tele du spermatozoide de la plupart des especes, y compris celle de Mystromys. est falciforme
mais il existe des differences dans l’organisation interne. Chez Mystromys la tele est longue et line et un grand segment
acrosomien apical est present. Les tetes falciformes de Saccostomus et Malacothnx sont plus larges & la base mats ont u
meme organisation h l'apex. alors que la tete de Deomys est trbs petite, aplatie bilateralement, et sans crochet. La tete de
Gerbillurus sc termine en un apex triis effile, alors que chez Tatera la tete est rondc il 1 apex et une invagination prolonde
existe dans la region nuclcaire caudale. Chez Otomys la tete est falciforme el 1 organisation du perforatorium et de
1' acrosome est similaire & celle de la plupart des Rongeurs Muridae. a part Acomys et Uranomys. .Cette etude suggere que le
spermatozoide de type falciforme est la condition ancestrale pour le clade Dendromurinae-Cricetomyinae-Otomyinae-
Murinac. Les spermatozoides de Deomys et de Tatera sont trbs divergents. et ceux des Otomyinae et Murinae sont ires
differents de ceux des espbees des autres sous-familles. Comme cette morphologie ires similaire ou identique se trouve chez
la plupart des especes des Otomyinae et Murinae. cela suggere que ces deux sous-familles sont des groupes-treres, a
Pexclusion des autres sous-familles et d' Acomys et Uranomys.
Breed. W. G., 1995. — Variation in sperm head morphology of muroid rodents of Alrica: phylogenetic
implications. In: Jamieson, B. G. M., Ausio, J., & Justine. J.-L. (eds). Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 409-420. Paris ISBN : 2-85653-225-X.
410
W. G. BREED : AFRICAN MUR! DAE ( MAMMALIA )
The superfamily of muroid rodents is composed of over 1300 species of about 280 genera
with major radiations in both the New and Old Worlds. SIMPSON [37] proposed two major
families: the Cricetidae and the Muridae with the latter including the African climbing mice
(Dendromurinae), swamp rats (Otomyinae), as well as true rats and mice of the Old World
(Murinae) inclusive of the African pouched mouse Saccostomus. Subsequently it was suggested
that the Dendromurinae [26] and Otomyinae [28] were closer to the Cricetidae, and that
Saccostomus and other cricetomyines were also part of this family [31, 32], CHALINE et al. [14]
later erected several new families: the Dendromuridae, Cricetomyidae, Nesomyidae (which
included the otomyines), whereas REIG [34] proposed these groups as subfamilies within the
Cricetidae. CARLETON & MUSSER [12] expanded the family Muridae to embrace 15 subfamilies
including the Dendromurinae, Otomyinae, Murinae, Gerbillinae, Cricetinae and others. Over the
last 10 years palaeontological and biochemical data have suggested that the dendromurines,
cricetomyines, and gerbillines are probably more closely related to each other than any is to the
Cricetinae [11, 13, 18].
Apart from the unstable higher order taxonomy, it has also become apparent that at least one
genus placed within the Murinae, Acomys, may not be part of this group [36, 40]. Recent DNA-
DNA hybridisation studies have suggested that it, together with Uranomys and Lophuromys,
form a monophyletic clade that clusters with the Gerbillinae [15, 30], whereas dental features of
Acomys and Uranomys appear to be intermediate between this group and the Murinae [16].
Since the taxonomy and phylogenetic relationships between these major groups of African
muroids is controversial, it would seem timely to explore the use of other data sets for
hypothesising relationships. Over the last few years spermatozoal morphology has been used with
varying degrees of success as an independent character for suggesting relationships between
various marsupials [22, 24, 38], bats [23], American cricetids [27], and Asian and Australasian
murids [5, 7, 8, 10]. Here it is used to explore relationships between the subfamilies of African
murids and, in particular, between members of the Murinae, Dendromyinae, Gerbillinae,
Cricetomyinae and Otomyinae subfamilies. As an outgroup comparison, the sperm morphology
of Mystromys (an archaic African species usually placed in the Mystromyinae) is considered. To
date there have been only a few investigations on morphology of spermatozoa of African murid
rodents [e.g. 1, 3, 6, 21, 39], and there appear to be no studies using it to determine the
relationships between the murid subfamilies. This is the aim of the present study.
MATERIAL AND METHODS
Sperm morphology was investigaied by light (LM) and. where possible, transmission electron microscopy (TEM)
from: Mystromys alba (subfamily Mystromyinae), Prionomys batesi, Stcatomys parvus, Dendromus mystacalis, D.
mesomelas, Deomys ferrugineus, and Malaeothrix typica (subfamily Dendromurinae), Saccostomus campestris (subfamily
Cricetomyinae), Otomys irroratus (subfamily Otomyinae), and Gerbillurus paeba and Tatera leucogaster (subfamily
Gerbillinae). The sources of animals were: M. alba and S. campestris from the South African Institute for Medical
Research, Sandringham, Johannesburg, one O. irroratus from near Pietermaritzburg and another from Ngare Forest, Natal
Province. G. paeba came from near Swakopmond, and a T. leucogaster from Etosha pan, Namibia. A second T. leucogaster
was collected in northern Quazulu. The M. typica specimen came from near Hutchinson, Cape Province (Transvaal Museum
No 36676), and material from the other dendromurines was from specimens at the Natural History Museum, London which
included D. ferrugineus from Kivu Province. Zaire. D. mystacalis from Babaka, Isai River, Huri, Zaire, D. mesomelas from
Uganda, and S. parvus from Swaziland.
Caudae epididymides were immersed in either 10% phosphate buffered formaldehyde or 3% paraformaldehyde/3%
glutaraldehyde made up in 0.1 M phosphate buffer, pH 7.4. For LM, spermatozoa were extruded from the ducts and placed
on microscope slides. For TEM the tissue was rinsed in phosphate buffer, fixed in osmium tetroxide, rinsed again
dehydrated by passing through a graded ethanol series, and embedded in Araldite. Thick and thin plastic sections were cut.'
the latter stained with lead citrate and uranyl nitrate, and observed with a JEOL 100S and/or Philips CM 100 EM.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
411
Fig. I
— a-g: Sperm of Myslromys alba, a, b: Head is falciform, c: An electron-dense nucleus (N) is capped by a large
acrosomc which has a narrow equatorial (ES) and wider principal segment (PS); d: a postacrosomal dense lamina
(PADL) occurs in the posterior region of sperm head; e, f: anterior region of nucleus is bilaterally flattened and
capped by a massive apical acrosome segment; g: TS of rostral tip indicates perforatorium (P) surrounded by
acrosomc (Ac) except midventrally. a. x 8 200; b, x 900; c. x 19 300; d. x 20 100; e. x 17 100; I.
x 26 800; g, x 97 000.
Source : MNHN . Paris
412
W. G. BREED : AFRICAN MUR! DAE I MAMMALIA )
RESULTS
Terminology used for the planes of section follows that of LALLI & CLERMONT [25]; thus
in a falciform sperm head the convex surface is referred to as dorsal and concave as ventral. The
space between the inner acrosomal membrane and outer nuclear envelope is the subacrosomal
space and, when extensive, the perforatorium. The taxonomy proposed by CARLETON &
MUSSER [12] is, in general, used although the family name of Cricetidae [37] is also referred to.
Mystromyinae
Mystromys alba. The sperm head is falciform with a length of 8-9 |im, maximum breadth of
only 1.5 |im, hook length of 5 (im, and connecting piece of tail attaches midbasally with
midpiece length of 38 jim and principal and end piece of about 100 pm (Fig. la, b).
The bilaterally flattened head has a homogeneous, electron-dense, nucleus that passes into
the apical hook (Fig. la) which is capped by an acrosome that has a thicker principal than
equatorial segment caudal to which is a postacrosomal dense lamina (Fig. lc, d).
In the anterior region of the sperm head there is a massive apical acrosomal segment that
extends at least 1 pm beyond the convex nuclear margin. The acrosomal matrix is more electron-
dense medially (Fig. le, f), and anterior to the convex nuclear surface, beneath the inner
acrosomal membrane, a subacrosomal space with electron-dense material is present (Fig. If).
The acrosome and perforatorium extend beyond the rostral tip of the nucleus. Cross
sections demonstrate that the acrosome is bilaterally flattened and surrounds the perforatorium,
except mid ventrally (Fig. lg); the perforatorium is spear-shaped in cross section and the anterior
region of acrosomal segment widens slightly before tapering apically (Fig. lg).
Gerbillinae
Gerbillurus paeba. Sagittal (Fig. 2a, b) and frontal (Fig. 2c) TEM sections of the sperm
head show that it is broad basally and narrows apically. The connecting piece of the sperm tail
attaches to an off-centre basal position. There is a large apical acrosomal segment with a narrower
principal segment over the anterior part of the nucleus posterior to which the equatorial segment
passes down over much of the convex margin of the nucleus with the postacrosomal dense lamina
passing around the posterior caudal margin (Fig. 2a). On the concave surface the equatorial
segment is restricted anteriorly (Fig. 2b). Frontal sections through the anterior region of the sperm
head indicate an invagination of the inner acrosomal membrane within which electron-dense
material of the subacrosomal space occurs (Fig. 2c).
Taler a leucogaster. The sperm morphology of this species is highly divergent. Longitudinal
TEM sections indicate that sperm head is bilaterally flattened (Fig. 2d), and has a deep
invagination posteriorly (Fig. 2d, e, f). The nucleus tapers apically and its anterior four-fifths is
capped by a nearly symmetrical acrosome (Fig. 2d) most of which is composed of a principal
segment with the equatorial segment forming a girdle around the posterior nuclear region. The
postacrosomal dense lamina is very short. A modest subacrosomal space is present with a small
apical extension (Fig. 2d). A complex structural organisation of the connecting piece occurs with
the basal plate running parallel to the long axis of the sperm head, and laterally an extension of the
nuclear envelope within which material that exhibits light and dark banding occurs (Fig. 2f). The
capitulum of the connecting piece passes vertically up into the implantation fossa and narrows
apically (Fig. 2f).
Source : MNHN , Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
413
FlG. 2. — Gerbillurus paeba and Tatera leucogasier. a-c: Sperm of G. paeba. a: Sperm head narrows apicaliy.
b, c: anterior to the nucleus (N) there is a perforatorium (P) covered by part of the acrosome (Ac),
d-f: T. leucogasier. d: spermatozoa have bilaterally flattened nuclei capped by a nearly symmetrical acrosome
with small equatorial segment (arrow), e, f: connecting piece is unusual in having vertically projecting basal
plate and partly bordered by fold of nuclear envelope (NE). a, x 9 100; b, x 15 000; c, x 31 600; d, x 18 200;
e, x 11 200; f. x 17 400.
Source : MNHN. Paris
414
W. G. BREED : AFRICAN MURIDAE ( MAMMALIA )
Cricetomyinae
Saccostomus campestris. The sperm head is falciform, has a length of 7 |im, maximum
breadth basally of 3 pm, and apical hook, which curves sharply caudad, of about 6 pm (Fig.
3a). The sperm tail connects to the midbasal region, and has a midpiece length of about 25 pm
and principal and end piece of about 100 pm.
The acrosome is largely restricted to the convex (Fig. 3b, c) and upper lateral surfaces of the
sperm head with the posterior region forming a narrow equatorial segment (Fig. 3e, f). Anteriorly
the inner acrosomal membrane is invaginated medially within which electron-dense material of the
subacrosomal space occurs (Fig. 3f). Transverse sections of the apical hook show that basally it
is bilaterally flattened and contains a nuclear extension which is completely surrounded by the
acrosome except midventrally (Fig. 3g). Most of the hook does not contain nuclear material but
the acrosome passes along its length and distally becomes triangular in cross section (Fig. 3h, i).
In addition, within the apical hook, a small midventral extension of the subacrosomal space
occurs which is in contact with the plasmalemma (Fig. 3h, i).
Dendromurinae
Prionomys batesi, Steatomys parvus, Dendromus mesomelas, D. mystacalis, Malacothrix
typica and Deomys ferrugineus. Only material from M. typica was available for TEM. LM
indicates significant interspecific differences in sperm head morphology. In P. batesi and
S. parvus a small triangular sperm head is evident that is broadest basally and tapers apically to a
hook that is flexed caudad. The connecting piece of the tail attaches midbasally (Fig. 4a, d).
Sperm heads of D. mystacalis and D. mesomelas (Fig. 4b, c) are similar in form, have a lateral
face of about 2 pm in diameter, and a 3-5 pm hook that extends from the convex surface and
projects caudally, and on the ventral margin a small anteriorly-projecting spike. The sperm tail
attaches off-centre basally and is about 90 pm long.
The third sperm type of Deomys ferrugineus (Fig. 4e) has a very small, 5 pm, bilaterally
flattened head that lacks an apical hook. Anteriorly there appears to be a somewhat ridged, cap¬
like structure, presumably the acrosomal region, and posteriorly the sperm head tapers a little
towards the connecting piece of the sperm tail which has a midpiece of 22 pm and principal and
end piece of about 70 pm.
TEM of Malacothrix typica sperm shows that its head is largely composed of an electron-
dense nucleus which does not appear to extend into the long apical hook (Fig. 4g, h). The
acrosome is present along much of the convex surface and makes up most of the material in the
apical hook which becomes triangular in cross section. There is a typical subacrosomal space
between the inner acrosomal membrane and outer nuclear envelope that extends into the apical
hook and is in contact with the plasmalemma midventrally.
FiG. 3. — Sperm ol Saccosiomus campestris. a, b: Sperm head is falciform with midbasal attachment of tail,
b, c: apical hook is long and largely composed of acrosome (Ac), d: posterior to the acrosome is the
postacrosomal dense lamina (PADL). e: acrosome has a very short equatorial segment (ES). f: frontal sections
show perforatorium extends apically. g: TS through apical hook shows nucleus basally distal to which the hook
becomes triangular and composed of acrosome except for small perforatorial extension, h, i: small perforatorial
extension, a, x 900; b, x 1 1 400; c, x 17 000; d, x 26 600; e, x 33 000; f, x 35 800; g, x 22 700; h.
x 50 100; i x 25 300.
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
415
Source : MNHN. Paris
416
W. G. BREED : AFRICAN MURIDAE I MAMMALIA >
Otomyinae
Otomys irroratus. Light microscopy indicates that the sperm head is falciform with the
connecting piece of the sperm tail attached on the lower concave surface (Fig. 4f).
TEM shows that a typical bilaterally flattened, electron-dense, nucleus extends into the
apical hook which is triangular in cross section. Within the hook the acrosome splits into a dorsal
and dorsolateral crest over the convex region and a much smaller, flat, head cap segment close to
the ventral margin. Most of the material in the apical hook is composed of the perforatorium that
has three prongs, one that is close to the medial convex margin and the other two, much larger
ones, which occur ventrolaterally.
Fig. 4. — Sperm from dendromurine and otomyine species, light microscopy, a: Prionomys bales:; b: Dendromus
mystacalis\ c: Dendromus mesomelas\ d: Steaiomys parvus ; e: Deomys fefrugineus ; f: Oiomys irroratus.
g, h: Malacothrix typica. TEM of sperm head, x 700; b, x 1 600; c, x 1 600; d. x 700; e, x 1 100; f,
x 600; g. x 9 100; h, x 1 1 000. a-d, phase contrast; e, f, Nomarski optics.
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
417
DISCUSSION
Most eutherians have a spatulate sperm head nucleus that is capped by an acrosome of
variable size, but in most murine rodents the sperm head is falciform with the tail attached to the
lower concave surface [6-10, 20]; there is a very large and elaborate apical perforatorium which
passes back over the anterior nuclear region as three prongs [25, 33], The sperm head of the
golden hamster (a member of the Cricetinae) is also falciform, but its ultrastructure is very
different with the acrosome occurring as a large cap over the dorsal and upper lateral nuclear
surface, a bilaterally flattened apical hook, and the sperm tail attached to the head midbasally [19,
29, 41]. The ultrastructure of the sperm head of Mystromys alba in general resembles that of the
golden hamster except that the sperm head is a little narrower and there is a much larger apical
acrosomal segment.
In Saccostomus campestris the ultrastructural characteristics of the acrosome,
perforatorium, and site of attachment of the connecting piece of the sperm tail are somewhat
similar to those of the golden hamster and Mystromys alba although the perforatorium and
acrosome are less extensive apically. This similar internal structural organisation in these three
species, two of which come from subfamilies outside the dendromurine-cricetomyine-otomyine-
murine clade, suggests very strongly that it represents the ancestral type for this group.
Amongst the dendromurines Deomys has sometimes been separated from the others and
even placed within its own subfamily [17]. The present data on sperm morphology support the
view that Deomys is highly divergent, whereas the ultrastructural characteristics of sperm head
and site of tail attachment of Malacothrix typica are similar to those of Saccostomus campestris.
The light microscopical observations suggest that 5. parvus and P. batesi sperm are also similar.
The ultrastructure of M. typica sperm does not resemble that of most of the murines [8, 9, 25]
which probably have a derived sperm type (see below). Thus there is no support for close affinity
of these groups from these data.
The position of the gerbils (subfamily Gerbillinae) has changed over the years. SIMPSON
[37] placed this subfamily within the Cricetidae, but subsequent biochemical data suggested that
the Gerbillinae could be the sister-group of the Murinae [13]. The present study indicates that the
spermatozoon structure of both Patera leucogaster and Gerbillurus paeba is highly derived and
quite unlike that of any other genus outside the Gerbillinae; no phylogenetic inferences can thus be
drawn.
Over the last 10 years it has become apparent that Acomys , previously thought to be a
typical murine, may not in fact be part of this large African murid group [36, 40]. It has
subsequently been suggested that Acomys, Uranomys and Lophuromys form a clade that is either
an early offshoot of the Murinae or closer to the gerbils [13, 15, 16]. The ultrastructure of the
sperm acrosome and perforatorium of both Acomys and Uranomys is more similar to that of
Saccostomus campestris and the golden hamster than to that of typical murines. However, unlike
sperm of S. campestris, dendromurines, and cricetines, the sperm tail attaches to the lower
concave surface of the sperm head similar to that of the falciform sperm of murine rodents [5],
This trait is thus shared between most members of the Murinae and Acomys-Uranomys to the
exclusion of the other groups except the Otomyinae [2], These data support the view that the
Acomys-Uranomys (and perhaps Lophuromys, which has a highly derived sperm type [6]) clade
may have branched off from the base of the murine-otomyine radiation [4, 16, 30]. Furthermore,
the present study extends the recent observations on the sperm of Otomys [2, 5] in confirming
that the head is falciform and showing that the structure of the acrosome and perforatorium, as
well as the site of attachment of the sperm tail, is identical to that of murines [1, 3, 5-10, 25],
These ultrastructural characteristics of the sperm head do not occur in species from any other
subfamily nor do they occur in Acomys and Uranomys. They are thus likely to be a
synapomorphic character shared by most members of the Otomyinae and Murinae to the exclusion
of Acomys and Uranomys and other subfamilies. The findings thus support the previous
418
W. G. BREED : AFRICAN MUR! DAE ( MAMMALIA )
suggestion [5] that these two subfamilies are sister-groups and that, in spite of a recent claim to
the contrary [35], the falciform sperm head is the ancestral condition within the Murinae.
The following tentative phylogenetic conclusions can be drawn from this study:
1 . Saccostomus is not part of the Murinae.
2. Within the Dendromurinae, Deomys is highly derived and may not be close to the
other members of this group.
3. The Dendromurinae is not particularly close to the Murinae.
4. The Otomyinae is the sister-group of the Murinae.
5. The Acomys-Uranomys lineage diverged from the base of the Murine-Otomyine
clade.
Clearly further insight into the phylogenetic relationships of African muroids could be
achieved by carrying out electron microscopy on spermatozoa of more species when, or if, they
become available; sperm from more genera of dendromurines and gerbillines need to be
investigated. In this study there was no material available from any of the Malagasy rats and mice
(subfamily Nesomyinae), maned rats (subfamily Lophiomyinae), or rock and climbing swamp
mice (subfamily Petromyscinae). Spermatozoal morphology of these species needs to be
investigated before a full appreciation of its significance to the understanding of the evolution of
the rats and mice of Africa can be ascertained.
ACKNOWLEDGEMENTS
I thank Prof. M. Isaacson and Ms. P. Hawkins of SAIMR, Prof. M. Perrin of Department of Zoology, University
of Natal, Pietermaritzburg, and Mr. G. Bronner of the Transvaal Museum, Pretoria for material. Ms. P. Jenkins of the
Natural History Museum, London, kindly allowed me access to the dendromurines. 1 should like to thank Mr. C. Leigh and
Mr. R. Murphy of the University of Adelaide for technical assistance, and Esther Breed for typing the manuscript.
Material for this study was obtained whilst the author was on study leave from The University of Adelaide.
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38. Temple-Smith, P. D., 1987. — Sperm structure and marsupial phylogeny. In: M. Archer. Possums and Opossums:
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Source : MNHN. Paris
Comparative Sperm Structure in Bats (Chiroptera):
Some Taxonomic and Adaptive Implications
Takayuki MORI
Zoological Laboratory,
Faculty of Agriculture, Kyushu University, 46-06, Fukuoka 812, Japan
ABSTRACT
In Megachiroptera, the spermatozoa of Pteropodidac have a wide spoon-shaped head, a large acrosome, a rounded nucleus
and a tail with a long midpiece. In Microchiroplera, the spermatozoa of Rhinolophidae have a spatulate head with a large
acrosome, being generally uniform in morphology at the specific level, but the midpicce varies in length with species. In
Vespertilionidae examined, two subfamilies of Vespertilioninae and Murininae are separable from another subfamily
Minioptcrinae in having a small acrosome and long midpiece. Furthermore, in the vespertilionine bats, greater uniformity
in sperm structure prevails in the tribe Myotini (Myotis) and the tribe Plecotini ( Plecotus and Barbas tellci) when compared
with the tribe Pipistrellini ( Pipistrellus , Nyctalus and Vesperiilio). The sperm nucleus of the tribe Myotini s. lat. ( Myotis ,
Plecotus and Barbastella ) is longer than that of Pipistrellini. and the midpiece is obviously distinctive at the generic and
specific levels. The midpiece in Myotis nattereri, Myotis macrodactylus and Pipistrellus abramus is longer than that in the
other vespertilionine bats examined, and their mitochondria are large in number and in size. The present study
demonstrates the potential usefulness of sperm morphology in taxonomic studies of Chiroptera. In addition, the presence
of a well-developed midpiece with a unique arrangement of abundant, large mitochondria in spermatozoa of hibernating
bats is explained as an adaptive phenomenon for prolonged survival of spermatozoa.
RESUME
Structure comparee du spermatozoide chez les Chauve-souris (Chiroptera): quelques implications
pour la taxonomie et Fadaptation
Chez les Megachiroptera, le spermatozoide des Pteropodidae a une tete large en forme de cuillere, un grand acrosome, un
noyau arrondi et une queue avec une piece intermediate longue. Chez les Microchiroplera, le spermatozoide des
Rhinolophidae a une tete spatuiee avec un grand acrosome qui est generalement de morphologie uniforme au niveau
specifique, mais la longueur de la piece intermediate varie selon les especcs. Chez les Vespertilionidae qui ont ete
examines, les deux sous-familles des Vespertilioninae et Murininae peuvent etre distinguecs de 1’ autre sous-famille
Minioptcrinae grace k leur petit acrosome et leur longue piece intermediate. De plus, chez les Vespertilioninae.
1’ uniformity de la structure du spermatozoide est plus profonde dans les tribus Myotini ( Myotis ) et Plecotini ( Plecotus et
Barbastella) que dans la tribu Pipistrellini ( Pipistrellus , Nyctalus and Vespertilio). Le noyau du spermatozoide dans la tribu
Myotini s. lat. ( Myotis , Plecotus et Barbastella) est plus long que celui des Pipistrellini, et la piece intermediate permet
de distinguer de maniere evidente les genres et les esp&ces. Chez Myotis nattereri, Myotis macrodactylus et Pipistrellus
abramus, la pi£ce intermediate est plus longue que celle des autres Vespertilioninae examines et les mitochondries sont
grandes et nombreuses. Cette £tude d£montre futility potentielle de la morphologie du spermatozoide pour les dtudes
taxonomiques sur les Chiroptera. De plus, la presence d’une pi£ce intermediate bicn developpee avec une disposition
originale de mitochondries grandes et abondantes chez les chauve-souris qui hibement est expliquee comme un phenomene
adaptatif pour prolonger la survie des spermatozo'ides.
Mori, T., 1995. — Comparative sperm structure in bats (Chiroptera): some taxonomic and adaptive implications.
In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (cds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem.
Mus. natn. Hist, nat., 166 : 421-429. Paris ISBN: 2-85653-225-X.
422
T. MORI : CH1ROPTERA ( MAMMALIA )
The phylogenetic system must be built up on conclusions obtained from various fields of
study. The morphology of spermatozoa is believed to be a good tool for better understanding of
phylogenetic relationships, because of their conservatism against environmental factors. In this
connection, there have been some light microscopical studies on the morphology of chiropteran
spermatozoa [2-5], On the other hand, electron microscopic studies have been made to analyse
sperm survival mechanisms in hibernating bats from the viewpoint of physiological adaptation
[12, 17, 19, 22], The ultrastructure of the sperm of pteropodids has also been described [18]. The
aim of the present study is to examine with the electron microscope the comparative morphology
of epididymal spermatozoa in 25 bat species (Table 1), and to discuss some taxonomic and
adaptive implications of sperm fine structure.
Table 1. — Species examined.
Fig. I. — Electron micrographs of sperm heads of bat species (a, frontal section; b, sagittal section). The sperm head of
Pteropodidae has a spoon-shaped head, a large acrosome (A) and a rounded nucleus (N). The spermatozoa of
Rhinolophidae have a spaiulate head with a large acrosome. In Vespertilionidae, two subfamilies of
Vespertilioninae and Murininae are separable from another subfamily Miniopterinae in having a small acrosome.
1: Pteropus dasymallus\ 2: Rhinolophus cornutus\ 3: Myotis macrpdactylus\ 4: Barbastella leucomelas\
5: Pipistrellus abramus\ 6: Vespertilio superans\ 7: Mur in a leucogaster, 8: Miniopterus schreibersii.
Source .
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
423
Source : MNHN. Paris
424
T. MORI : CH1ROPTERA ( MAMMALIA )
MATERIAL AND METHODS
The order Chiroptera is divided into two suborders; Megachiroptera, including a single family Pteropodidae, and
Microchiroptera, consisting of the remaining families. Bat specimens examined were as follows: four genera and four
species representing the family Pteropodidae, one genus and five species of the family Rhinolophidae, and eight genera
and sixteen species of the family Vespertilionidae (see Table 1). The cauda epididymidis of each species was fixed in cold
3% glutaraldehyde buffered with 0.1 M-phosphate buffer (PBS) at pH 7.4, then rinsed thoroughly with the same PBS and
postfixed in cold 1.3% osmium tetroxide in the PBS, dehydrated with alcohol and embedded in Epon 812 (Taab). Sections
(60 nm) were doubly stained with uranyl acetate and lead citrate before examination in an Hitachi HS-9 or an Hitachi H-
600A electron microscope (75 kV).
OBSERVATIONS
In Megachiroptera. the Pteropus dasymallus spermatozoon, when cut frontally, has a
dorsoventrally flattened, wide spoon-shaped head with convex lateral contours. The head is 6 (im
in length, of which the posterior 3.2 |im is occupied by the rounded nucleus, being about 3.4 (im
in width (Fig. I la). When cut sagittally, the nucleus has a convexity in the posterior region, and
the acrosome closely fits into an anterior concavity; thus, the nucleus appears rocket-shaped,
being thicker at the base, and tapering to the tip (Fig. I lb). The acrosome protrudes for a length
of 2.8 (im from the tip of the nucleus. The equatorial segment of the acrosome occupies the
posterior one-fourth of the nucleus. Thus, the postacrosomal region is shorter than that in
Microchiroptera (cf. Fig. I 2-8). The inner acrosomal membrane is serrated, while it is smooth in
Microchiroptera. The sperm tail is very long, because of the conspicuously long midpiece with a
length of 22.0 (im (Fig. II la, b). Mitochondria are large in number, but small in size (Fig. II la,
3a). The arrangement of mitochondria in Pteropus is similar to that in common mammalian
species, in which mitochondria are disposed in a helix with their tapering ends overlapping or
abutting end-to-end. The characteristics of the Pteropus dasymallus spermatozoon resemble those
of the other three genera examined in Pteropodidae (Fig. II lb).
In Rhinolophidae, of the Microchiroptera, the spermatozoon of Rhinolophus cornutus has a
dorsoventrally flattened, spatulate head with parallel sides and a narrow nucleus. The nucleus
occupies 4.1 pm of the head with a total length of 6.8 pm, being about 2.1 pm in width (Fig.
I 2a). When cut sagittally, the nucleus appears extremely slender and wedge-shaped, being
thicker at the base, and tapering to a point (Fig. I 2b). The large acrosome extends anteriorly
beyond the leading edge of the nucleus for a length of 2.7 pm. The equatorial segment of the
acrosome is situated at about the middle portion of the nucleus (Fig. I 2b). Thus, the
postacrosomal region is longer than that in Megachiroptera. The midpiece (12.0 pm) in this
species is very short compared with the midpiece (15.0 pm) with abundant mitochondria in
R. ferrumequinum (Fig. II 2a, d). In Rhinolophus , the sperm head is generally uniform in
morphology at the specific level, but the midpiece varies in length with the species (Fig. II 2a-e).
In Vespertilionidae, two subfamilies of Vespertilioninae and Murininae are separable from
the subfamily Miniopterinae in having a small acrosome and long midpiece. Furthermore, in the
vespertilionine bats examined, greater uniformity in sperm structure prevails in the tribe Myotini
( Myotis ) and the tribe Plecotini ( Plecotus and Barbastella) when compared with the tribe
Pipistrellini ( Pipistrellus , Nyctalus and Vespertilio). The sperm nucleus of the tribe Myotini ,v. /at.
(Myotis, Plecotus and Barbastella) is longer than that of Pipistrellini (Fig. I 3-6). For example,
the sperm nucleus occupies 4.9 pm of the head (5.1 pm in length) in Myotis macrodactylus, and
3.7 pm of the head (4.0 pm in length) in Pipistrellus abramus. At the specific level, the
spermatozoa of Pipistrellus endoi and P. javanicus have a narrower head and shorter midpiece
than the P. abramus spermatozoon.
In Vespertilionidae, the midpiece is clearly distinctive at the generic and specific levels. The
midpiece in Myotis nattereri, Myotis macrodactylus and P. abramus is longer than that in the other
vespertilionine bats examined, and their mitochondria are large in number and in size (Fig. II 3-
6). The arrangement of mitochondria is such that each turn of the flat helix is made up of just two
Source :
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
425
Fig. II. Semischematic reconstruction of the midpiece in bats, based on electron microscopic observations,
la: Pteropus dasymallus ; lb: Macroglossus minimus ; 2a: Rhinolophus cornutus ; 2b: Rhinolophus
monoceros ; 2c: Rhinolophus imaizumii ; 2d: Rhinolophus ferrumequinum\ 2e: Rhinolophus luctus;
3a: Myotis hosonoi ; 3b: Myotis nattereri\ 4: Plecotus auritus ; 5: Barbasiella leucomelas, 6a: Pipistrellus
endoi\ 6b: Pipistrellus javanicus\ 6c: Pipistrellus abramus ; 7: Murina leucogaster ; 8: Miniopterus
schreibersii.
large mitochondria whose end-to-end junctions are directly opposite on the dorsal and ventral
aspects of the midpiece (Fig. Ill b, c). The two opposite mitochondria are conspicuously thick
and ovoidal in M. nattereri and M. macrodactylus (Fig. Ill b), and rhombiform in P. abramus
(Fig. Ill c).
In Murina leucogaster , in the Murininae, the sperm nucleus occupies 4.0 pm of the spatulate
head (4.7 pm in length), and is about 2.0 pm in width (Fig. I 7a), being wedge-shaped in sagittal
section and surmounted by a small acrosome (Fig. I 7b). The spermatozoon of this species
resembles that of Myotis in morphology and in size. In Miniopterus schreibersii , in the
426
T. MORI : CHIROPTERA (MAMMALIA)
Fig. III. — Electron micrographs of the midpiece (transverse section), a: Pteropus dasymallus ; b: Myotis nattereri\
c: Pipistrellus abramus'y d: Miniopterus schreibersii. In Myotis nattereri and Pipistrellus abramus , there are just
two large mitochondria to each turn of the sheath: the end-to-end junctions of mitochondria are in line along the
dorsal and ventral aspects of the midpiece.
Miniopterinae, the spermatozoon has a large, spatulate head. The nucleus occupies 5.0 pm of the
head (8.0 pm in length), being about 2.8 pm in width (Fig. I 8a). When cut sagittally, the head is
lanceolate, tapering to a tip (Fig. I 8b). The large acrosome, tipped with a swelling, protrudes
anteriorly beyond the leading edge of the nucleus for a length of 3.0 pm. The midpiece in this
species is very short compared with that in the other subfamilies examined, and mitochondria are
conspicuously small in number and in size (Figs. II 8, 3d).
DISCUSSION
The spermatozoa of the two subfamilies examined, Pteropodinae and Macroglossinae in the
Pteropodidae, belonging to the Megachiroptera, are similar in possessing a flattened, wide spoon¬
shaped head with convex lateral contours, a large acrosome, a rounded nucleus and a tail with a
long midpiece. The fact that Pteropodidae share such similarities in sperm morphology with
Soricidae which is a group of the basic insectivores retaining primitive characters within
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
427
eutherians, strongly supported MILLER'S concept [11] that Megachiroptera should be allocated to
the lowest taxon within the Chiroptera from the viewpoint of adaptation for flight.
In Microchiroptera, the spermatozoon of Emballonura furax, belonging to the
Emballonuridae, has a spatulate head according to the figure presented [6], although the authors
do not refer to such a morphological character. The spermatozoa of Phyllostomatidae have a broad
head as well as those of Megachiroptera, and are reasonably constant at the generic and specific
levels [3], differing from those of Rhinolophidae and Vespertilionidae in morphology. In
Rhinolophus, the sperm head, with a large acrosome. is uniform in morphology at the specific
level. In R. ferrumequinum, the midpiece, with abundant mitochondria, is very long compared
with the other four species examined. This species copulates in autumn, and the spermatozoa are
stored in the female reproductive tract until the next spring (sperm storage type) [19]. Thus, the
well-developed midpiece in R. ferrumequinum may be an adaptation for prolonged survival of the
spermatozoa. The reason why the midpiece in the other four species of Rhinolophus is shorter
than that in R. ferrumequinum is unknown as their reproductive patterns have not yet been
examined in detail.
It has been suspected on the basis of morphological characters that the vespertilionid
ancestor was a Myotis- like bat. According to ANDO [1], the karyotypes of Myotis are
characterized by having a prototypic karyotype for the Vespertilionidae, and the karyotypes of
Myotini and Plecotini (Myotini .v. lat.), Pipistrellini and Nycticeini seem to be derived from the
ancestral karyotype of the Vespertilionidae. As to Myotis, the prototypic karyotype is said to have
remained unchanged at or even after divergence, and consequently their karyotypes resemble each
other at the specific level. Also from sperm morphology, the spermatozoa of Myotis are similar in
general structure at the specific level, showing a typical structure in Vespertilionidae in possessing
a spatulate head and small acrosome. The morphology of the sperm heads is consistent in Myotini
(5. lat.), thus being in good agreement with the phylogenetic relationships. However, there are
great differences in number and in size of mitochondria; for example, the midpiece in Myotis
macrodactylus, M. nattereri and M. lucifugus is very long, and their mitochondria are large in
number and in size compared with the other three species of Myotis. It seems to be an adaptive
phenomenon for prolonged sperm survival that the well-developed midpiece is present in
M. lucifugus in which clear evidence has been obtained for the prolonged fertilizing life of the
spermatozoon [21].
In Pipistrellini, taking distinct differences with regard to the karyotype and the baculum
morphology between Pipistrellus abramus abramus and P. javanicus javanicus into consideration,
they should be separated as a good species from each other, although they have been classified at
a subspecific level, P . a. abramus and P. a. javanicus [1], This assumption of specific
distinctness is supported also from differences in fine structure of spermatozoa, particularly in
number and in size of mitochondria. Further, the well-developed mitochondrial sheath in
P. pipistrellus [16, 17] and P. abramus [7, 8, 20] with unusual reproductive habits (sperm
storage type) may be concerned with an adaptation for prolonged survival of spermatozoa, as in
M. lucifugus mentioned above. The sperm nucleus of Pipistrellini ( Pipistrellus , Nyctalus and
Vespertilio) is shorter than that of Myotini ( s . lat.), and the above three genera are consistent in
sperm morphology. As for Murininae, the subfamily has been regarded as a conservative taxon
with primitive characters, being Myotini-like in sperm morphology.
In Miniopterus schreibersii, in the Miniopterinae, copulation is followed closely by
ovulation in mid-October [12-15], Embryonic development, however, proceeds very slowly
during the long hibernation period (delayed implantation type) [9], Correspondingly, the
M. schreibersii spermatozoon differ conspicuously from the other subfamilies examined
(Vespertilioninae, Murininae) in having a spatulate head surmounted by the remarkably large
acrosome, and a very short midpiece whose mitochondria are conspicuously small in number and
in size, compared with the other subfamilies. Such great differences in sperm morphology clearly
reveal subfamilial distinctness. On the basis of the dental formula [10], it has been suggested that
428
T. MORI : CH1ROPTERA ( MAMMALIA )
the genus Miniopterus be placed in a separate family Miniopteridae. The fact that the
spermatozoon has the very short midpiece, mentioned above, may be associated with the fact that
the spermatozoon does not need to retain its viability for as long as 5-7 months unlike the sperm
storage type; thus, M. schreibersii is in a striking contrast to bats belonging to the sperm storage
type with a well-developed midpiece.
In conclusion, the present study has demonstrated the potential usefulness of sperm
morphology in taxonomic studies of Chiroptera. The presence of a well-developed midpiece
(unique arrangement of abundant, large mitochondria) of spermatozoa in hibernating bats is
explained as an adaptive phenomenon for prolonged survival of spermatozoa, and the details of
the reproductive pattern in bats yet to be examined are needed to analyse adaptive implications of
sperm morphology.
ACKNOWLEDGEMENTS
The author thanks Emeritus Professor Teru Aki Uchida of Kyushu University for comments on the manuscript. I am
indebted also to Professor Koichi Ando of Kyushu Sangyo University for collecting the male reproductive organs of
Indonesian and Taiwanese bats.
REFERENCES
1. ANDO, K., 1982. — Karyotypic evolution and its phylotaxonomic implication in Chiroptera. Thesis, Kyushu
University, Japan: 1- 359 (in Japanese with English summary).
2. Bishop, M. H. W. & Austin, C. R., 1957. — Mammalian spermatozoa. Endeavour , 16: 137-150.
3. Forman, G. L., 1968. — Comparative gross morphology of spermatozoa of two families of North American bats.
University of Kansas Science Bulletin, 47: 901-928.
4. Forman, G. L., Baker R. J. & Gerber J. D., 1968. — Comments on systematic status of vampire bats (Family
Desmodontidae). Systematic Zoology, 17: 417-425.
5. Mirth, H. F., 1960. — The spermatozoa of some North American bats and rodents. Journal of Morphology. 106:
77-83.
6. HILL, J. E. & Smith, J. D., 1984. — Bats : a Natural History. Austin, University of Texas Press: 1-243
7. Hiraiwa, Y. K. & Uchida, T. A., 1956a. — Fertilization in the bat, Pipistrellus abramus abramus (Temmink). III.
Fertilizing capacity of spermatozoa stored in the uterus after the copulation in the fall. Science Bulletin of the
Faculty of Agriculture, Kyushu University , 15: 565-574. (in Japanese with English summary).
8. Hiraiwa, Y. K. & Uchida, T. A.. 1956b. — Fertilization in the bat, Pipistrellus abramus. A successful example of
artificial insemination with epididymal spermatozoa in autumn. Science, Tokyo , 26: 535. (in Japanese).
9. Kimura, K. & Uchida, T. A., 1983. — Ultrastructural observations of delayed implantation in the Japanese long¬
fingered bat, Miniopterus schreibersii fuliginosus. Journal of Reproduction and Fertility , 69: 187-193.
10. Mein, P. & Tupinier, Y. 1977. — Formule dentaire et position syst6matique du Minioptere (Mammalia, Chiroptera).
Mammalia, 41: 207-211.
1 1 . Miller. G. S., 1907. — The families and genera of bats. Bulletin of United States National Museum of Washington’.
1-282.
12. Mori, T. & Uchida, T. A., 1980. — Sperm storage in the reproductive tract of the female Japanese long-fingered
bat, Miniopterus schreibersii fuliginosus. Journal of Reproduction and Fertility, 58: 429-433.
13. Mori, T. & Uchida, T. A., 1981a. — Ultrastructural observations of fertilization in the Japanese long-fingered bat,
Miniopterus schreibersii fuliginosus. Journal of Reproduction and Fertility. 63: 231-235.
14. Mori, T. & Uchida. T. A., 1981b. — Ultrastructural observations of ovulation in the Japanese long-fingered bat,
Miniopterus schreibersii fuliginosus. Journal of Reproduction and Fertility, 63: 391-395.
15. Mori, T. & Uchida, T. A., 1982. — Changes in the morphology and behaviour of spermatozoa between copulation
and fertilization in the Japanese long-fingered bat, Miniopterus schreibersii fuliginosus. Journal of
Reproduction and Fertility , 65: 23-28.
16. Racey, P. A., 1973. — The viability of spermatozoa after prolonged storage by male and female European bats.
Periodicum Biologorum, 75: 201-205.
1 7. Racey, P. A. & Potts, D. M., 1970. — Relationship between stored spermatozoa and the uterine epithelium in the
pipistrelle bat (Pipistrellus pipistrellus). Journal of Reproduction and Fertility \ 22: 57-63.
18. Rouse, G. W. & Robson, S. K., 1986. — An ultrastructural study of megachi ropteran (Mammalia: Chiroptera)
spermatozoa: implications for chiropteran phylogeny. Journal of submicroscopic Cytology 18: 137-152.
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
429
19. Uchida, T. A. & Mori, T., 1987. — Prolonged storage of spermatozoa in hibernating bats. In: M. B. Fenton, P.
Racey & J. M. V. Rayner, Recent Advances in the Study of Bats. Cambridge, Cambridge University Press: 241-
255.
20. Uchida, T. A., Mori, T. & Son, S. W., 1988. — Delayed capacitation of sperm in the Japanese house bat,
Pipistrellus abramus. Journal of the Mammalogical Society of Japan, 13: 1-10.
21 . Wimsatt, W. A., 1944. — Further studies on the survival of spermatozoa in the female reproductive tract of the bat.
Anatomical Record , 88: 193-204.
22. Wimsatt, W. A., Krutzsch, P. H. & Napolitano, L. 1966. — Studies on sperm survival mechanisms in the female
reproductive tract of hibernating bats. I. Cytology and ultra-structure of intrauterine spermatozoa in Myotis
lucifugus. American Journal of Anatomy, 119: 25-60.
Source : MNHN, Paris
Source : MNHN , Paris
Molecular and Ontogenic Analysis
of the Human Sperm Tail Fibrous Sheath
Ali J AS SIM
Department of Immunology,
London Hospital Medical College, Turner Street, London El 2AD, United Kingdom
ABSTRACT
In the past few years, a considerable amount of data has been obtained regarding the biochemical and antigenic structure
of the human sperm tail fibrous sheath (FS). The development of a method for the FS isolation enabled its biochemical
characterization, and several polypeptides with MW ranging between 25 and 97 kDa were identified. These were
antigenic, and their dissection with monoclonal antibodies (MoAbs) showed the presence of bioactive groups e.g.
phosphates and sugars. The bioactive groups were incorporated into the proteins late in spermatogenesis, following the
FS assembly, as additional steps of post-translational modifications; this ensures structural maturity of the sperm tails
prior to the sperm release to the epididymis. This chapter is a description of these results and their significance.
RESUME
Analyse moleculaire et ontogenetique de la gaine fibreuse de la queue du spermatozoide humain
Au cours des dernieres ann6e$, une quantite considerable d’ informations a ete obtenue a propos de la structure
biochimique et antigenique de la gaine fibreuse de la queue du spermatozoide humain (GF). Le developpement d’une methode
d' isolation de la gaine fibreuse a pcrmis sa caracterisation biochimique, et plusieurs polypeptides, de poids moleculaire
entre 25 et 97 kDa ont et6 identifies. Ces polypeptides sonl antigeniques. et leur analyse grace a des anticorps
monoclonaux a montre la presence de groupes biologiquement actifs tels que des phosphates et des sucres. Les groupes
biologiquemenl actifs sont incorpores dans les proteines & un stade tardif de la spermatogenese, apres 1’ assemblage de la
GF, grace a des etapes additionnelles de modifications post-traductionnelles. Ceci garantit la maturity structurale de la
queue du spermatozoide avant la liberation du sperme dans Tepididyme. Ce chapitre dccrit ces r£sultats et leur signification.
The fibrous sheath (FS) is a unique cytoskeletal structure of the sperm tail which occupies
the principal piece. Its two longitudinal columns run dorso-ventrally along the outer dense fibres
numbers 3 and 8, and terminate by attaching to axonemal microtubular doublets numbers 3 and 8.
These columns are interconnected by large numbers of transverse ribs [9]. During
spermiogenesis, the FS assembly of the human spermatozoon begins at the fourth Sc stage
spermatid by the formation of a network of filamentous tubules [8] which are later obliterated
through the deposition of electron dense granular material.
Until recently, nothing was known of the molecular or antigenic structure of the human
sperm tail FS despite its involvement in various flagellar abnormalities which are associated with
sperm immotility and infertility [6, 18, 19, 21]. Molecular studies, therefore, are important to
JASSIM, A.. 1995. — Molecular and ontogenic analysis of the human sperm tail fibrous sheath. In: Jamieson,
B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist,
nat., 166 : 431-436. Paris ISBN : 2-85653-225-X.
432
A. JASSIM : HUMAN FIBROUS SHEATH
understand the biological significance of the FS in sperm motility and to unravel the molecular
basis for its anomalous formation. Furthermore, an understanding of the protein synthesis and
assembly could lead to the development of new methods of contraception through identifying
reagents capable of disrupting these processes without affecting other tissues. Therefore, in the
past few years, the main interest of the author was focused on this subject, and the results of these
studies are described below.
RESULTS AND DISCUSSION
Biochemical and antigenic analysis of the FS
The development of a method for isolating highly purified human FS preparations enabled
biochemical analysis of the FS [22]. As in rodents [5, 10, 26], the SDS-PAGE of human
preparations revealed several major protein bands (Fig. 1 ) with MW ranging between 25 and
97 kDa [22]. The presence of several polypeptide bands is a reflection of the FS structural
complexity, although some of these peptides could be degradation products. It is possible that
different proteins are utilized in the construction of the various FS components such as the
transverse ribs and longitudinal columns, which develop independently [13] and are made up of
different sized tubules [9, 28], and the electron dense material deposited in these filaments. In this
respect, the FS is similar to neurofilaments whose structure involves the use of three different but
closely related polypeptides which are encoded by three genes [30]. However, amino acid
sequencing of the FS proteins is essential to determine any sequence homology.
Fig 1. — SDS-PAGE of purified human sperm fibrous
sheath (FS) obtained from four normozoospermic
donors showing several protein bands, lanes (b-
e); a, MW markers. From [22], reproduced by
permission of Human Reproduction .
The FS polypeptides are highly immunogenic and express antigenic epitopes which are not
shared by several somatic tissues. This was demonstrated by using mouse xenoantisera (MAFA)
which were raised against purified human sperm FS and stained seven major protein bands (Fig.
2) with MW ranging between 25 kDa and 97.4 kDa by Western blotting [23]. In humans,
MAFA did not react with other sperm tail cytoskeletal elements, but in rats, a cross-reaction
between the FS and outer dense fibre proteins was reported [26],
In addition to xenoantisera, MoAbs have also been used to investigate the FS antigens. AJ-
p97 was the first polypeptide to be identified using RT97 MoAb [14] which was later found to be
phosphorylated [20]. In rats, the 80 kDa phosphoprotein reported by amino acid analysis [5]
could be analogous to AJ-p97, although amino acid sequencing is required. Other MoAbs
included GDA-J/F3 which detected a non-collagenous asialo-glycoprotein [16], AJ-FS1 and AJ-
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
433
FS9 MoAbs which recognized several FS-specific antigens [15, 17]. However, unlike other
MoAbs, the AJ-FS9 detected masked antigens which required proteolytic enzymes treatment for
their exposition; this indicates that the FS assembly involves the deposition of proteins mutilayers
[15]. In Western blotting, the reaction of AJ-FS1 and AJ-FS9 with multiple protein bands did
suggest the presence of shared epitopes in these polypeptides and possibly some structural
homology. Another group of non-glycosylated human FS antigens has recently been described by
others using MoAbs [3], The alpha-spectrin [31] and actin previously identified in the human
sperm tail principal piece [11] are more likely to be involved in the subcytoplasmic membrane
cytoskeleton [4] than the FS structure.
a b
200 kD
Fig. 2. — a: Western blotting and immunostaining of
purified FS with MAFA xenoantisera; b: negative
control. From [23], reproduced by permission of
Journal of Reproductive Immunology.
During the immunogold EM investigations to localize the target antigens for GDA-J/F3,
RT97, AJ-FS1, AJ-FS9 and MAFA, the gold labelling was seen to be restricted to the outer FS
surface, i.e. the inner FS surface was not stained [14, 15, 17. 19, 23], This antigenic polarity of
the FS surfaces is determined by the locality of the antigenic epitopes which may incorporate
bioactive groups e.g. phosphates or sugars [20, 16]. The presence of these groups together with
the intimate association of the FS to the cytoplasmic membranes [24] may indicate some molecular
interaction between these two structures during sperm motility.
Ontogeny and post-translational modifications of the FS antigens
The monoclonal and polyclonal anti-FS antibodies were useful for assessing the ontogeny
of their target antigens during spermatogenesis. Thus, the restricted reaction of the GDA-J/F3,
RT97, AJ-FS 1 , AJ-FS9 and MAFA with the mature sperm tails and lack of staining of the earlier
germ cell stages indicated the late expression of the antigenic epitopes which followed the FS
assembly [14, 15, 17, 19, 23], These data are in contrast to those reported in mice [29] and rats
[7, 10, 27], In mice, for instance, the target antigen for K32 MoAb was first detected in the
cytoplasmic matrix of stage 14 and 15 spermatids which then became localized to the FS at stage
434
A. JASSIM : HUMAN FIBROUS SHEATH
16 [29]. This discrepancy could be due to the degree of maturation of the cells used in
immunization; the epididymal and/or testicular germ cells preparations of rodents are likely to
contain cells which are less mature than the ejaculated human sperm samples.
Following the human FS structural assembly, some of its proteins undergo post-
translational modifications including phosphorylation [20], glycosylation [16] and disulphide
bonding [2], The phosphorylation and glycosylation were demonstrated by using RT97 and
GDA-J/F3 MoAbs which recognized phosphorylated and glycosylated antigens respectively.
These two processes take place in the testis prior to the release of spermatozoa to the epididymis
and continue thereafter, whereas the disulphide linking of the FS protein occurs in the epididymis
[2], Although the biological importance of these post-translational modifications is yet to be
established, their late occurrence following the FS assembly indicates their involvement in the FS
molecular maturation to ensure a functional structure. Phosphorylation of the AJ-p97, for
instance, is thought to contribute to the elasticity of the FS through the creation of electrostatic
repulsion between the negatively-charged phosphate groups [20], whereas the FS rigidity could
be due to the S-S bonding of the FS proteins [2],
Fig. 3. — Illustration of the FS function. The attachment
of the longitudinal columns (LC) to the axonemal
microtubules (AM) ensures transmission of the
microtubule linear sliding thrust to the FS which
bends due to its spring-like features and produces
a wave. AN, Annulus; EP, PP, and MP, end-,
principal- and middle- piece of sperm tail; TR,
Transverse ribs.
The biological function of the FS and its analogy to intermediate filaments
Because of their failure to react with anti-keratin, -vimentin and -neurofilaments antibodies,
the human germ cells were previously reported to lack intermediate filaments [1, 12, 25],
However, despite the lack of the conventional antigenic determinants of intermediate filaments,
the FS still shares several features with these cytoskeletal elements including: tissue specificity of
its antigens [14-17], disulphide cross-linking [2], phosphorylation [20], and insolubility in
vanous detergents and chemicals [22], Furthermore, the FS develop as filamentous structures
although their lumens are obliterated during development [8], The diameters of the FS transverse
rib and longitudinal column tubules are 5-6 nm [28] and 15-20 nm respectively [9], whereas that
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
435
of the somatic intermediate filaments is 10-15 nm [30], All these features indicate that the FS
could represent a modified form of intermediate filament. The AJ-p97 which shares a number of
features with the neurofilaments including phosphorylation could, therefore, be the FS
intermediate filament protein [14, 20], However, amino acid and/or DNA sequencing is essential
to establish its relationship to other intermediate filaments.
Although sperm motility is attributed to the axonemal microtubules, the presence of an
additional structure is essential to convert the nonprogressive microtubular sliding into wavy
movement. This structure is likely to be the FS due to its anatomical and structural properties. The
FS is characterized by its spring-like morphology, rigidity and flexibility, and its location in a
confined space; its anterior end starts at the annulus whereas its two longitudinal columns
terminate posteriorly by attaching to microtubule doublets 3 and 8. This direct attachment to the
microtubules ensures the transmission of the microtubular sliding thrust to the FS, and because of
its presence in a confined space and spring-like features, the FS is likely to bend; consequently,
the linear movements of the microtubules are converted into waves (Fig. 3). During bending, the
proximation of the negatively-charged groups, e.g. the phosphates of AJ-p97, might ultimately
lead to electrostatic repulsion and reversal of the original act [20]. In ciliated epithelium, the lack
of the FS could be the reason for the inability of cilia to produce waves despite the presence of
other axonemal structures.
Current investigations
The lambda gtl 1 human testicular cDNA libraries have recently been screened with AJ-FS1
MoAb, and two clones identified; their DNA sequence is currently being investigated
(unpublished data). This is essential to assess any DNA sequence homology with other
cytoskeletal proteins within the sperm tail and somatic cells. Future transfection of suitable host
cells with the encoding genes could provide some clues to the synthesis and assembly of the FS
proteins. Similar work of amino acid and/or DNA sequencing should be carried out in other
species to evaluate their phylogenetic relationship.
ACKNOWLEDGEMENTS:
The Autoimmunity Charitable Trust is acknowledged for financial support.
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spermatozoa. Biology of Reproduction, 39: 169-182.
27. Oko, R. & Clermont, Y., 1989. — Light microscopic immunocytochemical study of fibrous sheath and outer dense
fiber formation in the rat spermatid. Anatomical Record. 225: 46-55.
28. Olson, G. E., Hamilton, D. W. & Fawcett, D. W., 1976. Isolation and characterisation of the fibrous sheath of rat
epididymal spermatozoa. Biology of Reproduction, 14: 517-530.
29. Sakai, Y., Koyama, Y.-I., Fujimoto, H., Nakamoto, T. & Yamashina, S., 1986. — Immunocytochemical study of
fibrous sheath formation in mouse spermiogenesis using a monoclonal antibody. Anatomical Record, 215:
119-126.
30. Steinert, P. M. & Roop, D. R.. 1988. — Molecular and cellular biology of intermediate filaments. Annual Review
of Biochememistry, 57: 593-625.
31. Virtanen, I., Badley, R. A., Paasivuo, R. & Lehto, V.-P., 1984. — Distinct cytoskeletal domains revealed in
sperm cells. Journal of Cell Biology. 99: 1083-1091.
Source : MNHN. Paris
Diversity of Avian Spermatozoa Ultrastructure
with Emphasis on the Members
of the Order Passeriformes
Lawrence D. KOEHLER
Department of Biology, Central Michigan University, Mt. Pleasant, MI 48859, USA
ABSTRACT
The spermatozoa of passerine birds have ultrastructural features which are common to the sperm of members of the other
orders of the class Aves. The sperm are filiform, the acrosomc is conical in shape, and the nucleus is about the same
diameter as the acrosomc and midpiece. The helical shape of the passerine sperm, the presence of a helical membrane
extending laterally from the acrosomc, the 9 peripheral dense fibres (accessory fibres) which extend from a juxtanuclear
body along the doublets of the axoneme, and the presence of a single elongated helical mitochondrion are
synapomorphies of the passerines. Since avian spermatozoa exhibit considerable variation in gross morphology and in
ultrastructure, these variations may be useful indicators of phylogenetic relationships. It is necessary to have details of
the fine structure in order to develop a tree of these taxonomic relationships. The Class Aves is divided into a number of
orders based on a number of features including morphology, and behaviour. The order Passeriformes contains 5712 (59%)
of the 9672 species of recent birds. Most of the passerine birds are small land dwelling birds that tend to exhibit similar
morphological features thus making their subdivision into categories below suborders difficult. This study compares some
of the ultrastructural differences in sperm of selected members of the order Passeriformes.
RESUME
Diversity de I’ultrastructure des spermatozoides d’Oiseaux, en particular des Passeriformes
Les spermatozoides des Oiseaux Passeriformes ont des caracteristiques ultrastructurales qui sont communes aux
spermatozoides des membres des autres ordres de la classe Aves. Les spermatozoides sont filiformes, Pacrosome est
conique et le noyau est approximativement du meme diametre que Pacrosome et la piece i n termed iai re. La forme helicoidale
du spermatozoide, la presence d’une membrane helicoidale qui s’etend lateralement a partir de Pacrosome, les 9 fibres
denses p£ripheriques (fibres accessoires) qui partent d'un corps juxtanucleaire le long des doublets de Paxoneme, et la
presence d’une mitochondrie unique, allongee et helicoidale sont des synapomorphfes des Passeriformes. Comme les
spermatozoides des Oiseaux montrent des variations considerables de morphologie generate et d' ultrastructure, ces
variations peuvent etre d’utiles indicateurs des relations phylogeniques. II est necessaire de connaitre les details de
P ultrastructure pour cr£er un arbre des relations taxonomiques. La classe Aves est divisSe en plusieurs ordres bases sur de
nombreux caractdres incluant la morphologie et le comportement. L’ordre des Passeriformes contient 5712 (59%) des
9672 esp&ces d'oiseaux recents. La plupart des Passeriformes sont de petits oiseaux terrestres qui tendent h montrer des
caracteristiques morphologiques similaires, ce qui rend difficile leur classification au niveau infraordinal. Cette etude
compare certaines des differences ultrastructurales des spermatozoides parmi des membres selectionnes de Pordre
Passeriformes.
Koehler, L. D., 1995. — Diversity of avian spermatozoa ultrastructure with emphasis on the members of the order
Passeriformes. In: Jamieson, B. G. M., Ausio. J.. & Justine, J.-L. (eds), Advances in Spermatozoal Phylogeny and
Taxonomy. Mem . Mus. natn. Hist, run., 166 : 437-444. Paris ISBN : 2-85653-225-X.
438
L. D. KOEHLER : AVES
About 200 million years ago, Pangaea was breaking up to form Laurasia and
Gondwanaland. The dominant terrestrial life forms were, it is believed, the dinosaurs. The
radiation of the birds probably began about this time. A number of traits are used to construct the
taxonomic and phylogenetic relationships between the various members of the class Aves, but it
has been difficult to resolve the taxonomic relationships. According to SIBLEY & AHLQUIST [32]
most passerines are small land dwelling birds that feed primarily on insects, seeds, fruit or nectar.
The morphology of the syrinx is used to separate the members of the suborders Tyranni and the
Passeri. As birds evolved many have undergone convergent evolution to allow distantly related
groups to cope with similar environmental demands while others have undergone divergent
evolution so that related groups may look very different. Homologies or similarities due to
common ancestor must be recognized to demonstrate taxonomic relationships. It has been known
since the works of BALLOWITZ [7] & RETZIUS [29, 30] that morphological differences are
exhibited by the spermatozoa of various members of the Class Aves. Each noted especially the
differences in the sperm of passerine and non-passerine sperm. More recent studies of
spermiotaxonomy and spermiocladistics [19] have been useful in resolving taxonomic
relationships in other groups of organisms.
Since avian sperm exhibit considerably morphological variability it will be useful to examine
the ultrastructure of sperm as it relates to the phylogeny. A number of studies have given
descriptions of the ultrastructure of spermatozoa of representatives of orders of non-passerine
birds. The literature contains a few reports of ultrastructure of passerine spermatids and sperm in
sections of testis. There is currently very little information on the ultrastructure of mature
passerine sperm. The Order Passeriformes contains approximately 59% of all of the species of
living birds, and the order is divided into two suborders and generally into 45 families [31, 32], It
is of interest to examine and compare the ultrastructure of the mature sperm of some representative
members. Reconstructing the phylogenetic history of members of the order is a major challenge.
In this paper I will follow the classification of SIBLEY et al. [32] in which the order Passeriformes
is divided into the Suborders Tyranni (= Suboscines; Oligomyodi) and Passeri (= Oscines;
Polymyodi). The suborder Passeri includes the parvorders Passerida (which includes three
superfamilies and 21 families. The suborder Corvida includes three superfamilies and 15 families.
MeFARLANE [23, 24] noted that the avian sperm exhibit considerable species variation in both
size and morphology, thus it is here considered that the structure of sperm should yield
phylogenetic information. ASA & PHILLIPS [3] described some differences between the oscines
and sub-oscines.
The sperms of non-passerine birds are generally nearly linear in the head region while the
sperms of passerine birds are helically coiled. Tripepi and Perotta [35] examined oscine
spermiogenesis and divided the oscines (Passeri) into two types based on the presence of a
granular body and the ratio of length of the nucleus to the length of the acrosome. The present
work will describe the morphological variation of the sperm of various passerine birds and will
compare these sperm with those of some non-passerine birds.
MATERIALS AND METHODS
Sperm specimen were obtained from various species of birds of the Order Passeriformes. All birds were from the
suborder Passer., and represented five different families. Species included Agelaius phoeniceus, Molothrus aier, Quiscalus
quiscuta, Sturnus vulgaris, Cardinahs cardinalis, and Passer domesticus. Male birds were obtained during the breeding
season, euthanased. and sections of the distal portion of the vas deferens and/or the seminal glomeruli were removed, fixed
in 2% glutaraldehyde in phosphate buffer, pH 7.2, post fixed in 1% osmium. For TEM specimen were dehydrated in
acetone, embedded, sectioned, stained with uranyl acetate and lead citrate, and examined with a Philips 300. For SEM the
fixed specimen were dehydrated, critical point dried sputter coated and examined with an AMRAY 1200.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
439
RESULTS
The sperm of each species is characterized by being long and cylindrical in overall
morphology, with the diameter of the acrosome, nucleus and midpiece being nearly the same
Table 1 gives the various features and measurements of the sperm.
The sperm possess a helical shape, especially in the region of the acrosome and nucleus
rnmcfo/m J ^ hehcal c°ding endin§ at the neck. The degree of coiling and the length between
turns of the helix is variable in sperm of different species. The sperm also possess a helical
membrane starting near the anterior tip of the acrosome and extending over the length of the
acrosome to the anterior end of the nucleus. The helical appearance of the midpiec! and tail
generally is due to the coiling of an elongated granular mass followed posteriorly by the
endpiehcendna Surroundmg the neck and midpiece, and extending posterioriy to the
The acrosome at the anterior end of the head is characterized by having an electron dense
core surrounded by a more electron lucent region. Considerable differences are noted in the length
tou the 1(fngth of the nucleus. In some species there is an anterior
nuclear fossa of variable depth to 1.1 pm. A perforatorium was not observed in any of the
The TCf °f the SpCrm ?° n0t appear t0 have typical distinct centrioles. The
lagella are composed of an axoneme having a 9+2 arrangement, surrounded by 9 dense
nuclei flbrJf exten.d,ng from the neck t0 the Posterior end of the principal piece. A posterior
ni, ! f do^s "ot aPPear to occur in the sperm of these passerines studied. Morphology of
passerine sperm differs greatly from that of the sperm of non-passerine birds.
Table 1. — The sperm of passerine birds exhibit considerable variation in size and
scanning and transmission electron micrographs are presented.
morphology. Measurements from
440
L. D. KOEHLER : AVES
Table 1, continued
DISCUSSION
General
The overall length of the sperm of passerine birds is highly variable, the extr’emes noted in
those studied are 48 pm for the Quiscalus quiscalus sperm to 285 pm for the Tachycineta
thalassina [24] sperm. The overall appearance ranges from having a helical structure only in the
acrosomal region to having a helical acrosome, nucleus and a helical midpiece.
The acrosome of all of the passerine birds in this study is a solid, helical apical body with a
helical membrane generally running from the acrosome tip to the posterior end of the acrosome.
The helical membrane is a lateral extension of the acrosome and forms a left handed helix. The
size of this helical membrane is highly variable. Helical membranes or helically elongate nuclei are
also recorded in a variety of invertebrates such as the cephalopod and gastropod molluscs [14],
chilopods [18] and leeches [12]. The overall length of the acrosome is highly variable ranging
from 2.5 pm in the sperm of Sturnus vulgarus to a long structure that dominates the head and is
several times as long as the nucleus in the sperm of M. crintus. The acrosome generally appears to
be covered by an outer acrosomal membrane which lies just beneath the plasma membrane. The
acrosomal matrix generally has two different components that can be recognized by their density
in the electron beam. The acrosomes have a dense inner core and a less dense outer cortex, there
is not a membrane or a space separating these regions. This is in contrast to the acrosome of the
domestic fowl sperm in which there is a dense outer cortex and a less dense inner core. The less
dense outer material is continuous with the space in the helical membrane. McFARLANE [24]
described membrane-limited vacuoles in the contents of the acrosome of C. carolinus. Vacuoles
were not observed in any sperm in the present study.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
441
Fig. 1
— Sperm of four species of passerine birds were fixed on filters, dehydrated, critical point dried, coated with gold-
palladium, and examined with a scanning electron microscope. The figure shows the acrosomal region. Note that
some sperm, have a nearly linear acrosomal axis while others have a spiral shaped axis; all have a helical
membrane, a: Quiscalus quiscula , Common grackle. x 8 200. b: Agelaius phoeniceus , Red-winged Blackbird,
x 10 500. c: Passer domesticus. House Sparrow, x 11 200. d: Cardinalis cardinalis , cardinal x 11 200.
Source : MNHN , Paris
442
L. D. KOEHLER : AVES
Nuclei
The nuclei of the sperm of passerine birds tend to be continuous with the helical shape of
the acrosome and midpiece; the wavelength of the nuclear axis is highly variable and is probably
characteristic of the family [22, 24], As an example, the nucleus of the sperm of Passer
domesticus makes one complete helical revolution over its total length. The nucleus is enclosed in
a nuclear envelope which is associated closely with the plasma membrane. The chromatin
condenses during spermiogenesis. In the mature sperm the nuclear material is rarely
homogeneous, usually containing a number of small irregular cavities, randomly distributed,
which appear to result from incomplete condensation of the chromatin.
In passerine sperm the nucleus may show a distinct helical shape. The nucleus of most non¬
passerine sperm is nearly straight although it may demonstrate a slight curvature, as in Gallus
domesticus , but it is not helically coiled.
The sperm of passerine birds may have a small anterior nuclear fossa, a cone shaped
depression into which the posterior extent of the acrosome extends. There are no endonuclear
canals present in passerine bird sperm.
Midpiece
The centriolar region has been called the juxtanuclear apparatus by SOTELO & TRUJILLO-
CENOZ [33],
The components in the neck of the passerine sperm are not easily distinguished. SOTELO &
Trujillo-CENOZ [33] gave a partial description of the ultrastructure during spermiogenesis for
the house sparrow. There are some modifications in the neck as the spermatids mature. Passerine
sperm generally appear to lack a proximal centriole. They have a juxtanuclear body which is
sometimes referred to as a modified distal centriole. This juxtanuclear body in the mature sparrow
sperm appears as a striated, inverted conical structure when viewed in longitudinal sections. In
cross sections the juxtanuclear body appears as irregular fibrils attached to the inner margins of
the accessory fibres. The axonemal axis does not extend into the juxtanuclear body.
The axoneme is composed of the classical 9+2 arrangement of microtubules, originating a
short distance posterior to the juxtanuclear body. The axoneme is surrounded by nine outer
accessory fibres. The presence of these accessory fibres in passerine sperm is a feature that
distinguishes them from the sperm of non-passerine birds. The accessory fibres have a tear drop
shape in cross section with the point toward the associated pair of axonemal microtubules. The
anterior ends of the accessory fibres form a connecting piece at the base of the nucleus, and
surround the juxtanuclear body [10, 21, 24, 25]. Nine extremely short accessory fibres have been
described in the sperm of certain primitive birds, located adjacent to the axoneme in the region of
the annulus. These primitive bird sperm also contain a dense fibrous sheath which is located
immediately posterior to the annulus and which surrounds the accessory fibres [5], Very short
accessory fibres have also been described in sperm of Galliformes [34] and the mallard duck [17],
These fibres are greatly reduced in both diameter and length when compared with the accessory
fibres ol passerine sperm. Outer accessory fibres are also found in the sperm of mammals [8, 26]
of I uatara [16], and in molluscan sperm [15]. In passerine birds the fibres are all of similar size
and shape whereas the fibres in molluscs are of various sizes in the same sperm and in reptiles the
doublets at position 3 and 8 are enlarged [16]. These outer accessory fibres in passerine sperm
appear to be relatively rigid. When living passerine sperm are observed in a physiological saline
or in water they appear to maintain a rigid straight morphology. The movement appears to be due
to the circular motion of the rigid spermatozoon.
^,SP1™1 ™ass 1S present in the anterior region of the midpiece of some passerine
sperm ft /, 21, 24, 33J. In sperm of some species this granular mass forms a spiral mass located
anterior to the nitochondnon, as observed in Passer domesticus, with the anterior end of the
mitochondrion being a few micrometres caudal to the nucleus. In contrast, in other species the
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
443
anterior end of the mitochondrion is at the neck and the granular mass is located around the
mitochondrion and the axoneme for a short distance.
All passerine sperm appear to have a single very elongated mitochondrion that may extend
almost to the end of the midpiece. In the mature sparrow sperm the mitochondrion extends nearly
to the end of the tail and the principal piece is nearly non-existent. The mitochondrion appears to
attach to the accessory fibres associated with the axoneme at each end. The mitochondrion shows
a gradual decrease in its diameter near the posterior end of the flagellum. The cristae 'in the
mitochondrion are tubular in form and show a random arrangement when sectioned material is
viewed.
An annulus was not observed in any of the passerine sperm studied. An annulus is present
during spermiogenesis in the grackle [21] but it has not been observed in the mature sperm. The
sperm of psittaci forms also lack an annulus [20] but they show a clearly demarcated midpiece-tail
junction. In the passerine sperm the posterior end of the midpiece is demarcated by the end of the
single helical mitochondrion.
If the sperm of passerine birds are compared with those of the non-passerine birds the
notable differences include the presence of the helical membrane in the passerines (representatives
in two orders of non-passerine birds do possess helical sperm, the Charadriiformes and
Procellariformes, but it is believed that this membrane arose independently of the membrane in
passerine sperm), the helical shape of the nucleus, the lack of a perforatorium , the lack of the
endonuclear canals, the lack of distinct centrioles, the presence of the nine accessory fibres
connected to a juxtanuclear apparatus and the lack of an annulus.
Passerine sperm differ significantly from the sperm of the non-passerines but an
ultrastructural examination of spermatozoa of many more passerine species is necessary to reveal
phylogenetic relationships between the passerine birds.
ACKNOWLEDGEMENTS
Supported in part by a grant from the Central Michigan University Faculty Research and Creative Endeavours
Committee.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
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I 1. Furieri. P., 1963. — La morfologia degli spermi di Fringilla coelebs L. e di Tarentola mauritanica L. studiata
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Source . MNHN. Paris
Proteins
Source : MNHN. Parts
Source : MNHN. Paris
Histone HI and the Evolution of the
Nuclear Sperm-Specific Proteins
Juan AUSIO
Department of Biochemistry and Microbiology,
University of Victoria, Victoria, British Columbia, V8W 3P6, Canada
ABSTRACT
The chromosomal proteins of the sperm exhibit an enormous structural variability. During the last ten years, important
progress has been made in the chemical and physical characterization of these proteins over a wide range of organisms.
The data emerging from these studies indicate that the nuclear sperm-specific proteins are not as heterogeneous as
originally thought. Indeed, from the compositional point of view they can be arranged in a discrete number of basic types:
H Type (histone), P Type (protamine) and PL Type (protamine-like) consisting of proteins with an intermediate
composition between histones and protamines. Research on the PL Type has been carried out mainly in molluscs. This
taxonomic group is of special interest because it contains organisms with nuclear sperm-specific proteins that can be
considered representative of each of the three protein types. Work carried out with bivalve molluscs suggests the
possibility that all members of the PL Type have evolved or are related in one way or another to a PL-1 protein with
structural characteristics of an HI histone. PL-I proteins have been recently identified in both protostomes and
deuterostomes. The structural links existing amongst PL proteins, their arginine-rich composition and their relationship
to chromosomal proteins of the histone HI family suggest an evolutionary relationship amongst the three basic protein
types. On this basis, an evolutionary pathway starting from an early histone precursor and leading to the protamine type
which is present in the most evolved organisms of the deuterostome and protostome branches is proposed.
RESUME
Histone HI et evolution des proteines nucleaires specifique des spermatozoides
Les protyines chromosomiques des spermatozoides montrent une immense variability structural. Pendant les dix
dernieres annees, d’importants progres ont etc faits dans la caracterisation chimique et physique de ces proteines dans des
organismes tr£s varies. Les resultats emergeant de ces Etudes montrent que les proteines specifiques des spermatozoides ne
sont pas aussi h£tyrog£nes que ce que Lon pensait originellement. En fait, elles peuvent etre classes par leur composition
en un nombre fini de types: type H (histone), type P (protamine) et type PL (proche dcs protamines) correspondant ci des
proteines ayant une composition intermediate entre les histones et les protamines. La recherche sur les proteines proches
des protamines a porte principalement sur les Mollusques. Ce groupe taxonomique est d'un interet particular car il
comprend des organismes avec des proteines specifiques des spermatozoides qui peuvent etre considerees comme
repr£sentant chacun des trois types. Les travaux sur les Mollusques Bivalves suggerent la possibility que toutes les
proteines du type PL ont evolue ou sont reliees d*une maniere ou d’une autre avec une proteine PL-I avec les caracteristiques
structurales d’une histone HI. Les protyines PL-I ont ete recemment identifies chez les Protostomiens et les
Deuterostomiens. Les liens structuraux existant entre les protyines PL, leur composition riche en arginine et leurs
relations avec les protyines chromosomiques de la famille des histones HI suggerent une relation yvolutive entre les trois
types de proteines. Sur cette base, un chemin evolutif commen^ant avec un precurseur histone et menant au type protamine
qui est pisent chez les organismes les plus evolues des Deuterostomiens et des Protostomiens est propose.
Ausio, J., 1995. — Histone HI and the evolution of the nuclear sperm-specific proteins. In: Jamieson, B. G. M.,
Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, not.,
166 : 447-462. Paris ISBN : 2-85653-225-X.
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JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
During spermatogenesis, chromatin from the stem cells undergoes several dynamic
transitions which are often associated with important changes in its composition and structure. In
most instances, the composition of the chromosomal proteins at the onset and in the final stages of
spermatogenesis is quite different. These compositional changes significantly alter the structure of
chromatin. As a result, chromatin becomes highly compacted and gene expression is completely
shut off in the spermatozoon. The ways in which all this is achieved can be mediated by a wide
spectrum of apparently diverse chromosomal proteins [17, 40] which are mirrored by the
morphological diversity of the mature sperm cell [15]. The structure of chromatin arising from the
protein-DNA interactions in each particular situation is in most instances poorly understood and
the evolutionary relationship amongst these proteins still remains obscure.
This chapter reviews the classification of the nuclear sperm-specific proteins in the light of
new biochemical data that have been gathered in recent years. With the information on the
chromosomal sperm-proteins expanding over a wider range of taxonomic groups from both the
protostome and deuterostome branches as well as in lower phylogenetic taxa, it is also possible to
envisage and outline an evolutionary relationship for these proteins.
RESULTS AND DISCUSSION
Structural variability and compositional homogeneity of the nuclear sperm-specific proteins. The
classification of the nuclear spenn- specific proteins, 25 years after.
In 1969, David BLOCH [17] published the first comprehensive catalogue of the nuclear
sperm-specific proteins. Despite the attempt at classification proposed in his paper (Fig. 1), the
author concluded: “...the variability (non-conservatism) of the protein reflects an evolutionary
indifference to a relatively unimportant protein in an inert nucleus”. This somehow pessimistic
comment reflects BLOCH’s difficulty in finding a phylogenetic relationship among the different
groups of chromosomal sperm proteins. This was mainly due to the fact that even though proteins
belonging to the same group in the classification had a similar amino acid composition, they
exhibited an enormous structural variability. In addition, the patchy cytochemical and biochemical
information, available at that time, came from organisms usually belonging to phylogenetically
distant groups.
The availability of techniques such as high performance liquid chromatography and protein
microanalysis has allowed us to extend this information to a much broader spectrum of organisms
in recent years. More importantly, it has been possible, for the first time, to gain information on
organisms from phylogenetically relevant/related taxonomic groups [59, 60].
The global picture emerging from these studies is that despite their enormous structural
variability, the nuclear sperm-specific proteins can be grouped, from the compositional point of
view, into three basic categories (Fig. 1). Type H (Histone type) consists of proteins with amino
acid compositions that, although specific for the germ line, are structurally and compositionally
similar to those of the somatic histone type (Table 1). This grouping is equivalent to the Rana type
of BLOCH’s classification [17] (see also Fig. 1). At the other end of this classification, type P
(protamine type) consists of proteins of low molecular mass (4000 to 10 000 daltons), that are
arginine rich (arginine content > 60%). The P type is defined here irrespective of the presence or
absence of cysteine in the protein molecule. Thus it brings together under a common name the
monoprotamine and stable protamine type of BLOCH’s classification [17] (Fig. 1). In the course
of spermatogenesis, these proteins almost completely replace the histone counterpart of the stem
cells (see [53] for a recent review on these proteins, and Table 3). The PL type (protamine-like)
consists of proteins with an amino acid composition intermediate between the previous types. The
arginine and lysine content usually amounts to at least 35-50% of their amino acid composition,
but occasionally it can be higher. Although the relative ratio of these two amino acids may vary
over a wide range, it usually stays constant for a given taxonomic group (Table 2). In
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
449
A
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C BASIC TYPES
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0. Relatively non-baslc proteins (Crab type)
1. H type (Histones) 1. Typical histones (Rana type)
2. PL type (protamine/histone like) 2. Intermediate proteins (Mydlus type)
3. P type (protamines) 3. Monoprotamines (Salmon type)
4. Stable protamines (Mouse type)
— A: Schematic representation of the changes in the nucleus in ram spermiogenesis (modified from [42]). TP =
transition proteins. B: Nuclear protein transitions in the four basic types of spermiogenesis. C: Classification
of the nuclear sperm-proteins. The classification used in this paper is compared to that proposed by Bloch [17],
most instances, these proteins can be structurally related to histone HI (as will be discussed later)
and as in the case of the P type these proteins replace to a large extent (>80%) the somatic
histones during spermiogenesis. In the final stages of spermatogenesis, PL proteins exhibit a
significant degree of structural heterogeneity, with molecular masses in the 5 000-30 000 dalton
range. This group corresponds to the type of intermediate proteins ( Mytilus type) of BLOCH’s
classification [17] (see also Fig. 1).
The phylogenetic relevance of this later group of proteins was already anticipated by BLOCH
[18], He noted “... the similarities between the ‘evolutionary intermediate’ proteins of the mature
sperm of mussel and the ‘developmentally intermediate’ proteins of the spermatids of Loligo
450
JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
Table 1. — Amino acid composition (mol %) of the H-type proteins from the sperm of several representative organisms
in comparison with somatic type H proteins from calf thymus.
LP = Limulus polyp/iemus [48], PM = Petromyzon marinus [60], CA = Carassius auratus [49] and CT = calf
thymus [45],
tr = trace amounts.
HI H2A
H2B H3 H4
(squid) [16] and salmon'’... (which both contain proteins of the P type in their mature sperm)...
“suggest a relationship between ontogeny and phylogeny”. Although the precise relationship (if
any) between the “developmentally intermediate” proteins and" PL proteins has yet to be
established, BLOCH’s hypothesis provides an excellent model for the biochemical classification of
spermatogenesis within a phylogenetic context (Fig. IB). Thus, from the point of view of the
nuclear sperm-specific proteins which are present in the mature sperm and the protein transitions
undergone by chromatin during the differentiation process, four basic types can be defined. The
first group (type I) of this classification consists of those organisms which lose their
chromosomal protein complement (histones) during spermiogenesis. As a result, DNA appears
essentially naked in the mature sperm cell, which exhibits anuncondensed nucleus. In Type II,
the somatic type (H type) of histones is maintained throughout the whole spermatogenesis. In
most instances histones undergo an important turnover during which the somatic histones from
the stem cells at the onset of spermatogenesis are partially or completely replaced by sperm-
specific histones (Table 1). In the third group of this classification, the somatic and/or the
germinal histone type of proteins (H type), is replaced to a large extent by proteins of the PL type.
I hese are the proteins that are found in the mature spermatozoa. Finally, in group IV, at the onset
of spermiogenesis, the histones of somatic and/or germinal type are initially displaced by a set of
intermediate proteins “transition proteins” and finally replaced by arginine rich proteins of the
protamine type.
With the exception of group I, which has been described so far only in some members of
the decapod crustaceans [72], all the other types of this classification are widespread in both
deuterostomes and protostomes.
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
451
Table 2. — Amino acid composition (mol %) of some representative proteins of the PL-type,
tr = trace amounts.
Presence of an Hi-like PL-protein in the sperm of Molluscs
The study of the nuclear sperm-specific proteins from molluscs is of special interest for
several reasons. First, this taxonomic group consists of organisms that can be considered
representative of any of the protein types described in the previous section. Also, this is the group
from which the organism Mytilus (mussel) was selected by BLOCH to name the protein type
corresponding to PL-type of the protein classification described in the preceding section (Fig. 1).
With regard to the first point, all the organisms of the subclass Pteriomorphia (oysters and
scallops) of the class Bivalvia analyzed to date belong to the H type [2, 4, 52, 76], All the
members of the class Cephalopoda seem to contain chromosomal proteins of the P-type (Table 3),
some of which may even contain cysteine, as in the case of Eledone [68], Cysteine is also present
in the mammalian P-type proteins. However, the most common proteins present in the sperm of
the organisms from this phylum are by far the proteins from the PL-type. From a pragmatic point
of view, these latter proteins can be classified into two major categories: those PLs which have a
molecular mass smaller than 15,000 daltons and those with higher molecular mass. Although this
division may seem arbitrary, it has profound structural implications, as will be described next.
452
JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
A
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PL-Im
H5
i — mmmm—
. 1
Hie
PL-Im
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H5
PL-Im
H5
? W i
6 6 Gs
4
oM
*0 4C eo 70
Y I L ANN - K-G — INTSR- -LGSAMKLAPAKG-L
Y V A A - HSSLK-GAVLH- PR - LRRALAAG-L
YIKS-HY — KVG — HN-ADLQIRLSIRRLLAAGVL
QZZZ1 - f, - 3-m
B
C
me « _ t_j _ ?, t, r _ f
y y v i b e l
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PL-Im ksgvlvrpktsagasgatgsprvg
PL-ls KSGALAHPKGSAG- - WVLVPKK
H5 KQT - KGV-GASG - SPRLAKSDK
PL-Im - MM® -
H5
Hlc
!1
6G GASG ^
H5
Fig. 2. — A: Sequence comparison between the trypsin-resistant cores of two PL-I proteins from bivalve molluscs (PL-Im
from Mytilus californianus, PL-I$ from Spisula solidissima ) and the core histone H5 from chicken erythrocyte. The
shaded boxes shown below identify the a-helix (light) and B-sheet domains as determined by combination of
secondary structure prediction and the experimental analysis shown in B [39]. Also shown are the major conserved
features for the core of the histones of the HI family [27]. In this representation, the black dots correspond to
conserved hydrophobic residues. Conserved positively and negatively charged amino acids are also identified.
B: Fourier-transform Infrared Spectroscopy (FTIR) analysis of the trypsin-resistant core of PL-I from M.
californianus . The deconvolution and curve-fitting of the region corresponding to the amide I band of the infrared
spectrum is shown. The different frequencies (peaks) of the spectrum can be assigned to different secondary
structures [39]. a-helix (lightly shaded), B-sheet (darkly shaded), other (not shaded). The areas under the different
Irequencies (peaks) are proportional to the amount of the corresponding secondary structure. C. Tertiary structure
organization of the trypsin-resistant globular core of histone H5 determined by X-ray crystallography [55]. By
comparison, an idealized hypothetical model for the trypsin-resistant core of PL-I from M. californianus is also
shown. The model is based on the conservation of the primary structure and on the secondary structure assignments
shown in A.
In order to account for the large molecular mass of some of the PL-proteins found in
molluscs such as Spisula solidissima (surf clam) (which has a PL protein with Mr = 27 000),
SUBIRANA et al. [68] had earlier proposed a mechanism of “gene polymerization” from a PL
precursor gene of lower molecular mass. In 1986 we decided to test this hypothesis [12] by
Source . MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
453
Table 3. — Amino acid composition (mol %) of several representative proteins of the protamine type P.
* average composition from the PI and P2 components from (70]
tr = trace amounts.
conducting a detailed structural and biochemical analysis of the PL protein of Spisula. The results
surprisingly showed that, like somatic histone HI, the major PL protein component from Spisula
sperm: 1) was not insoluble in dilute PCA (5%) [4], 2) could be digested with trypsin to produce
a 75 amino acid trypsin-resistant core, 3) this trypsin-resistant core had a globular conformation,
4) the primary structure of this globular core exhibited a high degree of sequence similarity with
the globular domain of somatic histone HI and it fulfilled the constraints imposed by the
consensus sequence established for the globular trypsin-resistant domain of this histone [27] (Fig.
2). When this was taken together with the fact that the trypsin-resistant core of histone HI was the
most conserved region of this molecule [26], it was clear that, despite the high lysine and arginine
content of Spisula PL (Table 4), this molecule was a member of the histone H 1 family.
Extension of these analyses to PL proteins from other bivalve molluscs [20, 33, 34, 39]
indicates that the extent of sequence similarity of the globular domain of these molecules and that
of other histone H 1 members is in the 30-40% range, with higher values in the case of the sperm-
specific histone HI proteins. This value should be considered high, taking into account the high
evolutionary variability of the histones of the HI family [37], We call these proteins PL-I
proteins. We define them as PL proteins (Fig. 1C) which have an internal trypsin-resistant
454
JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
Table 4. — Amino acid composition (mol %) of the PL-I proteins of different organisms,
tr = trace amounts.
globular core with structural and compositional similarity to the globular counterpart of the protein
members of the histone HI family. Unlike the sperm-specific HI histones found in echinoderms
or in the bivalve group Pteriomorphia [2, 54] (and like protamines), PL-I proteins replace most of
the core and linker histones present at the onset of spermiogenesis. They account for > 80% of the
chromosomal protein of the mature spermatozoon.
The N and C terminal non-globular domains of these molecules are extremely variable.
Most of the basic residues are found in these regions and in many instances the arginine residues
are arranged in clusters similar to those found in the proteins of the protamine type [4],
All PL proteins with Mr > 15,000 analyzed to date are members of the PL-I protein group.
When similar structural analyses as those carried out with these molecules were extended to other
PL proteins with Mr < 15,000 [3], no trypsin-resistant peptide could ever be detected in these
molecules, regardless of their overall compositional similarity to PL-I. This could already be
anticipated from the complete absence of hydrophobic amino acids (Phe, Tyr, Leu...), which are
usually present in low amounts in PL-I proteins.
Chromatin Structures
During the last twenty years important progress has been made in our knowledge of the
chromatin organization of the nucleus of somatic cells [71]. In contrast, our knowledge of
chromatin organization in the sperm cell has progressed at a slower pace but has benefited greatly
from the information gained in the former as will be discussed later.
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ADVANCES IN SPERMATOZOA!. PHYLOGENY AND TAXONOMY
455
Fig. 3. — A: Change in the length of sperm nucleoids as a function of the ethidium bromide concentration. Figure
redrawn from [56]. 1. Sperm nucleoids consisting of protein of the 11 type ( Rana ccitesbeiana) exhibit a bipharic change.
As a result of the interaction of the H type proteins (histones) with DNA, chromatin adopts a nucleosomal organization
(see C). Nucleosomes stabilize DNA in a negatively supercoiled configuration. As the ethidium bromide concentration
increases, the native negative superhelicity is increasingly lost (ethidium bromide intercalation twists DNA in a positive
sense), DNA becomes more relaxed and the size of the nucleoid, therefore, keeps increasing until all negative supercoils
have been removed (equivalence point ~8 pg/ml ethidium bromide). Further increase in the ethidium bromide beyond this
point induces positive supercoiling. 2, 3. In contrast, sperm nucleoids consisting of protein of the PL ( Xenopus
laevis ), 2, and P type (Bufo fowled ), 3. exhibit only a decrease in their dimensions as the concentration of ethidium
bromide increases. This indicates that DNA has a relaxed conformation in the nucleoprotein structure arising from its
interaction with these kinds of proteins. The 5% acetic acid, 2.5 M urea polyacrylamide gel electrophoresis (PAGE) shows
the protein composition of the sperm nuclei of 1) Rana catesbeiana, 2) Xenopus laevis. 3) Bufo sp B: Average diameter
of the chromatin fibres from advanced spermatids of marine bivalve molluscs with different H- and PL-type protein
compositions (modified from [22]). A 5% acetic acid, 2.5 M urea PAGE is also shown, a) Ensis ensis, b) Callisia chione.
c) Donax irunculus , d) Pecteti maximus , and e) Mylilus edulis. The thick arrows point to the PL-I proteins and the thin
arrows point to other PL protein components of smaller molecular mass. C: Upon interaction with histone HI, the
polynucleosome filament folds into a higher order structure. This results in the 30 nm fibres observed in the sperm of
organisms containing H-typc chromosomal proteins. D: Hypothetical arrangement of the PL-DNA nucleofilamcnts in
the (25-50 nm) chromatin fibres observed in advanced spermatids (as in B) of the organisms with a nuclear PL-type
protein composition. HI, H2A, H2B, H3, H4. histone types, N. nucleosome, P, protamine.
456
JUAN AUS10 : EVOLUTION OF THE CHROMOSOMAL PROTEINS
Chromatin organization (somatic, H-type). Chromatin resulting from the interaction of DNA
with proteins of the histone type (H-type) is organized in discrete nucleosome subunits. In the
nucleosome, about 200 base pairs of DNA are wrapped in approximately two negative
superhelical turns about a histone core octamer (consisting of “core histones” H2A, H2B, H3 and
H4). Thus, DNA is stabilized by nucleosomes in a negative supercoiled state, which poises
eukaryotic chromatin for genetic activity (replication, transcription). In addition to the “core
histones”, “linker histones” (proteins of the HI histone family) bind to the linker DNA regions
connecting the neighbouring nucleosome subunits and condense chromatin into higher order
structure fibres of about 300 A in diameter (Fig. 3C). Although electrostatic interactions play an
important role in the maintenance of this organization, only about half of the negatively charged
DNA phosphates are neutralized by the arginine/lysine side chains. This makes the nucleohistone
complex very sensitive to environmental ionic conditions [65] and amenable for interaction with
other regulatory proteins.
The realization that histones are not only passive structural blocks, but also functional
elements [35] represents one of the most important landmarks in our understanding of the
function-structure relationships of somatic chromatin. Despite the lack of DNA sequence
specificity of the histone-DNA interactions, the resulting nucleosome structures may play, by
themselves or in conjunction with other regulatory proteins [43, 75], a very important role in the
modulation of the genetic activity of the chromatin complex [6]. The ability of different histone
HI (linker histones) subtypes to condense the chromatin fibre to different extents, and thereby
possibly to be involved in its genetic activity, has also been postulated [25], As will be discussed
at the end of this chromatin section, the latter consideration is important in understanding the
presence of proteins of the H type in the sperm of some organisms. All these functional and
structural aspects of the H-type chromatin organization may explain why somatic histones have
been conserved so invariably through evolution, in contrast with the proteins of the sperm.
Chromatin organization resulting from the interactions between the PL-P type proteins with
DNA. I am next going to highlight briefly what I consider to be the most recent significant
advances in chromatin organization resulting from the interaction of DNA with proteins of the PL
and P type.
Although PL and P proteins usually coexist with a small amount of histones in the sperm
nucleus [2, 14, 32], the structure of the nucleoprotein complexes arising from the interaction of
these proteins with DNA lacks the nucleosomal organization of the somatic chromatin type, as can
be envisaged by X-ray diffraction [7, 10, 63].
The overall negative superhelicity of DNA is lost [56] (Fig. 3A), most likely as a result of
the topoisomerase II activity associated with the histone displacement/replacement by these PL or
P proteins [46, 57]. Thus, the nucleohistone-nucleoprotamine (protamine-like) transition leads to
a complete reorganization of chromatin, while possibly maintaining the specific three dimensional
organization of DNA and its DNA loop domain structure [73],
The detailed molecular structures of the nucleoprotein (P, PL) complexes are still
controversial. Both PL and P proteins interact electrostatically with DNA (which basically retains
a B conformation [63]) to form fully saturated complexes, unlike the somatic nucleohistone [1,
10, 13], In these complexes, the PL and P proteins have been postulated to adopt an a-helical like
configuration [51, 66, 74], The positioning of these proteins in the major or minor groove of
DNA has not yet been experimentally settled [65]. Nevertheless, recent raman spectroscopy
analysis suggest that tish protamine may adopt an unusual l->3 y turn (non a helical) structure
interacting with the major groove of the DNA [36],
At the higher order level of organization, it has been shown recently [22] that PL groteins
(Fig. 3B), like H type proteins, can organize the nucleoprotein complexes into 250-500 A fibres
regardless of the particular PL composition and the absence of nucleosome-like structures. This is
an important finding because it indicates that the higher order structures of the nucleoprotein
complexes are mainly determined by the ionic nature of the interactions involved [31, 67], rather
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
457
than the particular structure of the proteins (H, PL or P) itself. Intermediate 300-400 A fibres have
also been described during the process of chromatin condensation in the sperm of cephalopods
[44], salmon [77], lizards [19] and humans [62], all containing P type proteins.
Sperm chromatin variability
After analyzing the different types of chromatin structures, the next question that arises is,
what is the reason for so much chromatin variability in the sperm? Although there is no clear cut
answer to this question, in what follows I will discuss several points which may provide a useful
hint into the problem.
The only functional theme that all the sperm chromatin types I through IV, in Fig. IB, have
in common is the complete shut off of the genetic activity in the mature spermatozoon.
Since H type is the protein type which is always present at the onset of spermiogenesis in
any of these groups, regardless of the final nuclear protein composition, it is clear from what has
been mentioned earlier, that the silencing of the genetic activity at this point can be achieved in
several different ways.
One possible way (type I of spermiogenesis) would be the complete erasing of the starting
chromatin structure (including nucleosomes, and other chromatin associated regulatory proteins).
An alternative way could be by increasing the ratio between linker histones and core histones or
by using specific linker histones [54] that lock chromatin in a functionally inert structure (type II,
Fig. IB ). A third possibility would be the replacement of the H type proteins by highly charged
sperm-specific PL or P proteins (types III and IV of Fig. IB ) which remove the nucleosomal
organization and negative superhelicity of DNA. Thus, in addition to the genetic repressive effect,
types I, III and IV also have in common the erasing of the nucleosome imprinting of the stem
cells. It is not clear yet whether or not in the case of type II a reorganization (randomization) of the
nucleosome positioning takes place upon binding of the specific linker histones, which could
produce a similar erasing effect.
While we do not know exactly why type P has been increasingly selected throughout
evolution, several arguments can be made. Although the four types appear to be equally efficient
from the two previous points of view, it is obvious that type I leaves the genome more exposed to
possible damage by physical/chemical mutagenic agents. Types II, III and IV differ mainly in the
extent of chromatin compaction achieved. Substitution of histone by PL (and removal of the
otherwise unnecessary nucleosomes) and finally by P leads to an increasingly more compacted
nucleus that may have finally been selected by the constraints imposed by the mechanisms of
fertilization [40] or by other selective pressure mechanisms yet to be established.
Evolution of the nuclear sperm-specific proteins
From what has been discussed in the previous sections, it is clear that the PL-type
represents an intermediate type both from the structural and functional point of view. Whereas
protamines have only been found at the tips of the most evolved groups from both the protostome
and deuterostome branches, PL proteins are already found in the eukaryote groups preceding this
branching. A rather exhaustive analysis recently carried out in our laboratory on several
organisms belonging to different groups of the phylum Cnidaria has revealed that a primitive Hl-
PL-I protein is the major chromosomal protein found in the sperm nucleus of these organisms
(unpublished results, but see Fig. 4A). Evidently the intermediate structural features of the PL
proteins, discussed earlier, must reflect their intermediate evolutionary position.
All this, provides support for the early hypothesis put forward by SUB1RANA several years
ago that sperm-specific nuclear proteins, including protamines, may have evolved from a common
histone ancestor [64]. In the light of the experimental information presently available, we propose
the evolutionary pathway which is shown in Fig. 5A. Accordingly, all the chromosomal sperm
458
JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
A
B
Fig. 4. — Widespread distribution of PL-I proteins in the eukaryotic kingdom. A: Urea acetic acid polyacrylamide gel
electrophoresis of (a) calf thymus somatic histone HI, (b) chicken erythrocyte histone H5, (c-f) nuclear proteins
from the sperm of (c) Mullus surmuletus (Fish), (d) Chelysoma production (tunicate), (e) Spisula solidissima (clam)
and (0 Metridium senile (anemone). (The arrows point to the PL-I protein components.) B: Comparative
schematic representation of the teriary structure (circle = globular core, linear regions = N, C terminal domains) of
the protein PL-I from different organisms and somatic histones HI. 115. The letter symbols designate the same
species as in (A). CH = chicken erythrocyte histone standard.
proteins would have arisen from a primitive histone precursor, presumably the same from which
the somatic lysine-rich histone HI lineage would have also originated. The enormous structural
variability and the rapid evolution [37] of the proteins from the HI family make it very difficult to
trace the origin of such a precursor and its identification with any protein previous to the metazoan
organization (see KASINSKY, this volume).
The next step in the evolution of these proteins in the case of the sperm lineage would
involve an increase in the arginine contents of this originally lysine-rich precursor. In the early
stages such protein would still coexist with a full complement of the somatic-type of histones
(histone H type of spermiogenesis, see Fig. IB). The sperm-specific HI histones of the sperm of
echinoderms provide a good example of such histones. A further increase in the arginine content
would allow these molecules to displace and replace the somatic histones, as occurs with the PL-I
molecules found in the sperm of many bivalve molluscs [2, 4, 5, 13]. It is important to point out
here that PL-I is not only restricted to molluscs. As shown in Fig. 4A, PL-I proteins have been
identified in many phylogenetic groups, including chordates [59] and vertebrates [61].
In the next evolutionary step the PL-I proteins would lose their globular core, giving rise to
smaller PL proteins either by processes of post-translational cleavage (Fig. 5B), such as in
Mytilus [21], or by other mechanisms yet to be described, such as alternative splicing. Evidence
for alternative splicing has already been provided for the bovine protamine 2 gene [4 1 ].
1 he small PL proteins may have then evolved into the arginine-rich protamines, which are
found in the upper phylogenetic levels of the deuterostome and protostome branches. In the
process of selection, arginine may have been selected over lysine because of its higher potential to
form hydrogen bonds [11],
The evolutionary pathway from histones to specialized sperm-specific histone HI, PL-I. PL
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
459
HISTONE
PRECURSOR
I
HISTONE HI
(specialized)
I
PL-I PROTEINS
1
PL PROTEINS OF
LOW MOLECULAR WEIGHT
(spermatid transition proteins?)
PROTAMINES
PL-I
Pig. 5. — A: Proposed evolutionary relationship for the nuclear sperm-specific proteins. B: Some of the low molecular
weight PL proteins (Mr < 15000 Da) might have arisen from post-translational cleavage of a larger PL-1 precursor
[21].
and protamines appears in repeated instances within different phyla both in protostomes [69] and
in deuterostomes [60]. The presence of the most primitive protein types, H and PL, decreases as
the P type increases in the most evolved groups of the phylogenetic tree. Thus, the scheme shown
in Fig. 5A represents, in fact, the “mode” rather than the “tempo” [30] of evolution followed by
the chromosomal sperm proteins.
The selection of an Hl-related protein type at the base of this evolutionary pattern in each
different phylum may have occurred initially by a process of evolutionary convergence due to the
intrinsic ability of this molecule to condense chromatin and possibly to lock it in an “inert"
structure. This could have happened at the expense of increasing the arginine content of this
protein and/or the stoichiometric ratio of this molecule with respect to the nucleosome subunit. In
this process the tripartite organization of histone H 1 and most of the secondary structure of its
globular core have been significantly conserved and therefore both functional and structural
convergence most likely occurred in this case. At the other end of this evolutionary pattern, all
protamines exhibit a highly arginine-rich composition with very similar primary sequences
consisting of arginine clusters (see KASINSKY, in this volume). The possibility therefore exists
that this could represent a convincing case of genuine sequence convergence [29] starting from
different PL-I proteins. It should be pointed out, however, that such a possibility is hard to prove
considering the small size (30-80 amino acids) of protamines, which excludes the possibility of
long range cladistic analysis [28].
The evolutionary “mode” of the nuclear sperm-specific proteins presented here represents an
alternative to the “retroviral hypothesis” proposed for the origin of protamines [38]. This
hypothesis was initially put forward in order to account for the sporadic distribution of the protein
types H and P in fishes. As a matter of fact, a recent thorough reexamination of the distribution of
the three protein types H, PL, and P in the sperm of bony fish [61] has not been able to provide
any support to the retroviral origin of protamines in this vertebrate class.
ACKNOWLEDGMENTS
I would like to thank my wife for her skillful computer assistance in the preparation of the figures. I am very
grateful to Maree RooMF. for her patience and careful typing of the manuscript. I am also thankful to Phil Rice for carefully
reading the manuscript. This work was supported by NSERC grant OGP0046 399.
460
JUAN AUSIO : EVOLUTION OF THE CHROMOSOMAL PROTEINS
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Source : MNHN. Paris
Evolution and Origins of Sperm Nuclear Basic Proteins
Harold E. KASINSKY
Department of Zoology,
University of British Columbia, 6270 University Boulevard, Vancouver, B.C., V6T 1Z4, Canada
ABSTRACT
Although the DNA-binding, basic nuclear proteins of sperm (SNBPs) are highly diverse, constraints on their diversity
in particular taxa suggest that they may play an adaptive role in such taxa and are not merely randomly distributed. For
example, in the subphylum Vertebrata, internally fertilizing tetrapods have arginine-rich protamines or keratinous
protamines that condense sperm chromatin to a greater extent than in externally fertilizing frogs or fish having sperm
histones. In such taxa “R-CLUES," relatively constant / argest units of evolutionary similarity, are broader
phylogenetically for mammals (for example, infraclass Euthcria) than for frogs (for example, genus Bufo). The evolution
of SNBPs appears to be saltatory rather than continuous. Consequently, the phylogenetic breadth of R-CLUES may assist
in the classification of particular taxa where SNBPs are either protamine-like or histone-like amongst particular genera of
frogs ( Xenopus versus Silurana) or subfamilies/families of fish (Salmoninae versus Sparidae). SUBIRANA & COLOM (1987),
AusiO el al. (1987), and Chiva et al. (1991) have proposed that protamines in molluscs may have evolved from very
lysine-rich HI histones. This is supported by the presence of protamine-like SNBPs with trypsin-resistant cores in
cnidarian sperm. The presence of protamine-like SNBPs in lower plants may be a case of convergent evolution. Highly
condensed chromosomes in the eukaryotic dinoflagellate Crypthecodinium cohnii and the highly condensed nucleoid in
the bacterium Chlamydia trachomatis, both have lysine-rich basic proteins similar to portions of histones H5 or HI,
respectively. This suggests possible additional cases of convergent evolution amongst basic proteins of condensed
chromatin preceding the origin of the primitive type of sperm in metazoans.
RESUME
Evolution et origines des proteines basiques nucleaires des spermatozoides
Bien que les proteines nucleaires basiques lites & l’ADN des spermatozoides (PNBS) soient extremement diversifiees, les
contraintes pesant sur leur diversity dans des taxons particuliers suggerent qu’elles pourraient jouer un role adaptatif dans
de tels taxons et qu'elles ne sont pas simplement distributes au hasard. Par exemple, dans le sous-embranchement
Vertebrata, les Tttrapodes a fecondation interne ont des protamines riches en arginine ou des protamines keratineuses qui
condensent plus intensement la chromaline du spermatozoide que chez les Amphibiens ou les Poissons a fecondation
externe possedant des histones dans le spermatozoide. Dans de tels taxons, les plus grandes unites relativement constantes
de similarite evolutive (“R-CLUES” en Anglais) ont une plus grande etendue phylogenique chez les Mammiferes (par
exemple, 1’infraclasse Eutheria) que chez les Amphibiens (par exemple, le genre Bufo). L’evolution des PNBS apparait
saltatoire plutot que continue. De ce fait, l'ampleur phylogenique des “R-CLUES” peut etre utile pour la classification de
taxons particuliers chez lesquels les PNBS sont proches des protamines ou proches des histones, parmi des genres
particuliers d’ Amphibiens ( Xenopus versus Silurana ) ou des families ou sous-famillcs de Poissons (Salmoninae versus
Sparidae). Subirana & COLOM (1987), Ausio et al. (1987), et Chiva et al. (1991) ont propost que les protamines chez les
Mollusques aient tvolut h partir d’histones HI tres riches en lysine. Cette hypothtse est confortte par la prtsence de PNBS
proches des protamines avee des domaines rtsistants cl la trypsine dans les spermatozoides des Cnidaires. La prtsence de
PNBS chez les plantes inferieures pourrait etre un cas devolution convergente. Les chromosomes hautement condensts du
Kasinsky, H. E., 1995. — Evolution and origins of sperm nuclear basic proteins. In: Jamieson, B. G. M., Ausio,
J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 463-
473. Paris ISBN : 2-85653-225-X.
464
H. E. KASINSKY : EVOLUTION OF BASIC PROTEINS
Dinoflagelle Eucaryote Crypthecodinium cohnii et les nucleoides tres condenses de la Bacterie Chlamydia trachomatis ont
tous deux des proteines basiques riches en lysine, respectivement similaires h des portions des histones H5 et HI. Ceci
sugghre l'existence de cas supplementaires devolution convergente parmi les proteines basiques de la chromatine
condcns6e, anterieurs it 1’ apparition du type primitif de spermatozoide chez les Metazoaires.
Sperm basic proteins (SNBPs) that bind to DNA in animals and plants are highly diverse
[12], in sharp contrast to the evolutionarily conservative nucleosomal histones that characterize all
other cell types. SNBPs range from low molecular weight arginine-rich protamines in which these
basic residues are clustered repeatedly, as in the sperm nucleus of the cartilaginous fish
Scyliorhinus canicula [17], to replacement by a sperm-specific variant of histone HI, as in the
frog Rana ridibunda [34]. In sperm of the goldfish Carassius auratus [40], the entire complement
of histones is retained along with the nucleosomal organization of chromatin. Keratinous
protamines with disulfide bonds also can be found in the sperm of Scyliorhinus [17]. Some
protamine-like SNBPs show similarities to very lysine-rich histones, as in bivalves like the surf
clam Spisula solidissima [7] and the mussel Mytilus edulis [8]. Other protamine-like SNBPs have
intermediate compositions containing lysine and sometimes histidine along with arginine, as in the
turtle Chrysemys picta [20], some frogs like Ascaphus truei [33] and Bufo japonicus [60], and in
some bony fish, including sticklebacks like Pungitius pungitius [37], Some crab species have no
basic proteins at all in their sperm [12].
DISCUSSION
Internal fertilization as a constraint on SNBP diversity
Is this diversity of SNBP type due to randomness or adaptation? In BLOCH's classical paper
he came down on the side of the former when he stated, “It is proposed that the variability (non¬
conservatism) of the protein reflects an evolutionary indifference to a relatively unimportant
protein in an inert nucleus” [12, p. 107]. In the same paper, he also indicated that "... although a
phylogenetic relationship is often apparent from the similarity of proteins within tightly defined
taxonomic groups (e.g. , the clupeids, or the eutheria), there seems to be no evolutionary trend.
Most of the classes of sperm proteins are represented within most of the broad taxa.” [12, p. 99].
In fact, analyses [30-32, 53] of the distribution of SNBPs in animals indicates (Fig. 1, left) that
the mode of fertilization acts as a constraint on SNBP diversity, such that either protamines,
keratinous protamines or protamine-like SNBPs are present in sperm of internally fertilizating
taxa. Thus, sperm of the honey bee Apis mellifera [5] and the barnacle Balanus nubilus [21] are
not exceptions to this rule, as thought by BLOCH [12], but appear to contain protamine-like
SNBPs upon gel electrophoresis, while cytochemical analysis [53] indicates the presence of
protamine-like SNBPs in sperm of the platyhelminth Notoplana and the nematode Thelastoma
periplaneticola. The only exception to this rule known to date is the deep sea bony fish Cataetyx
laticeps (order Ophidiiformes, family Bythitidae). Recently, SAPERAS et al. (58) have discovered
that internally fertilizing sperm of this species contain histones and an additional sperm-specific
protein that is compositionally similar to erythrocyte H5 from grass carp. Perhaps this anomaly is
a consequence of the fact that internally fertilizing deep sea fish such as brotulas have relatively
short larval stages [41, p. 469], a reproductive strategy designed to enable a small number of
young to settle in suitable habitats in their benthic environment. Such a progenetic tendency [39]
in this viviparous fish might require the utilization of histone-like genes, rather than the more
usual protamine-like genes to condense DNA, as the first sperm might have to be made after
fewer cell divisions following fertilization in such a precociously maturing organism [35]. It may
be possible to test this experimentally by examining the relationship between SNBP type and
progenesis in the five species of bythids that are confined to shallower freshwater or weak
brackish water environments [41, p. 226],
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
465
Internal fertilization as a constraint on SNBP diversity is most clearly observed in the
tetrapods. In the subphylum Vertebrata (Fig. 1, right), internally fertilizing eutherian mammals
possess keratinous protamines [10, 42], metatherians have non-keratinous PI -like SNBPs [63],
birds contain protamines [19, 42] and reptiles have protamine-like SNBPs [20, 30]. In some
frogs with external fertilization, like Rana, sperm-specific histones HI and H2B are present in the
nucleus. Here [50], sperm chromatin is not as tightly condensed as in amniote species with
protamines or protamine-like SNBPs. Thus, there is a trend in the tetrapods from variability of
SNBP type in externally fertilizing anurans to relative constancy of protamine type in internally
fertilizing urodeles and amniotes [30, 31]. This can also be observed in cartilaginous fish [17, 30]
and in bony fish [12, 30], with the sole exception of Cataetyx laticeps [58] noted above. In
molluscs [18, 23], internal fertilization also constrains SNBP diversity in mesogastropods,
neogastropods and cephalopods. Here protamine-like SNBPs or keratinous protamines are found.
R-CLUES as phylogenetic measures of SNBP diversity.
If we define a new term, “R-CLUES,” as the “relatively constant, largest units of
evolutionary similarity [32], then amniotes have phylogenetically broader R-CLUES than
externally fertilizing vertebrates like anurans and most bohy fish by virtue of the constraint of
internal fertilization. For example, Table 1 shows that similar PI keratinous protamines occur
throughout the infraclass Eutheria, but similar protamine-like intermediate SNBPs are confined to
particular genera of Australian frogs, like Litoria [33], and stickleback fish, such as Gasterosteus
[37],
The acronym “R-CLUES” is also intended to denote the search for “clues” for “R,” the
arginine content of SNBPs in particular taxa. Thus, a low arginine content of 4.5 mole percent in
the lysine-rich protamine PL-IV of the mussel Mytilus edulis [8] is typical for R-CLUES at the
family level (Mytilidae) in the class Bivalvia. In the genus Bufo, R-CLUES for these
representatives of the order Anura are delineated by a much higher arginine composition, for
example 42.3 mole percent, in Bufo japonicus protamine PI [60].
Is internal fertilization the only constraint on SNBP diversity in animal sperm? Apparently
not. As seen in Table 1, echinoderms are all external fertilizers. Constancy in the marine
environment probably accounts for the relative constancy of SNBP type in this taxon. All of the
echinoderms have sperm-specific histone HI [44], with R-CLUES showing variation for
different orders [30]. In externally fertilizing tunicates, the constant marine environment also
appears to maintain the relative constancy of SNBPs. A protamine-like PI SNBP that resembles
HI histone is the principle protein for all species studied thus far [22, 56], with some variation for
R- CLUES for the genus Styela. Therefore, from R-CLUES indicated in Table 1, we see that
internal fertilization is a particular kind of constraint for certain taxa on land, like amniotes, and in
the marine environment, like mesogastropods, neogastropods, cephalopods and cartilaginous
fish. I agree with Saperas [55, p. 330] that internal fertilization tends to fix the type of SNBP,
insofar as the more specialized the biology of reproduction, the more such variation impacts
negatively on SNBP function.
As can be seen in Fig. 1 and Table 1, the evolution of SNBPs appears to be saltatory rather
than continuous, in the sense that these proteins can differ quite markedly between related taxa. In
the case of eutherian mammals, PI keratinous protamines are sufficiently similar to constitute a
family of related proteins [17], yet they are amongst the most rapidly diverging polypeptides
studied [47], evolving at rates close to that of fibrinopeptides. Nevertheless, these proteins can
easily be distinguished from the protamines of metatherian mammals [63] and the arginine-rich
protamines of birds [19, 42], both of which lack cysteine.
466
H. E. KASINSKY : EVOLUTION OF BASIC PROTEINS
Table 1. — R-CLUES of representative animal taxa.
i. Classification modified from [11, 28, 41, 65], 2- See legend of Fig. 1 for definitions of P, KP. PL and H HI = sperm-
specific histone HI, with higher arginine content than somatic histone HI. H2B = sperm - specific histone H2B.
3 R-CLUES (relatively constant /argest units of evolutionary similarity) are in italics. , Int. - internal;
Ext. = external. 5, Ter. = terrestrial; Mar. = marine; FW = freshwater, , PI, P2a, P2b, = keratinous
protamines. 7, Reference. 8, Taxonomic ranking as follows: phylum > subphylum > class > infraclass > superorder
> order > suborder > family > subfamily > genus [11, 28, 41]. 9, Silurana = proposed genus [14] to contain former
species Xenopus iropicalis (2n=20) and X. epiiropicalis (2n=40). 10, Some primitive urodele species are external
fertilizers [30]. 11 , Some species are anadromous; i.e., marine, but breed in freshwater [37]. , Ascidia is a
facultative internal fertilizer [22] in this suborder. 13, Includes SNBPs from 3 of the 15 living families of suborder
Lacertilia (Sauria) but from only one family (Colubridae) of 15 in suborder Serpentes [65]. , In order
Carcharhiniformes. May also include SNBPs of Squalus acanthias [30], family Squalidae, order Squaliformes and R-
CLUES may therefore be broader phylogenetically than indicated here. 15, May also include SNBPs of Sepia
officinalis [23] in order Sepoidea.
Source . MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
467
Amongst the vertebrates, the saltatory nature of SNBP evolution can best be seen in frogs
and bony fish. Table 1 shows that different genera of frogs can have either histone-like SNBPs or
arginine-rich protamine-like SNBPs, whereas some bony fish can show a similar alteration at the
familial level (subfamily Salmoninae versus family Sparidae). This alteration appears to be of a
sporadic nature [58], Analysis of such R-CLUES may assist us in determining whether protamine
histone or histone -> protamine transitions may have occurred in closely related taxa. The
clearest example we have to date of the success of such an analysis is the ability of R-CLUES to
distinguish between frogs of the genus Xenopus [61] and morphologically similar frogs of the
genus Silurana [14]. In the polyploid genus Xenopus, there is a lineage of frogs with diploid
chromosome numbers of 36 -» 72 -> 108 that includes laevis and another lineage with 20 -» 40
diploid chromosome numbers that includes tropicalis and epitropicalis. Only SNBP type can
clearly distinguish these two lineages biochemically. Thus, R- CLUES can distinguish two
separate genera, since the former lineage has intermediate type, protamine-like SNBPs, while the
20 -» 40 line contains only histones, with one additional spermatid/sperm-specific protein [38].
Recently, on the basis of morphological criteria, CANNATELLA & TRUEB [14] have split the
genus Xenopus and have placed the discordant tropicalis and epitropicalis species into the genus
Silurana, which they have resurrected from GRAY (1864). -Thus, amongst the five genera of pipid
frogs [65, p. 364], the two species of Silurana are more closely related to Hymenochirus and Pipa
than to species of Xenopus. As we predicted the electrophoretic profile of Silurana epitropicalis
SNBPs solely on the basis of chromosome number before doing the actual experiment [38], it
appears that R-CLUES may be useful characters for systematic studies, along with more
traditional analyses.
Origins of SNBPs.
What is the origin of the SNBP pattern in the deuterostomes? Recently, SAPERAS et al. [57]
presented two models, both of which take into account the difference between histones present in
echinoderm sperm and protamine-like SNBPs in urochordates and the cephalochordate
Branchiostoma floridae. The lamprey Petromyzon marinus also has somatic-like histones in its
sperm [57], However, cartilaginous fish have protamines and keratinous protamines [17],
Alternation between protamines and histones can be seen in different families of bony fish and
frogs, whereas urodeles and the amniotes have only protamines, protamine-like SNBPs or
keratinous protamines.
From these data, SAPERAS et al. [57] concluded that both models would have histones at
the base of the SNBP pattern in deuterostomes. However, examination of Fig. 1 (left) indicates
that at the root of deuterostome phylogeny are taxa that show protamine-like SNBPs, either by
cytochemical criteria, as in nematodes and platyhelminths [53], or by a combination of
cytochemical and biochemical analysis, as in cnidaria [6, 53]. In the case of the sea anemone
Metridium senile, AUSIO [4] has shown that the SNBP belongs to the PL-I type, related to HI
histones, with a peptide core that is trypsin resistant. It would seem, therefore, that another model
could place protamine-like SNBPs at the base of deuterostome phylogeny, with reversions to
histones occurring in particular taxa like echinoderms and agnatha. This might be due to the loss
of protamine genes or gene expression in deuterostome evolution [57], or perhaps due to shifts in
developmental timing, such that an earlier onset of spermiogenesis in particular taxa might require
selection for histone rather than protamine gene expression [35]. Such an argument was made in
the previous section for the lack of protamines in the internally fertilizing deep sea fish Cataetyx
laticeps [58]. Perhaps it might also apply to the histone-like SNBPs in sessile echinoderms
evolved from motile ancestors [11, p. 838].
What might be the origins of protamine-like SNBPs in the lower metazoa? SUBIRANA &
COLOM [59], AUSIO et al. [9] and CHIVA et al. [18] have proposed that protamines in molluscs
468
H. E. KASINSKY : EVOLUTION OF BASIC PROTEINS
MAMMALIA7
Fig. 1. — Diversity of SNBPs in animals. (Cladogram on left has been modified from [13, pp. 873, 882] and on right
modified from [28, pp. 607, 628, 669, 684. 707, 736]. 1, Phylum; 2, SNBP type: H = somatic like-histones; PL =
intermediate protamine-like SNBPs; P = protamines; KP = keratinous protamines; 0 = no SNBPs present in sperm
nucleus; 3, Thick line indicates some or all species in this taxon are internally fertilizing; 4, Position of Nematoda
is not indicated in [13, p. 882]; 5, SNBP type based on cytochemical data [12, 53] or 6, on electrophoretic and
cytochemical data [6, 53]; 7, Class; 8, Order; 9, Subphylum.
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
469
CHLAMYDIA PLANCTOMYCES-
TRACHOMATI S HC1 CHLAMYDIA GROUP
BACTERIA
EUGLENA
GRACILIS HI
TETRAHYMENA
THERMOPHILA
EUGLENOIDA
CILIOPHORA
KINGDOM
PROTOZOA
CRYPTHECODINIUM
COHN I I
(dinof 1 age Hate)
HCc2
DINOZOA
METRIDIUM SENILE
(anemone) PL
CNIDARIA
DROSOPHILA
MELANOGASTER KP?
ARTHROPODA
KINGDOM
ANIMALI A
EUCARYA
SWIFTOPECTIN
SWIFTI
(scallop) PL
HOMO SAPIENS KP
MOLLUSCA
CHORDATA
ZEA MAYS H
CHARA CORALLINA
(stonewort) PL
TRACHEOPHYTA
KINDOM
PLANTAE
CHAROPHYTA
Fig. 2. — Possible outgroups of animal SNBPs amongst nuclear basic proteins of plant sperm and protists. Cladogram is
modified from Cavalihr-Smith's 18s rRNA phylogcny for 150 eukaryotes [16] and Worse's 16s rRNA phylogeny
for bacteria [64].
may have arisen from very lysine-rich HI histones by the evolutionary route HI histones ->
protamine-like SNBPs -> protamines. The presence of PL-I basic proteins in cnidarians [4, 6]
supports such an evolutionary pathway. Also, as can be seen in Fig. 2, plant SNBPs [51] provide
an outgroup for patterns of SNBP diversity in animal phyla since they arose from different
protistan ancestors than did animals. Therefore, the presence of protamine-like SNBPs in
biflagellate, motile sperm of green algae, such as the stonewort Chara corallina [49], and in
bryophytes, like the liverwort Marchantia polymorpha [48], along with the presence of Hl-like
histones in condensed nuclei of the nonmotile male gamete in a higher plant such as Lilium
Source
470
H. E. KASINSKY : EVOLUTION OF BASIC PROTEINS
longiflorum [62], indicates that the histone H l-> protamine-like SNBP transition may be a case of
convergent evolution [24]. This attests to the importance of the connection between sperm motility
and the condensation of sperm chromatin in the origin of SNBPs, a connection that is further
emphasized if we consider the nature of the basic proteins in highly condensed nuclei of
chromosomes in protists [46, p.10]. Thus, the eukaryotic dinoflagellate Crypthecodinium cohnii
[54], which has condensed chromosomes, lacks histones and nucleosomes but contains two
lysine-rich base proteins, one of which, HCc2, shows 38% of residues identical with the 53
residues near the carboxyl terminus of the erythrocyte-specific histone H5 of duck. More
primitive dinoflagellates have histones, whereas in Nocliluca [15], a transitional form, histones
are present in the giant vegetative cells, but not in the small sexual swarmer stages. As
Cavalier-Smith [15, p. 350] points out, “It could be that histone loss in the noctilucean
swarmers is adaptive, analogous to the replacement of histones by protamines or other basic
proteins in many animal sperm to allow more compact sperm nuclei.” The histone HI in nuclei
with condensed chromatin in Euglena gracilis [29] is unique and in the condensed,
transcriptionally inactive, germ-line micronuclei of Tetrahymena thermophila , [2], proteolytic
processing of histone HI gives rise to three HI -like polypeptides, a, p and y. GOROVSKY [26]
notes that “like some other transcriptionally inert nuclei such as nucleated red blood cells [25] and
some histone containing sperm [1], micronuclei have different linker-associated (HI -type)
histones than their transcriptionally active counterparts.” RAIKOV [46, p. 7 1 ] calls the micronuclei
of many Ciliophora “spermal” as they “are filled with compact chromatin, just as the nuclei of
spermatozoa are.” In this regard it is interesting to note that the highly condensed nucleoid in the
elementary body of the prokaryotic bacterium Chlamydia trachomatis [27] contains a basic
protein, Hcl, that shows 34.9% identity in a 106-amino acid overlap with histone HI of the sea
urchin Lytechinus pictus. This is highly unusual for bacteria, however, where the nucleoid is
normally not so highly condensed [52] but may none the less contain some HI -like basic proteins
as in Pseudomonas aeruginosa [36], or protamine-like basic proteins as in Escherichia coli [3],
From the viewpoint of SNBP types in lower metazoans, there is a sense of deja vu with
regard to the role of HI -like histone and related basic proteins in condensing chromatin in some
protists and a bacterium. Perhaps it is the common need for tight packing of chromatin in motile
sperm of animals and lower plants and in the condensed nuclei or chromosomes of motile protists
that has given rise, probably by convergent evolution, to some common features within the
diverse spectrum of basic proteins binding to DNA in these different organisms.
ACKNOWLEDGEMENTS:
I wish to dedicate this paper to the memory of my sister, Joyce, who greatly encouraged these studies despite
suffering from prolonged and painful illness. I also thank my family, Vicki, Yuri, Jeremy and Leah, as well as Esther and
Kaz, for supporting both Joyce and myself during this period. My collaborators, Dave Kulak, Ellen Rosenberg, Mairi
Mackay, Niels Bols, Mike Lemke, Bill Byrd, Mike Risley, Juan Ausio, Manel Chiva, Nuria Saperas, Montse Daban,
Carlos Olivares, and Juan Subirana, have helped in many ways to see this work to fruition. I thank Sammy Lee for his
drawings and Linda Kachur for typing the manuscript.
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Source : MNHN , Paris
Male Germ Line Specific Histones
of Sea Urchins and Sea Stars
Dominic POCCIA
Department of Biology, Amherst College, Amherst, MA 01002, USA
ABSTRACT
Sea urchin sperm nuclei contain two classes of histone molecules specific to the male germ line: Sp HI and Sp H2B. Sea
stars have only Sp HI. These molecules contain repeated tetrapeptide motifs of serine-proline adjacent to two basic amino
acids in domains which are absent from somatic histones. The tetrapeptides are always phosphorylated except in mature
sperm nuclei, where they are correlated with unusual physical properties of the chromatin including high packing densities
and long nucleosomal repeat lengths. The occurrence of these molecules in the echinodcrms, their modulations during
spermatogenesis and following fertilization, their modes of interaction with DNA, and speculations concerning their
functions are discussed.
RESUME
Les histones specifiques de la lignee germinale male des Oursins et Etoiles de Mer
Les noyaux des spermatozoi'des des Oursins contiennent deux classes de molecules d' histones specifiques de la lignee
germinale male: Sp HI et Sp H2B. Les Etoiles de mer ont seulement Sp HI. Ces molecules contiennent des motifs repet£s
de tetrapeptides constitues de serine-proline adjacentes k deux acides amines basiques, dans des domaines qui sont absents
dans les histones somatiques. Les tetrapeptides sont toujours phosphoryies sauf dans les noyaux des spermatozoides murs,
ou ils sont correies avec des proprietes physiques inhabituelles de la chromatine telles que de hautes densites et de grandes
longueurs de repetition nucieosomiques. La discussion porte sur la presence de ces molecules dans les Echinodermes, leur
modulation pendant la spermatogen<ise et apres la fecondation, leurs modes d’ interaction avec I’ADN et des hypotheses sur
leurs fonctions.
The minimal eukaryotic chromosome consists of DNA and its associated basic proteins, the
histones. A histone octamer, in association with 146 bp of DNA, makes up the core nucleosome.
The core contains two molecules each of the H2A, H2B, H3 and H4 histones. The remaining
histone, HI, is almost always present and is associated with an additional ~20 bp of DNA to form
two DNA loops about the protein core. HI is also associated with a variable amount of linker
DNA between adjacent cores. The variable linker gives rise to chromatin of different average
nucleosomal repeat lengths.
The chromatin fiber may be folded into a solenoid of ~30 nm diameter or into other fiber
structures. Several higher orders of packing result in the formation of highly condensed mitotic
chromosomes or even more highly condensed sperm chromatin. Knowledge of these latter steps
of chromatin packaging is extremely limited.
POCCIA, D., 1995. — Male germ line specific histones of sea urchins and sea stars. In: Jamieson, B. G. M., Ausio,
J., & Justine. J.-L. (eds), Advances in Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 475-
489. Paris ISBN : 2-85653-225-X.
476
D. POCCIA : E CH1NODERM HISTONES
Histones are present in virtually all cell types of all eukaryotes. The histones are
distinguished by a high degree of evolutionary conservation of amino acid sequence. For
example, H4 of calf and pea differ by two conservative amino acid substitutions out of 102. H3
and H4 are the most conserved, H2A and H2B less so, and HI the most divergent. This high
degree of conservation implies that histone structure is critical for proper assembly of
nucleosomes and interaction with trans-acting factors affecting gene expression.
Not all nuclei contain histones. Among the earliest characterized chromatin proteins were the
protamines by MlESCHER in 1897 [31]. The protamines are more basic than the histones and are
not simply related to them in sequence. Their evolutionary relationship to the histones has been
discussed [82, 25, see AUSIO in this volume].
MiESCHER’s source of tissue was mature salmon sperm. It is now clear that sperm nuclear
proteins in general are free from the evolutionary constraints operating on somatic cell histones,
and the variety of non-histone structural proteins now known to be associated with sperm DNA
seems at least as great as the variety of shapes that sperm display (for reviews see [25, 42]). The
reason that these constraints do not apply may be that, once spermiogenesis is well underway,
neither transcription, replication, nuclear transport nor mitosis have any longer to be performed.
Instead, the main task is packaging of sperm DNA into a very condensed mass to be transported
to the egg. It follows that in the most extreme cases of substitution of histones by other nuclear
structural proteins during spermatogenesis, the transitions should take place late in
spermiogenesis, after normal nuclear function ceases.
Unlike salmonids or mammals, the sea urchins and sea stars retain histones in their mature
sperm cells and there are no traces of protamines or protamine-like molecules. In the urchins,
however, two of the five histones, Sp HI and Sp H2B, differ from their somatic counterparts in
ways discussed below. Similar Sp His (but not H2Bs) are found in sea star sperm. Sp histones
first appear in sea urchin spermatogenesis in the spermatogonia/spermatocytes where they
completely replace somatic histones of their classes. Thus unlike protamines, the non-conserved,
testis-specific echinoderm histones must function in typical nuclear processes.
In mature sperm, an additional role of Sp histones appears to be to confer unusual physical
properties upon the chromatin, in particular a very large amount of linker DNA which, for the sea
urchin, gives rise to the longest nucleosomal repeat lengths known, and also a high degree of
chromatin compaction. How this is accomplished and speculations on the structure and function
of these proteins is the subject of this article.
RESULTS AND OBSERVATIONS
Occurrence ofSp histones among echinoderms
Sp histones have been well-documented for sea urchins, established for a few sea stars and
may be lacking in sea cucumbers. Ophiouroids and crinoids have to my knowledge not been
investigated.
The time of radiation of the echinoderm classes including the echinoids, asteroids and
holothuroids may have been about 500-600 million years ago [32, 39, 55, 63, 64]. Resolution of
class relationships between echinoids, asteroids and holothuroids is not yet clear owing to
difficulties in resolving estimates based on different methods and a poor fossil record for the time
of their divergence [64],
All extant echinoids stem from a member of the order Cidaroida, Myocidaris [63]. The
cidaroids or “primitive sea urchins” diverged from the euechinoids or “modern” urchins about 200
million years ago. Sp histones have been found by sequence or electrophoretic analysis in all
species of sea urchin so far examined including the cidaroid Eucidaris tribuloides [81]. Eucidaris
Sp H 1 is about 9 amino acids shorter than that of the euechinoid Strongylocentrotus purpuratus
HI owing to a shorter C-terminal region. Their Sp H2Bs are of similar same size (see Table 1).
Source : MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
All
Table 1. — Relative sizes of Sp HI variants. Sizes given in number of amino acid residues.
Sea star sperm ( Aphelasterias japonica, Patiria miniata, Asterias vulgaris ) lack large Sp H2B
subtypes, but have large Sp HI molecules only one of which has been sequenced [16, 30, 81,
86]. Sea cucumber ( Holothuria tubulosa) sperm chromatin possesses somatic type histones, a
sperm specific HI, and a 78-amino acid molecule resembling the C-terminal region of somatic
His called <t>0 [3, 4]. The sequence of the sperm-specific HI is not known, and therefore its
relationship to Sp His is uncertain.
Sp histones in vivo
That sperm-specific histones of the HI and H2B classes were present in mature sea urchin
sperm was recognized with the advent of gel electrophoretic analysis [1 1, 37, 38, 72], The single
Sp HI and the two or three variants of Sp H2B appeared larger than their somatic counterparts. A
decade later, the first amino acid sequences of Sp histones were determined for Parechinus
angulosus [68-71]. These demonstrated that the larger size was due mainly to N-terminal
extensions on both Sp HI and Sp H2B (and a smaller C-terminal extension on Sp HI). The N-
terminal regions are rich in serine, suggesting the potential of multiple phosphorylation sites in
vivo. In addition, Sp HI also had a long alanine-lysine-rich region preceding the C-terminal
domain (Fig 1). As discussed below, Sp histones at first approximation can be considered
chimeric, a relatively conserved histone core altered by additions at the termini.
Subsequently it was found that of the five histone classes, only the Sp histones were
modified in male pronuclei immediately after fertilization [17, 44, 48, 49]. These modifications
were at least in part due to phosphorylation events which were mapped to serines on the N-
terminal regions of Sp H2B and the N- and C-terminal regions of Sp HI [17]. Thus sperm-
specific histones are specifically phosphorylated following fertilization. Subsequently they are
either displaced or diluted by histones from the egg during the first few cell divisions of the
embryo [49, 59].
The origin of the Sp histones during sea urchin spermatogenesis was also investigated.
Expression of the Sp H2B gene occurs in the earliest cells of the male germ lineage [51, 81], This
pattern of expression is different from that of a gene product utilized exclusively by the mature sea
urchin sperm such as bindin, a major acrosomal constituent, whose mRNA is detected late in
spermatogenesis [33]. This also contrasts with the expression of protamine genes in fish and
478
D. POCCI A : E CHINODERM HISTONES
* *HHH* *
Sp HI
a HI
* * * * *
Sp H2B-3
a H2B
Conserved core HI
Lys-ala rich region
: : Conserved region H2B
* Known phosphorylation sites
SPKK | KKSP ■ Polybasic region
Fig. 1. — Diagram of regions in sea urchin histone variants: Comparison of Sp and somatic histones.
Derived from sequences in the Protein Identification Resource database (accession numbers: Sp HI A02586, Sp
H2B-3 A02620) and in the GenBank/EMBL database (accession numbers: embryo somatic histones HI a, J01 171,
H2B a V01144).
protamines and transition protein genes in mammals which are expressed post-meiotically in
spermatids [34, 46]. The cells showing expression of Sp H2B are the spermatogonia and pre-
replicative spermatocytes, cells undergoing DNA synthesis. Indeed, the structure of the Sp H2B
gene suggests a cell-cycle regulated gene typical of somatic histones [51],
Protamine mRNAs are long-lived and their expression is translationally controlled, the
proteins appearing several developmental stages after their messages are transcribed [34, 46].
However, during sea urchin spermatogenesis, the Sp histones appear in the earliest cells of the
male germ line (spermatogonia/spermatocytes) suggesting they are under transcriptional control
[50], From their first appearance until the transition from late spermatid to spermatozoon, Sp
histones are phosphorylated [50], The major phosphorylation sites in S. purpuratus gonadal
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
479
histones were mapped to serines in the N-terminal regions of the Sp HI and Sp H2B [50], All
sites of phosphorylation were mapped in more detail using spermatid chromatin of E. esculentus
[23] and shown to occur on consensus amino acid sequences SPKK (see below). As shown in
Fig. 1, Sp HI has six of these sites on serines of the N-terminal region and three sites on serines
of the C-terminal region, only one of which was not in an authentic SPKK site (at SPQK). In Sp
H2B, the four sites were located in the N-terminal region, one of which is at the related sequence
SPSK [23],
From the parallel behavior of Sp HI and Sp H2B phosphorylation during spermatogenesis
and following fertilization, it was proposed that lack of phosphate in these histones was correlated
with extreme compaction and that phosphorylation permitted the molecules to function by
decreasing the affinity of the histone arms for DNA allowing the more conserved regions to
dominate function [17, 43, 50], It was suggested that the unphosphorylated arms serve to cross¬
link the chromatin into a compact form and stabilize it [17, 36, 82] and that this function is only
exerted in the mature sperm.
The identity of the kinase responsible for SPKK phosphorylation is uncertain. Clearly it
must act rapidly following fertilization. However, no reports of an endogenous sperm kinase with
SPKK specificity have appeared. Eggs contain kinases which will phosphorylate the SPKK
regions [14, 53, 54, 77], SPKK sites themselves are related to cdc2 kinase sites, and are almost
always present in multiple copies in C-terminal regions of somatic His, suggesting that they may
be modified by mitotic kinases. On the other hand, inhibition of male pronuclear decondensation
by amounts of the drug 6-DMAP which are sufficient to inhibit cdc2 kinase during mitosis in the
same cells has little or no effect on the phosphorylation of Sp histones after fertilization [52],
suggesting that the enzyme is different from cdc2, or cdc2 is differentially regulated immediately
after fertilization (during G1 of the sea urchin cell cycle). Kinases of the male germ line
responsible for Sp histone phosphorylation during spermatogenesis or phosphatases responsible
for dephosphorylation in late spermatids are as yet uncharacterized.
Whether phosphorylation is required for removal of Sp histones from male pronuclear
chromatin is not known. A protease which cleaves Sp HI has been isolated from sea urchin eggs
and suggested to be involved is such removal [76]. Its specificity for SPKK sequences is not
unequivocal however, and in addition there is no evidence for proteolytic degradation of Sp HI
taking place on chromatin in vivo.
Primary and secondary structure ofSp histones
Sea urchin Sp HI can be divided into four domains (Fig. 1), a conserved globular domain
characteristic of all HI molecules (amino acids 40-1 14), a long region rich in alanine and lysine
(120-185) , and the N- and C-terminal regions characterized by multiple tetrapeptides of serine-
proline adjacent to two basic amino acids (lysine and/or arginine) [43], a motif which has become
known as SPKK [73].
The Sp H2B molecules (present as two or three variants in each species) can be regarded as
having a well conserved C-terminal region, a 40 amino acid N-terminal region containing, in Sp
H2B-1, several SPKK repeats in the first ~20 residues and a glycine-rich stretch in the next 20
amino acids followed by a conserved polybasic region rich in arginine [43, 82].
Thus the Sp HI and Sp H2B molecules may be considered chimeric. The N-terminal
regions of both and the C-terminal region of Sp H 1 are unique domains related in sequence to one
another which are “added” to a more conserved core molecule. The long lysine-arginine motif of
Sp HI is present in other His where it is shorter, may contain proline and is punctuated by
SPKK motifs. Its length and lack of proline, though, make the long lysine-arginine region a
unique feature of Sp HI.
480
D. POCCIA : ECH/NODERM HISTONES
Table 2. — Repeating tetrapeptides in Sp HI and Sp H2b.
X7 = X 1 X2SPQKR where X\ = P or A; X2 = G or A.
“SPKK" sequences in bold. Sea star sequence from [30]. For original references to sea urchin sequences, see [45].
Histone Species N-Terminal sequence
Few sequences are available for sea star sperm histones. The complete sequence of H2B
from Asterias rubens sperm indicates a single variant lacking in SPKK repeats [29] consistent
with electrophoretic data indicating lack of sea star Sp H2Bs. Sequencing of the N-terminal
region of Asterias vulgaris Sp HI however shows at least two SPKK motifs are present [30]
separated by a proline (Table 2). Since the partial sequence includes only the first 1 1 residues,
additional SPKKs may be present.
The repeating amino acid sequences of Sp histones were originally described as
tetrapeptides for Sp HI and pentapeptides for Sp H2Bs [68—7 1 J. The two basic amino acids may
lie to either side of the serine-proline motif in different variants. Therefore the tetrapeptide serine-
proline adjacent to two basic amino acids is the minimal common pattern which incorporates the
repeated sequences present in Sp HI and all of the Sp H2B’s known (Table 2) [17. 43], This
grouping emphasizes the placement of a phosphorylatable serine near two basic amino acids.
SPKK, considered a subset of SPXX, is a unit of predicted secondary structure [43] in which
serine may interact with amino acids at positions 3 and 4 [73], The nature of the third and fourth
residues is not important as their interactions do not involve their side chains. The SPXX is not
always coincident with SPKK in Sp histones (Table 2). The N-terminal domains containing
multiple SPKK’s in all Sp histones are predicted as p-turn structures [17, 36, 43, 731 or extended
kinked helices [70].
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ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
481
In an early comparison of p-turn structures in a variety of proteins, 24 of 30 phosphorylated
residues from 14 different proteins were found in p-turn regions [62], As pointed out above this
is the case in vivo for SPKK regions of Sp histones which are both phosphorylated and predicted
p-tums. The four residues forming p-turns are often found at the surface of globular proteins and
greatly affect the conformation and specificity of antibody-combining sites and enzyme active
sites. They can be considered to represent cassettes which can be inserted by evolution or genetic
engineering to alter the geometry of proteins [24], SPKK “cassettes” are found in multiple copies
in a variety of nucleic acid binding proteins [47],
The tetrapeptide containing region at the N-terminus of Sp H2Bs is not its only region
predicted to have p-turn structure. The glycine-rich stretch between the SPKK repeats and the
polybasic region of Sp H2Bs is also predicted to have p-turn structure which therefore dominates
the first 40 residues of Sp H2B-1 [43].
SPKK tetrapeptides have been modelled by analogy with the nucleic acid binding drugs
neotropsin and Hoechst 33258 [73] and it has been suggested that they form p-tums stabilized by
hydrogen bonds between the OH and CO groups of serine and the NH amide groups of the third
and fourth amino acids of the tetrapeptide. More recently, a o-turn structure has been proposed to
co-exist as a conformational isomer with the p-tum in Sp HI [78].
Measurements in solution (see below) are consistent with p-tum structure, but ultimately the
question of secondary structure must be settled by crystallization of the protein both bound to and
free of DNA. The only crystal structure of an HI histone domain solved so far, for the globular
region of erythrocyte H5, is a helix-turn-helix structure likely to exist in the most conserved
domain of Sp HI as well [57],
The two tetrapeptides included in the peptide SPRKSPRK are equivalent turn structures in
exchange with a more extended structure by NMR analysis in solutions of DMSO [78]. The
peptide may exist in a conformation between a p-turn and a a-turn. In the latter, the amides of the
two serine residues are further away and fit binding to the minor groove of DNA less well. Model
building has not solved unequivocally the problem of how the binding occurs to DNA.
The long lysine-alanine stretch in Sp HI, free of proline, is predicted [43, 80] and found
15, 21] to have a high a-helical content. It has been suggested that the long a-helical stretch
accommodates the extra linker in sperm DNA [21], The long linker has been proposed to form
three equivalent superhelical turns per repeat, optimizing sperm chromatin packing efficiency in
some way [22]. Alternatively, the lysine-arginine-rich region can be considered a palindrome and
has been modeled as two.a-helices stabilizing an extra superhelical turn [75], This reverse loop or
figure-eight solenoid model would force the linker DNA into a right-handed superhelical loop
towards the centre of the solenoid structure.
Interaction of Sp histones and DNA.
Most evidence supports the idea that Sp histones bind to linker DNA and that the N-
terminal regions prefer the minor groove of AT-rich regions. HI histones are known to bind to
linker DNA, but Sp H2B may behave similarly. Linker binding was originally suggested on the
basis of indirect evidence [17, 86], but more recent measurements confirm the proposal.
Reductive methylation of lysines after their exposure following dissociation from linker DNA is
consistent with binding of Sp H2B through its N-terminal region [20], Crosslinking of DNA to
Sp H2B histone also demonstrates linker binding [7], Sp H2B in hybrid reconstituted
nucleosomes protects an additional 8 bp of linker DNA against micrococcal nuclease digestion in a
more efficient way than wheat H2A, which has a similar extension but lacks SPKK motifs [28].
Binding of Sp histones to linker DNA may involve short-range or long-range, intrastrand
or interstrand, interactions. Although evidence for cross-linking of chromatin fibers in vivo by Sp
histones is lacking, many studies demonstrate the unique abilities of Sp histones to bind strongly
to DNA resulting in aggregation. For example, Sp HI is more effective at aggregating
482
D. POCCIA : E CHINODERM HISTONES
superhelical DNA than somatic His such as calf thymus histone [36], In reconstitution of SV40
DNA with core nucleosomes, at intermediate ionic strengths, sea urchin core histones form large
intermolecular associations which have been attributed to the Sp H2B [35].
Current data favor a model of Sp histone binding to the minor groove of AT-rich regions
of DNA. Based upon binding competition studies of the N-terminal peptide of Sp HI and
Hoechst 33258, it was proposed that Sp HI binds to the minor groove [73], although others have
found no evidence for competitive inhibition [19]. Further support for binding to AT-rich regions
was obtained by hydroxyl radical footprinting [8] using a synthetic peptide of two SPKK’s. For a
more detailed discussion of this subject, see [9].
Binding of DNA to the synthetic octapeptide SPKKSPKK conjugated to anilino-acridine
was studied using DNase 1, OH-radical footprinting and osmium tetroxide. AT-selectivity of
binding supported a minor groove binding model [5] and was interpreted as SPKK recognition of
defined DNA sequences. Although a single tetrapeptide could unwind and extend DNA and
distort the double helix, two SPKKs seem to be minimally necessary for the AT-specific minor
groove binding [13].
Conformational changes in DNA have been noted upon binding of the octapeptide
SPKKSPKK. This peptide without the acridine binds to calf thymus DNA or various
polynucleotides. As assessed by circular- dichroism, only binding to poly (dA-dT)»poly(dA-dT)
induces major changes in the spectrum consistent with a <p-type condensation pattern [6]. Other
peptides containing TPKK alter DNA condensation giving <p-type CD spectrum and it was
concluded that the Hip-turn, although it becomes more rigid, remains a p-turn upon DNA
binding [12].
What role, if any, selectivity for AT-rich regions plays in sperm chromatin structure is not
clear. The stoichiometry of histone-DNA binding in vivo implies that Sp histones bind to all the
nuclear DNA and it would be surprising if binding is not essentially uniform.
Effect of Sp histone phosphorylation on DNA binding.
Phosphorylation of the serine of the SPKK repeat should have two important consequences
for its DNA binding. First the resultant addition of two negative charges per repeat should
effectively neutralize the entire structure thus drastically lowering its DNA affinity. Second, by
disrupting the serine hydroxyl group, the phosphate should interfere with the secondary structure.
Fewer studies have been made of the interactions of phosphorylated Sp histones with DNA
than with the unphosphorylated forms. But some direct measurements support the concept of
weaker binding of phosphorylated N-terminal regions to DNA. Intermolecular migration of Sp
HI which involves dissociation from the DNA is facilitated by phosphorylation [23],
Phosphorylated Sp HI binds less tightly to DNA by affinity column chromatography [23, 73].
Multiple phosphorylation in the N-terminal but not the C-terminal regions of Sp HI drastically
reduces DNA affinity [23], Purified N-terminal domains of Sp HI from sperm and male germ
cells were compared by several criteria including DNA cellulose binding, salt induced
precipitation, thermal denaturation of DNA and inhibition of HO 33258 binding which showed
that binding to DNA was always weaker for the phosphorylated peptides [19]. Unphosphorylated
Sp HI induces salt-dependent folding of long oligonucleosomes and promotes DNA self¬
association more effectively than phosphorylated [23],
Since sea urchin sperm chromatin has the longest repeat length known, it has been
suggested that Sp HI and Sp H2B arms might bind to and neutralize linker DNA [17, 18, 86] as
discussed above. The correlation of long linker DNA in sperm chromatin with dephosphorylation
and shorter linker in spermatogenic or pronuclear chromatin with phosphorylation suggested that
the arms of the Sp histones might bind to linker only when their phosphates were removed [17,
18, 60]. When unphosphorylated Sp histones are bound to linker, the DNA is more resistant to
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
483
micrococcal nuclease digestion than either pronuclear [60] or spermatid [18] chromatin linkers
which may be bound weakly if at all by the N-terminal arms.
Sp histones and the structure of sperm chromatin
Sperm nuclei containing unphosphorylated Sp histones have several properties which may
be dependent upon these special nuclear proteins. These include high chromatin compaction, the
shape of the nucleus, and the exceptional amount of linker DNA of the chromatin.
Sea urchin sperm heads are generally conical; sea star and sea cucumber spherical. Thus
conical sperm nuclear shape in the echinoderms is correlated with the presence of Sp H2B,
occurring only in the sea urchin. The transition from spherical to conical morphology occurs in
the late spermatid, when dephosphorylation of Sp histones takes place, and the sea urchin late
spermatid resembles the sea star mature sperm in nuclear shape. However, it is not clear whether
nuclear shape is dependent upon dephosphorylated Sp H2B.
Sea cucumber sperm chromatin is not as highly condensed as sea urchin or sea star
chromatin [40, 41]. Thus the highest degrees of chromatin compaction occur in the sea urchin and
sea stars which contain a Sp HI, but again the relevance of this has not been demonstrated. In
vitro , oligonucleosomes from sea urchin sperm seem to be more compact compared to somatic
chromatins by sedimentation criteria [83], In addition, circular dichroism measurements indicate
that sea urchin sperm chromatin behaves uniquely with respect to compactness as a function of
ionic strength [58].
It is impossible at present to compare in detail the higher order structure of chromatin in the
nuclei of echinoderm sperm because preparation artifacts and current methodology do not permit.
Sea star nuclei show regular ordered structures in minimally disrupted nuclei by electron
microscopy [16]. Possibly related higher order structures have been reported in sea urchins as
well [87, 88]. Chromatin fibre diameters vary between species but these are apparently not
correlated with repeat length [1, 65, 85].
Whatever the exact structure of chromatin in the mature sperm nuclei, certain correlations
can be made with the degree of compaction and Sp histone phosphorylation. Sea urchin sperm
chromatin is packed about as densely as mitotic chromosomes [17]. Phosphorylation of Sp
histones occurs prior to decondensation of the chromatin following fertilization [17]. If
decondensation of chromatin is blocked by the kinase inhibitor 6DMAP, Sp histones appear to be
normally phosphorylated [52], This indicates that phosphorylation is not sufficient to induce
decondensation, although it might still be necessary. Likewise it suggests that, in spite of the
similarity in target sequences, the Sp kinase is not the cdc2 mitotic kinase which is inhibited under
the same conditions.
The nucleosomal repeat lengths of sea star and sea cucumber sperm chromatins are shorter
than sea urchin but longer than typical somatic chromatin repeat lengths (Table 3) [10, 86]. Repeat
lengths increase in the sea urchin at the last stages of spermatogenesis when Sp histones are
dephosphorylated [18]. In contrast, repeat lengths are constant during sea cucumber
spermatogenesis [10]. Sea star and sea cucumber sperm chromatins each have repeat lengths
approximately 20 base pairs (bp) shorter than sea urchin [10, 86]. Analysis of sperm chromatin of
crinoids and ophiuroids is unavailable.
Thus the longest nucleosomal repeat lengths are correlated with the presence of both Sp
H2B and Sp HI. Given data demonstrating that both bind to linker DNA, this is not surprising.
However, sea cucumber sperm nucleosomal repeat lengths are about the same as those of sea
stars. We do not know enough about the chemistry of the special sperm HI and 4>0 in sea
cucumber to be able to speculate about their relationship to long linker.
It is not obvious whether the long linker is a consequence of Sp histone binding or is
controlled by some other factor. Sea urchin sperm HI is more efficient than somatic His at
484
D. POCCIA : E CH1N0DERM HISTONES
Table 3. — Average nucleosomal repeal lengths of echinoderm sperm chromatins
organizing nucleosomes at low packing densities in reconstitution systems in vitro using
polyglutamic acid and poly d(A-T)«poly d(A-T) [67]. Sperm core histones (containing Sp H2Bs)
are likewise more efficient at being recruited [67] . In this assay, Sp HI gives repeat lengths
approximately the same as those in vivo. It would be of great interest to determine if
phosphorylation of Sp histones shortens the nucleosomal repeat length, using in vitro chromatin
reconstitution systems.
DISCUSSION
Phylogenetic trees for core histones have recently been constructed [79], The H2A/H2B
histones which form dimers appear to have co-evolved and are 10 times more divergent than the
H3/H4 pair. Sea urchin Sp H2B was the only divergent core histone without a correspondingly
divergent H2A. It was suggested that sea urchin Sp H2Bs evolved more rapidly than other sea
urchin H2Bs or H2As and that they perform a function that is atypical or assemble nucleosomes
in a different way free of typical constraints. Based on the discussion above, it seems likely that
Sp H2B function in the unphosphorylated state is atypical, namely the packaging of sperm
chromatin, but that it can assemble proper nucleosomes when in the phosphorylated state when its
conserved regions dominate its function.
It has been suggested that the repeating structure of Sp H2B is related to the structure of
vertebrate protamines based upon (1) the similarity of basic residue spacing between proline and
hydroxyamino acids and the nature of the basic residues (arginines) when the repeat is considered
a pentapeptide of the form pro-X-X-X-ser (with two of three X’s basic), and (2) the similar size
of protamines and the Sp H2B N-terminal extension [82], In this view vertebrate protamine genes
would have evolved from the N-terminal region of Sp H2B toward greater basicity and more
efficient DNA packaging.
However, similar motifs are not unknown among invertebrates. For example, sperm-
specific protein $3 from the mussel Mytilus californianus has two SPKK motifs punctuating an
otherwise largely lysine-alanine-rich sequence [2], This molecule of 5 kD is predominantly a-
helical in structure and in some ways resembles the C-terminal region of Sp HI. In what way
should we imagine its evolutionary relationship to sea urchin Sp histones?
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
485
The scheme proposed by AUSIO in this volume takes a different approach to the origin of
protamines. It postulates a primitive histone precursor giving rise to somatic His and sperm-
specific basic nuclear proteins. Echinoderm Sp histones would result from an increase in arginine
content of the precursor and would co-exist with histones, whereas in other organisms
intermediate type protamine-like (PL) molecules would evolve and replace somatic histones. By
loss of their globular cores, these would become small PL molecules as found for example in
Mytilus. The small PLs might then evolve to protamines. This scheme would, however, have to
take into account the appearance of similar SPKK domains on the N-termini of both Sp His and
Sp H2Bs and the possibility of their assembly from scattered SPKK domains seen in somatic His
remains.
Another problem concerns speculation on evolutionary and functional relationships merely
on the basis of amino acid sequence similarities. A good example is the central globular domain of
HI histones which is highly conserved. The crystal structure of this domain of the HI variant H5
from erythrocytes was recently solved to 2.5 A resolution [56, 57, 61]. Its secondary structure is
remarkably similar to a liver-specific transcription factor HNF-3y in a helix-turn-helix motif.
However, sequence similarity between the two proteins is limited (12% of the structurally
equivalent residues). Each is a member of a large family of proteins highly conserved in sequence
and function. The choice of whether these proteins have diverged from a common ancestor or
converged to a common three-dimensional structure is uncertain.
The presence of very long nucleosomal repeat lengths and Sp histone molecules in sperm of
echinoids and asteroids suggests that these related molecular patterns were developed by a
common ancestor and conserved to the present day. From the functional standpoint, it is not
obvious why such long repeats are useful, except to allow binding of the extensions of the Sp
histones. That this binding may allow a special kind of compaction of chromatin in these
organisms has been discussed above. Certainly in a functional sense, the arms of Sp histones
might be considered to lend protamine-like qualities to the Sp histones.
The motif SPKK or its variants (threonine instead of serine, arginine instead of lysine) is
always present in the C-terminal domain of somatic HI molecules and could represent a cassette
borrowed by other molecules which would function in strong but reversible nucleic acid binding.
The occurrence of the multiple tetrapeptides in a variety of DNA and RNA binding proteins has
been noted [47]. It has also been argued that the related SPXX motif is found often in gene
regulatory proteins [74], Yet unfortunately too few data are available for comparisons to be made
among the sperm histones of echinoderms to generalize about similarities of function of
evolutionary relationships.
Since sperm cells compete for successful fertilization, they can be under great selective
pressure for the brief period following spawning. This may depend on events occurring before
fertilization, such as local environmental conditions, or after, such as the ability of the egg to
rapidly disassemble and reassemble male chromatin. It would seem likely that efficient packaging
of the sperm nucleus is an advantage in marine environments for motile sperm. However, co¬
evolution of egg mechanisms for formation of the male pronucleus from the sperm nucleus
following fertilization may limit the rate at which sperm nuclear structural proteins can evolve.
ACKNOWLEDGEMENTS
The author acknowledges the support of the National Science Foundation (Grant DCB 9004170) and an Amherst
College Faculty Research Award.
486
D. POCCIA : E CH/NODERM HISTONES
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Source : MNHN. Paris
Source : MNHN. Paris
The gene encoding the sperm-specific basic nuclear
protein 0O from sea cucumber
Eva PRATS & Luis CORNUDELLA *
Department of Molecular and Cell Biology,
Centro de Investigation y Desarrollo del CSIC, Jordi Girona 18-26, Barcelona, E-08034, Spain
* correspondence and reprints
ABSTRACT
The gene encoding the sperm-specific protein <j>0 from the sea cucumber Holothuria tubulosa has been cloned and
characterized. Sea cucumber sperm chromatin displays a somatic-like histone complement and is beaded with a constant
227 bp DNA linker length, second to the longest repeat of sea urchin chromatin. Protein <t>0, a small basic protein
reminiscent of the C-terminal tail of histone HI, appears at the onset of spermiogenesis and accumulates in ripe sperm.
The <>0 gene displays a coding frame interrupted by three large intervening sequences which combine to make it the longest
for a sperm-specific protein yet reported ( ca . 17.7 kb). The identified gene is present as a single copy and, in turn, encodes
a polyadenylated transcript. The protein «j»0 specified by this gene has 77 residues, reproducing unaltered the partial amino
acid sequence of the protein previously determined. The structural arrangement and content of the <j>0 gene are totally
unrelated to cell-cycle regulated histone gene structure. Instead, it combines several features common to replication-
independent genes coding for histone variants and even to protamine genes. Inference is made about the potential
implications of this divergence in gene arrangement as regards chromatin transitions and modulation of gene activity
occurring during spermiogenesis.
RESUME
Le gene codant pour la proteine basique nucleaire <{>0 specifique des spermatozoi'des chez
rholothurie
Le gene codant pour la proteine basique nucleaire <j>0 specifique des spermatozoides chez fholothurie Holothuria tubulosa
a 6l6 clone et caracterise. La chromatine du spermatozoTde d’holothurie presente un complement d' histone de type
somatique et forme un chapelet avec un intervalle constant de 227 paires de bases d’ADN, qui est la seconde plus longue
repetition de chromatine d’echinoderme. La proteine $0, une petite proteine basique qui rappelle l'extremite C-terminale de
I’ histone HI, apparait au d6but de la spermiogen&se et s’accumule dans les spermatozoides murs. Le gene <?>0 comprend une
region codante interrompue par trois grandes sequences intercalaires qui se combinent pour en faire le gene le plus long
connu actuellement pour une proteine specifique des spermatozoides (environ 17.7 kb). Le gene identify est present en
copie unique et code pour un transcrit polyadenyle. La proteine <>0 specifiee par ce gene a 77 residus parmi lesquels on
retrouve sans erreurs la sequence partielle d’acides amines precedemment determinee pour la proteine. L’organisation
structural et le contenu du gene <j>0 sont sans relations avec la structure des genes des histones regules par le cycle
cellulaire. Au contraire, ce gene combine plusieurs caracteristiques communes aux g5nes independants de la replication
codant pour des variants d' histones et meme aux genes de protamines. La consequence potentielle de cette divergence dans
(’organisation des g£nes en ce qui conceme les transitions de la chromatine et la modulation de l’activii^ du g£ne pendant
la spermiogen£se est discutee.
Prats, E. & Cornudella, L., 1995. — The gene encoding the sperm-specific basic nuclear protein <j>0 from sea
cucumber. In: Jamieson, B. G. M.. Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoa! Phylogeny and Taxonomy.
Mem. Mus. natn. Hist, nat., 166 : 491-500. Paris ISBN : 2-85653-225-X.
492
E. PRATS & L. CORNUDELLA : PROTEIN <pa GENE IN SEA CUCUMBER
Eukaryotic chromatin is a macromolecular nucleoprotein assembly essentially composed of
DNA and basic proteins. Histones, which are relatively conserved through evolution, are the
genuine protein components in chromatin of non-proliferating cells and are involved in the
organization of the chromatin fibre into various structural hierarchies (for full review see [42]). In
stark contrast, DNA in male germ cell lineages appears to be bound by widely diverse basic
proteins as regards chemical composition and number, giving rise to a seemingly striking variety
of protein molecules along the different zoological groups [32], spanning from those species
which retain histones that are close to the somatic types, to those in which they are fully displaced
by more basic proteins like nucleoprotamines [7]. The chemical data reported comprise a variety
of vertebrate species, from fishes [9, 17] to mammals [25], It is apparent that an enhanced
basicity of the sperm proteins favours a tighter packaging of DNA. This requirement in
spermatozoa seems obvious as a means of protecting the genomic complement. Likewise, it may
also be an evolutionary adaptation of sperm to endure long-term storage and transport in the
absence of DNA repair mechanisms. However, the functional significance of basic protein
diversity in sperm and its effect at the molecular level are still ill-defined. DNA packing may not
be the exclusive role. The actual existence of germ-line variants and sperm-specific protein types
argues for more discriminating assignments such as fine-structural transitions of chromatin related
to modulation of gene activity and its final quiescence during spermiogenesis [14].
The variability of sperm nuclear proteins is of unknown origin. A hypothetical evolution of
some of these proteins from histone HI has been put forward on the basis of the protein
composition of the sperm of some bivalve molluscs [4], Histone HI and its many subtypes
constitute the most heterogeneous of the histone classes [41]. Its largest evolutionary sequence-
variation appears confined to both the N-terminal and C-terminal extensions of the molecules
whereas the hydrophobic central globular core remains fairly well conserved [13]. This
asymmetric organization has led to the suggestion that the carboxyl-terminus is involved in the
higher order compaction of chromatin [1], Models to test that assumption are provided by marine
invertebrates, particularly echinoderms and molluscs. These organisms deserve particular mention
since their somatic histones apparently coexist with both sperm-specific variants and protamine¬
like molecules, different from fish or mammalian protamines [3, 32, 43]. These protein molecules
mostly fit into classes 3 and 4 of BLOCH's cytochemical categorization of nucleoproteins in
mature sperm cells [7], These two types have been ulteriorly combined into a rather
heterogeneous group of intermediary sperm-specific types, plausibly representing transitions from
histones to protamine-like molecules [32].
Although the prevalent cellular histones are encoded by a highly reiterated multigene family
whose expression is tightly coupled to DNA replication, histone-variant genes tend to be present
in single or few dispersed copies not subjected to cell cycle regulation [11, 23], Already
classification schemes based on regulatory correlations have been devised [43], Nonetheless, very
little is known about the organization of tissue-specific, variant-histone genes, along with the
evolutionary origin of nucleoprotamine and protamine-like genes [30].
It is important to address these questions and obtain new evidence concerning histone to
protamine transitions and the genes that encode them, aiming to understand at the molecular level
their differential function and its influence on the structural organization of sperm chromatin. Our
work has been involved in the analysis of chromatin from the germinal tissue of the echinoderm
Holothuria tubulosa. During sperm maturation there is no bulk replacement of the histone
complement, transitions being restricted to the addition of a sperm-specific, arginine-rich HI
variant [31] and the presence in ripe sperm of a small basic protein termed 4>0 [40]. The latter has
an ammo acid composition reminiscent of the carboxy-terminal region of sea urchin Hl-S,
provided that Arg is considered equivalent to Lys [2], Incorporation of protein $0 into chromatin
occurs in the terminal stages of spermiogenesis [10], representing about 4% of the histone moiety
ol the mature spermatozoa. Nucleosome organization remains invariable throughout sea cucumber
spermatogenesis with a constant DNA linker length of 227 bp [15] consistent with sea urchin
Source : MNHN , Parts
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
493
nucleosomal repeats, which exhibit the longest lengths ever measured. The isolation and sequence
determination of a cDNA for H. tubulosa protein <t>0 have been previously reported [33]. In the
present paper we describe the molecular cloning and characterization of the gene encoding this
protein specific to the sea cucumber sperm chromatin. This is the first gene coding for a histone-
to-protamine transition protein to be identified.
MATERIAL AND METHODS
Living organisms. Male specimens of the sea cucumber Holothuria tubulosa were collected periodically off the
catalonian shore during the breeding season, moved live to the laboratory in cold seawater and held in 8°C seawater until
used. Excision of gonads and sperm collection were performed as detailed elsewhere [36].
Isolation and purification of genomic DNA. High molecular weight genomic DNA was extracted from fresh sperm
suspensions essentially as described [15]. Briefly, suspensions were treated with proteinase K (50 pg/ml) overnight at
37°C. After incubation, samples were deproteinized by successive phenol and chloroform extractions and the aqueous
phases precipitated with ethanol. The DNA was further purified by Cesium chloride banding and subsequent dialysis.
Construction and screening of a sea cucumber genomic library. For construction of the H. tubulosa genomal
library, purified sperm DNA was subjected to a partial digestion with Mbol to generate fragments with flamHI-compatible
overhangs and subsequently size-fractionated by sucrose gradient centrifugation. DNA fragments in the size range 12-20
kb were pooled and ligated to dephosphorylated lambda-based Charon 35 (ACh35) replacement vector [26] linearized with
BamHl. Ligation reactions were carried out at a vector to insert molar ratio -of 2:1. Recombinant phages were encapsidated
and used to transform E. coli 555 recA' cells yielding a titre of 3xl05 plaque-forming-units (pfu) per |ig of ligated DNA.
The genomal library was screened by in situ plaque hybridization [5]. Plaques at a density of 104* pfu were replicated onto
nitrocellulose membranes and screened with a 441 bp long H. tubulosa (j>0-cDNA clone [33] labelled by random-priming
with the Klenow enzyme [21].
Positive plaques were purified by plating at decreasing densities and the isolated phages were grown by cascade
infection and banded onto ethidium bromide-containing CsCl gradients. The resulting DNA was purified by phenol and
chloroform extractions and used for further analysis. All recombinant DNA manipulations were carried out by standard
procedures [37] and conducted in accordance with established guidelines for recombinant DNA research.
Restriction analysis and Southern transfers. DNA from positive recombinant clones was digested with selected
endonucleases and pairwise combinations thereof. Where required, restriction fragments were electrophoresed on agarose
gels, transferred to nylon membranes by alkaline blotting [34] after partial depurination and screened with six different
regions of the «j>0-cDNA cloned insert as probes.
<P0 gene number. To assess the copy number of the <$»0 gene in the haploid sperm genome of H. tubulosa , genomic
DNA was digested, independently or in combination, with various endonucleases not cleaving inside the «j>0-cDNA
sequence. Restriction fragments were subsequently electrophoresed on 0.5% agarose gels, blotted onto a nylon membrane,
and hybridized to labelled probes prepared from the <j>0-cDNA clone. The $0 gene number was derived by comparison of the
number and intensity of the autoradiographic signals measured by densitometric analysis, with those from graded amounts
of the cloned (j»0-cDNA equivalent to integer-copies per haploid genome of the cDNA sequence. Standards of cDNA were
electrophoresed in parallel, supplemented with a mass excess of sheared calf thymus DNA to compensate for the amount of
restricted sea cucumber sperm DNA loaded on each gel slot.
Plasmid subcloning and nucleotide sequence analysis. Genomic DNA restriction fragments of appropriate size
identified by hybridization analysis as containing $0-cDNA sequence tracts were excised off the gel, purified further on
low-melt agarose, and ligated into the phagemid vector Bluescript +SK. Chimeric recombinants were used to transform
competent E. coli XL 1 -blue recA' cells and were selected as AmpRTcR:Lac' phenotypes. DNA inserts from the
recombinant plasmids were sequenced by the dideoxy chain-termination procedure [38] using the Sequenase system with
forward and reverse primers for both orientations. Computer analysis was performed using the MicroGcnie sequence
analysis software (Beckman, USA).
RESULTS AND DISCUSSION
Spermatogenesis in the sea cucumber H. tubulosa is a rather simple process. Chromatin
from ripe spermatozoa retains the five somatic-type histones in normal relative amounts
accompanied by the highly basic protein <j>0 (average Mr = 8640), structurally related to histone
HI [2], We have previously reported the characterization of a clone carrying a full length <j>0
transcript, isolated from a cDNA expression library made from the poly (A+) fraction of total
RNA extracted from immature gonadal tissue and screened with polyclonal anti-<j>0 antibodies
[33], The 441 bp cloned cDNA encompassed a continuous open reading frame for a basic
polypeptide of 77 residues whose sequence conformed to the partial amino acid sequence of 4>0
previously established. Likewise, poly (A+) selected RNA yielded a product electrophoretically
494
E. PRATS & L. CORNUDELLA : PROTEIN <p0 GENE IN SEA CUCUMBER
comigrating with protein <t>0 upon in vitro translation in wheat germ, cell-free extracts, whereas
Northern blot analysis detected only a 0.6 kb <))0-mRNA transcript homologous to the cDNA
probe.
Isolation of the sea cucumber <t>0 gene
The genomal library of H. tubulosa sperm DNA cloned into the ACh35 vector was screened
by hybridization with the <t>0-cDNA cloned insert. The screening of about 250 000 plaques
yielded four positive clones with inserts of -16.5, 14.4, 13 and 14.5 kb, named AHt7, AHt2,
AHtl and AHt8, respectively. The four recombinants were next subjected to endonuclease
restriction and Southern blot analysis using six cDNA-derived probes encompassing specific
regions of the <j)0-cDNA clone: (i) the two asymmetric segments resulting from the cleavage along
the EcoRl site internal to the <j)0-cDNA inseit, namely, the 248 bp leader (5' probe) and the 193 bp
trailer (3' probe) fragments, encoding the first 69 and last 8 amino acids of protein 4>0,
respectively; (ii) the 81 bp long EcoRl-Pstl fragment comprising the 5'-flanking region and the 42
bp sequence coding for the 14 amino acids inclusive of the initial methionine residue heading the
N-terminus (P probe); (iii) the Ddel-EcoRl fragment of 81 bp representing amino acids 43 to 69
(D probe); (iv) the two segments of 1 13 bp and 80 bp in length {XI and X2 probes) resulting
from the nibbling of the J'-probe at the single Xbal site. Probe XI contained the sequence for the
last 8 amino acids of the C-terminus plus 88 bp of the adjacent downstream extension whereas
probe X2 consisted of the final 80 bp of the 3'-noncoding region of the <j>0-cDNA clone. Those
restriction fragments shown to carry 4>0 sequences were subsequently subcloned for further DNA
sequence analysis.
Ht7
I -
- 1 Htl
Ht2
- - - 1 Ht8
Fig. 1. Isolation and characterization of the Holothuria tubulosa $0 gene. The restriction endonuclease map and
organization of the <t>0gene are shown. The four positive isolates picked out from the sperm DNA library of 10-20
kb Mbo\ partials cloned into the fiamHl replacement vector ACh35, were digested with several endonucleases and
combinations thereof. Restriction fragments carrying (|>0-cDNA sequences were identified by hybridization.
Posmvely reacting fragments were further purified, subcloned into the phagemid Bluescript +SK and sequenced by
the dideoxy chain-termination procedure of Sanger. Filled boxes indicate the relative positions of exons encoding
sequences consecutively numbered I to 4, and open boxes the 5' and 3’ flanking regions homologous to the
noncoding extensions of the cloned tj>0-cDNA. The hatched rectangular boxes highlight those regions of the gene
that were completely sequenced either on both DNA strands or repeatedly in one direction. D , D, E, H , P , Sl% Ss , and
X denote ligll, Dde\, Eco RI, Hindlll, Pstl, Sal I, Sstl, and Xbal restriction sites, respectively. The thin lines depict
the positions of the four strongly hybridizing genomic clones (AHt 1 - 8) used to map the <j>0 gene.
Source . MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
495
The results of the restriction mapping are shown in Fig. 1 and can be summarized as
follows. The entire <])0 gene appeared split into four distinct exon sequences scattered along the
lengths of the clones. The latter displayed partial overlaps differing in extension. AHt7 harboured
the 5’-proximal exons 1, 2 and 3. The central exons 2 and 3 were also present in AHt2 and AHt8.
In addition, the former contained the first 79 nucleotides of the fourth exon whereas the latter
spanned its entire coding sequence extending 831 nucleotides beyond the stop codon. The
shortest clone AHtl carried only exon 1 centrally positioned within the genomic DNA insert.
On the basis of these results it was feasible to correlate the four recombinants and to
conclude that the coding sequence of the sea cucumber <j>0 gene is interrupted by three long
intervening sequences which amount to 16.2 kb in total length (see Fig. 1). These unusually large
introns combine to make the §0 gene the longest for a sperm-specific protein ( ca . 17.7 kb) so far
reported. The overall organization of this sea cucumber gene appears to significantly diverge from
that of the intron-less histone genes [27] and also from the arrangement of mammalian, single-
intron protamine genes [30].
Sequence analysis of the ip0 gene
The nucleotide sequence of the sea cucumber <|>0-gene is shown in Fig. 2. The coding
sequence is discontinuous and encompasses an open reading frame for a basic protein of 77
residues, interrupted by three introns involving canonical splice junctions [24], specifically those
assigned to invertebrates [22], The first and second introns of 6.8 and 4 kb in respective length,
are inserted within codons 9 and 15, respectively. The third intron is 5.4 kb long and is
positioned contiguous to codon 41. The complete coding sequence of the <j>0 gene is identical to
that of the cDNA clone previously reported [33] but for six nucleotide substitutions (97.9%
homology). Five changes are conservative since they correspond to third-base degenerates of the
most common synonym triplets, involving C for T and A for G conventional exchanges with no
ensuing alteration of the assigned amino acid. The only relevant nucleotide substitution occurs at
codon 61 and involves a G for A replacement in the first base causing a change of amino acid
assignment. The overall level of sequence conservation observed argues for a stable organization
of this gene.
The deduced primary structure of the encoded protein corresponds exactly with the <J>0
sequence specified by the cDNA clone, exclusive of the noted single alteration at codon 61
(98.7% homology). The cDNA sequence contains the triplet GCC for alanine in this position
while the ACC counterpart in the gene codes for threonine. Most likely this difference arises from
the DNA polymorphism detected in echinoderms [12] which is reflected in the well-documented
intraspecific microheterogeneity found in a substantial variety of sperm variant proteins from
marine invertebrates [28] as well as in the protamines of trout [18].
The close sequence homologies between the <t>0-cDNA and the cloned gene are endorsed by
the identity of the deduced protein sequences which, in addition, reproduce wholly unaltered the
partial amino acid sequence of protein (J>0 previously established. The coincident similarities
observed sustain the conclusion that, in actuality, the cloned gene encodes the sperm-specific
protein ())0 in H. tubulosa.
Comparison of the nucleotide sequences in the noncoding regions of the <])0 gene with those
of replication-dependent histone genes reveals that the former lacks the conserved motifs defining
the S-phase regulated histone gene structure such as the downstream hairpin loop sequence at its
3' proximal purine-rich tract required for 3' processing of the histone transcripts [6]. Instead, the
leader and trailer regions surrounding the <J>0 gene combine several structural elements found in
both replication-independent histone variant as well as protamine genes (see Fig. 2).
The region upstream of the initiation codon contains an atypical TATA motif identical to the
TTCAAA box identified in the cell-cycle independent H2Ap histone gene that codes for an
extreme H2A variant in chicken, whose transcript is polyadenylated [16]. Significantly, both
496
E. PRATS & L. CORN UDELL A : PROTEIN tp0 GENE IN SEA CUCUMBER
elements are found similarly positioned 144 nucleotides upstream from the initiator triplet. There
is another potential TATA motif with perfect homology to the non-canonical TTAAAT element
present in both the chicken and duck H5 genes [19, 35]. This sequence starts at position -205, 65
and 28 nucleotides further upstream than the mentioned homologues, respectively. Another
general feature required for promotion of transcription by RNA polymerase II is a CAAT
sequence often located between 70 to 90 bp upstream of TATA sites [8]. In this regard, the leader
region of the <]>0 gene displays a potential CAAT motif (-221 to -218) located 73 nucleotides
upstream relative to the H2Ap-like TTCAAA box. Another motif shared with leader regions in
protamine genes is the TGACGTCA sequence found far upstream in the 4>0 gene (-489 to -482).
This c/s-acting element, usually referred to as a cAMP regulatory element (CRE), is strictly
conserved in all protamine genes and it is considered essential for the biological activity of cAMP-
regulated enhancers [30], Since spermatogenesis in echinoderms is known to be under hormone
control probably involving cAMP [39], such a regulatory signal might well represent a link
between hormonal signals and the expression of sperm-specific genes such that of protein <J>0.
The downstream extension of the <f0 coding sequence is devoid of the highly conserved
structural features of the replication-dependent histone genes required for the 3' end formation of
histone transcripts. Instead, similarities with the equivalent regions in protamine genes are
encountered. First, three potential polyadenylation signals are present, starting at positions 306,
392 and 396 respectively, 3' to the TAA stop codon. The last two elements consist of the
heptamer AAATAAA which appears repeated with a trinucleotide overlap. This heptameric
sequence motif bears a perfect homology with the conserved polyadenylation signal found in the
protamine genes from salmon and trout [29] . Nonetheless, the significance of the close
similarities encountered between the organization of the <J>0 gene and that of genes coding for
extreme histone variants or even for protamines remains to be unambiguously defined.
Genomic content of the <pa gene
The copy number of the q>0 gene was determined by Southern blot hybridization analysis of
sperm DNA restriction digests with the 81 bp long EcoRI-Psrl fragment (probe P) of the <t)0-
cDNA clone comprising the 5'-flanking extension and the initial 42 bp of the coding sequence,
labelled by random priming. A set of endonucleases lacking cleavage sites within the cDNA
sequence was selected for the single and double enzyme digestions. DNA restriction fragments
were electrophoretically resolved in conjunction with varying amounts of the cloned <)>0-cDNA
insert, diluted with a mass excess of sheared heterologous DNA to make up for those of restricted
sperm DNA loaded on the gel lanes. The amounts of the cDNA standards, equivalent to one and
four copies of the respective sequence per haploid genome, were inferred from the DNA content
of the haploid genome (C-value = 3 x 109 bp) of H. tubulosa sperm previously determined [36],
Hybridization patterns from both single and combined enzyme restrictions yielded in every
case only one size class of DNA fragment positively hybridizing with the cDNA probe (Fig. 3).
Fig. 2. — DNA sequence of the sea cucumber $0 gene and Hanking regions. The 5'->3' nucleotide sequence of the non-
transcribed strand (i.e. mRNA-like) is given along with the amino acid sequence derived from the coding region,
shown above the nucleotide sequence. The abbreviation ini and the asterisk mark the respective positions of the
initiator and the stop codons. The noncoding leader and trailer extensions are numbered with negative and positive
numerals begininng at nucleotides 5' adjacent to the initiation codon and 3' proximal to the stop triplet,
respectively. Coding triplets are denoted with numbers in italics. The slash symbols mark the normal donor splice
site junctions. Positions of the putative CRE, CAAT and TATA elements as well as the poly(A) addition signal
discussed in the text, are doubly underscored. Most of the extensive intron sequences have been removed for
clarity.
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
497
Exon 1
-700 -650
GACTGCTCACGAGTGATAGCGCCCAAACAAGCGTCCAAGAGGCCGAACAGACGCTAAGGCTCGCTACGGCGCGTTAGCAT
-600
GTACAAAATTGCCTCGGCGTTTTCACTAGTATACGCGTTTCTACGGCGATGGAATCGACTTGAGTAAGCTGACCTACATC
-550 -500
ACGTGACCTTCCATGATGGCTTCACAACAACACTGTGGGATGCGCCTTCCTGTTATATAGTTCAGTGGAGCCGATGCTTA
-450
I^ggXg^TAATTTGGTGTAGAAGTCTAGTCCACTGCATATTTCCAAACGTGAAGAAACGGTTTGAGACCGAATCAAATG
-400 -350
CATTTTTCTTACCAGCTTACCGCGCTCTGGAATACGATGAGTATGTATGCAATCCTCAGTTCAAAGCAAAGTGGACGCAC
-300 -250
TACGTTTGGACACTGTACCTAGCCTACATTATTCATGTTCTTGTTCCCTTTATGCACGTAAGGACGCAACCCGATGCGAC
-200
GTTTCTGAAACGGGCACTTAGACGCGCG£MXTTGTAATCACGTTTAAATCATAGTATTTGGTGCGCGTAGCTATAGGTG
-150 -100
CGTTTATGCGCCTCACTTTGTAAAAJT££££AAAATAACAAATATTGTTTTCAGTTTTTAAAGAACCGTTACTTGATCGC
-50
TTACAGCCGAGCAACTAAGACGTTGGTCCTACGCACTGCCCAGTTTTGATTCCCCCTTGTGTCGGAAATTCCAACTACAA
-1 5
TCAATAATC ATG GTA GCC AGA CGA CAA AC A AAG AAA G/GTAAATAAAGGGAACAATATATGCAAGGCGTT
ini Val Ala Arg Arg Gin Thr Lys Lys A
Exon 2
10 15
TTTTATTTCTTCTTTCTCACTTCACAG CT AGG AAG CCT GCA GCC AG/GTGAGTGAATACAATTTAAATTTTAT
la Arg Lys Pro Ala Ala Ar
Exon 3
20 25
TAACTCAAGACTTTAAATGCTTTGTCCTTCCTCCCCAG G AGA CGC AGC GCA GCC AAA CGC GCA GCC CCA
g Arg Arg Ser Ala Ala Lys Arg Ala Ala Pro
30 35 40
GCC GCG AAG AAG GCT GCG AGT CGC CGT CGC CCA AAG AGT GCT AAG AAG/GTAGGTAATAAGATGT
Ala Ala Lys Lys Ala Ala Ser Arg Arg Arg Pro Lys Ser Ala Lys Lys
Exon 4
45 50
AATTAAAAAAAGTCCTGACAATATATTTTTCTCTTTTCAG GCT AAG CCC GCA GCA AGG AGG CGC AGC AGC
Ala Lys Pro Ala Ala Arg Arg Arg Ser Ser
55 60 65 70
GTC AAA CCT AAA GCA GCA AAA GCA GCC ACC CAA GTC CGT CGC AGG AGC CGA CGA ATT CGC
Val Lys Pro Lys Ala Ala Lys Ala Ala Thr Gin Val Arg Arg Arg Ser Arg Arg lie Arg
75 + 1 +50
CGT GCG TCC GTG TCA AAG TAA TTCAATGGAAGACTGATCATTAAATCGTAACCCCTTCGAAAGATTAAACTTA
Arg Ala Ser Val Ser Lys *
+100
TCAAATTTCATTTTGTAGAACTGTCCAAATTTTCTAGAATATTGCAGAACTGAACATTTAAAACACATCCAAATTCGTAA
+150 +200
GCGAACAAGCAAGCAACGATGACCTACAATTTACAGTCGTTTCTTATTATTTCAAGTTTGCCTTTATTCAGTTTCAGTTT
+ 250
CAGTTTATTTACTCTTTAATACCTCCTCGGAGGTGTCAGAGTCAAAACATACAATTAGATACACAGAAATATACAAAAAG
+300 +350
CAGCAAGATCAAC^AX^^^CAAAAACAAAAAACAAAAATCATGCAAGCAAATCAGTACAATCAAAAAACTAACTTCAACC
>-400
+ 450
+ 500
TACGCACAACACAACGAGCAACCTACGTATGCACTAATGCCTAGCTATACACACTACATCATCAAATCAACTACGGCACT
+550 +600
CATTAACGATGATATACACTACAGAAGTGGCCAGGTTTCTTTGCACCTCCTTATTTCTAGACGTTAAAAGCTGGGCCATT
+ 650
TTTAAAGTATTTGGACCCACCCTATAATAATGCTTCAAATAGTATTTCCGATCTGAGTTGAAAGCCTTACATTGAAAAAG
+700 +750
ATAATGAAATTACATCTCCTATCAGCCCACTAGTACAAAGTTCACAAAGTCTATCATTAAAGTCAATATTAAGAAATCGT
498
E. PRATS & L. CORNUDELLA : PROTEIN <pl: GENE IN SEA CUCUMBER
In turn, each singly-reacting fragment appeared to hybridize with the probe to a similar extent as
revealed by the intensity of the autoradiographic signals estimated from densitometer tracings.
Comparison of the level of hybridization of the genomic fragments with the quantified signal
intensities of the cDNA standards yielded an average value of 0.92 copies of the (t>0 gene per
haploid genome. These results indicate that the H. tubulosa sperm 0O protein is probably specified
by a single copy gene.
m1234 5 678
2948 -
1830 -
998 -
519 -
435 -
Fig. 3. — H. tubulosa o0 gene copy
number. Autoradiogram of
Southern blots of sea cucumber
sperm DNA restriction digests
hybridized with P-probe of the
cDNA (see text for details). Sperm
DNA samples (20 jig) were
digested with: lanes I to 6 BamH\\
/y/ndlll; Kpn\\ Bam\\\ + Hind III;
Bam\\\ + Kpn\\ Hind III + Kpn\,
respectively. Graded amounts of
the cj>0-cDNA cloned insert
equivalent to 4 (lane 7) and 1
copies (lane 8) per haploid
genome, supplemented with a
mass excess of sheared calf thymus
DNA were co-electrophoresed as
hybridization standards. Sizes of
restriction fragments from the <|>0-
cDNA clone run in parallel as
migration markers, are given in bp
(lane m).
The structural arrangement and content of the <j>0 gene closely coincide with the common
features of a sizable number of post-meiotically expressed genes, typified by those encoding most
histone-to-protamine transition proteins as well as protamine genes [20, 23], Besides being
expressed in a replication-uncoupled manner, these genes usually generate polyadenylated
transcripts and most of them, although not all, are present as single copies containing coding
regions often interrupted by intervening sequences. The overall organization of these genes
becomes clearly divergent from that of the somatic histone genes. The functional implications, if
any, of this divergence in gene arrangement remains unexplained, although potential correlations
with chromatin transitions related to modulation and final arrest of gene activity during the
spermiogenic process should be taken into account. Further studies are underway to characterize
new genes encoding known sperm-specific protein variants.
Source MNHN. Paris
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
499
ACKNOWLEDGEMENTS
This work has been supported by grants to L. C. from the EEC SCIENCE Programme (contract n°. SC 1-CT9 1-0693)
and the Spanish Direction General de Investigation Cientifica y Tecnica (PB91-01 17)
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tubulosa . Experimental Cell Research, 63: 253-260.
41. Von Holt, C., 1986. — Histones in perspective. BioEssays, 3: 120-124.
42. WoLFFE, A., 1992. — Chromatin: Structure and Function. London, Academic Press: 1-213.
43. Zweidler, A., 1984. — Core histone variants of the mouse. In: G. S. Stein, J. L. Stein & W. F. Marzluff. Histone
Genes. New York, Wiley & Sons: 339-372.
Source : MNHN. Paris
Nuclear Basic Proteins from the Sperm
of Tunicates, Cephalochordates, Agnathans and Fish
Mattel CHIVA*, Nuria SAPERAS*, Carme CACERES* & Juan AUSIO **
* Departament d'Enginyeria Qufmica,
ETSEIB, UPC, Diagonal 647, Barcelona E-08028, Spain
** Department of Biochemistry' and Microbiology,
University of Victoria, Victoria, British Columbia, V8W 3P6, Canada
ABSTRACT
In this chapter we try to review and arrange the studies on sperm nuclear basic proteins (SNBPs) carried out in several
groups of deuterostomes (tunicates, cephalochordates, agnathans, chondrichthyans and osteichlhyans). Four general
points arise: 1. There are two main types of SNBPs: a. proteins similar to histones but with enhanced basicity, named here
”PL" (protamine-like) and b. very specialized proteins, named here "P”; 2. The"PL" proteins have appeared independently
several times during deuterostome evolution; 3. In some cases, “P” proteins may have arisen from "PL” proteins, but other
origins can not be ruled out for a particular P protein; 4. The classical evolutionary point of view about the appearance of
protamines (histones -> intermediate proteins -a protamines) is re-interpreted as histones -> PL proteins -a P proteins in
this paper. This transition seems to have repeatedly occurred during the evolution of different groups of deuterostomes.
Nevertheless, it should not be interpreted as a continuous evolutionary line of the sperm proteins of the whole
deuterostome line. In other words, there does not exist an apparent continuous evolutionary line relating the SNBPs of
echinoderms with the bony fish and tetrapod protamines.
RESUME
Les proteines nucleaires basiques des spermatozoides chez les Tuniciers, Cephalocordes,
Agnathes et Poissons
Nous essayons dans ce chapitre de faire une synthese des dtudes concernant les proteines nucleaires basiques des
spermatozoides (PNBS) qui ont etc effectuees sur plusieurs groupes de Deuterostomiens (Tuniciers, Cephalocordes.
Agnathes, Chondrichthyens et Ostdichthyehs). Quatre points generaux emergent: 1. II existe deux types principaux de
PNBS: a. des proteines similaires aux histones mais avec une basicite augmentee, nommee ici "PL" (proches des
protamines), et b. des proteines tres specialises, nommees ici “P”. 2. Les proteines "PL” sont apparues independamment
plusieurs fois pendant revolution des Deuterostomiens. 3. Dans certains cas, les proteines “P” peuvent etre apparues a
partir des proteines "PL”, mais d’autres origincs nc sont pas h exclure dans le cas de certaines proteines P. 4. Le point de
vue evolutif classique concernant Fapparition des protamines (histones -> proteines intermcdiaires -a protamines) est re¬
interprete dans cet article comme: histones -♦ proteines PL -> proteines P. Cette transition semble s’etre produite de
maniere rdpdtee pendant revolution des differents groupes de Deuterostomiens. Neanmoins, elle ne doit pas etre
interpretee comme une ligne devolution continue des proteines des spermatozoides dans Pensemble de la lignee des
Deuterostomiens. En d’autre termes, il n’existe pas de ligne devolution continue reliant les PNBS des Echinodermes aux
protamines des Poissons osseux et des T£trapodes.
Chiva, M., Saperas, N., Caceres, C. & Ausio, J., 1995. — Nuclear basic proteins from the sperm of tunicates,
cephalochordates, agnathans and fish. In: Jamieson, B. G. M., Ausio, J.. & Justine, J.-L. (eds). Advances in Spermatozoal
Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 501-514. Paris ISBN : 2-85653-225-X.
502
M. CHIVA ETAL. : NUCLEAR PROTEINS OF CHORDATES
In recent years, we have perfomed a series of studies on the SNBPs in deuterostomes
intended to link information already available from echinoderms [44], and from several groups of
fishes and tetrapods [reviewed in 22, 31-32], The sperm nuclei of echinoderms contain somatic
histones or specific histones with specialized N- and C-terminal domains. In contrast, the SNBPs
from fish (also referred to as “typical protamines”) are very short proteins (about 30 amino acid
residues), consisting mainly of arginine and a few other types of amino acid residues [31]. Two
hypotheses have been proposed for the evolution of SNBP in deuterostomes. One of them relates
the SNBPs from echinoderms to fish protamines [44]. The second one proposes a foreign origin
for the “typical protamine” of bony fish [21, 27], However, there was a complete absence of
information about the SNBPs of deuterostomes from intermediate taxonomic groups. In this
chapter we arrange the information already available on the SNBPs of these groups, which may
help to clarify some basic and general aspects of the evolution of these molecules. Yet there are
several major difficulties in establishing the evolutionary link amongst different SNBPs. They
stem from the remarkable variability of these proteins, the limited amount of information still
available and the lack of a general consensus about the phylogenetic relationship among different
groups of deuterostomes [12, 45].
RESULTS
SNBPs from Tunicates
In recent years, we have studied the SNBPs of several species from four different families
of ascidiacean tunicates [9-10, 36]. The main conclusion from these studies is the general
constancy in the SNBP pattern exhibited in each of them. Figure 1A shows the electrophoretic
patterns of four species, each one belonging to a different family. In all cases, they consist of a
major protein (PLasc) (asc = ascidiacean) which migrates close to the position corresponding to
histone H4. Each one of the patterns shown in Fig. 1 is representative of all the studied species
belonging to the same family, except in the case of the family Styelidae. Within this family, the
two species studied of the genus Styela (S. plicata and S. montereyensis) display a slightly more
complex SNBP electrophoretic pattern (Fig. IB). Styela species contain, in addition to the PLasc
protein, a significant amount of other proteins.
The PLasc proteins consist of about 150 amino acid residues, and are rich in arginine,
lysine, and relatively rich in glycine (Table 1). The studies carried out to date [35, 36] indicate that
PLasc exhibits the same tripartite structural organization which is characteristic of histone HI
[19]: An N- and a C-terminal tail flanking a central hydrophobic core (Fig. 1C). The N-terminal
region has only two arginine residues (NH2-R-R-). The trypsin-resistant core consists of 74
amino acid residues and has an amino acid composition (Table 1) and sequence [35] that
ressemble those of the trypsin-resistant cores from both HI histones [1] and some of the SNBPs
from bivalves [3] and gastropod molluscs [15]. The C-terminal tail is very basic (Table 1) and its
sequence does not display any similarity with the N- or C-terminal tails of the SNBPs from
echinoderms ([35] and unpublished results).
In the case of the genus Styela , the slow migrating protein (PLsty.l in Fig. IB) contains
Note:
In this article the PL proteins of small molecular weight resulting from post-translational cleavage of larger PL precursors
have been genetically referred to as P proteins. This nomenclature has been adopted to emphasize the structural
similarity existing between these proteins and protamines as discussed in the preceding article (Ausio, this
volume).
Abbreviations:
aa am,no acids* AUT = Acetic Acicl/ Urea/Triton Gel Electrophoresis, SDS = Sodium Dodecylsulfate
SNBPs = Sperm Nuclear Basic Proteins
Source MNHN. Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
503
A
st a b c
d
PL- ASC
c
B
d
MW
e f
P-STY2
Fig. i — A: Electrophoretic pattern of SNBPs from four ascidiacean tunicate species. Each species belongs to a different
family: a, Ascidia cdllosa (Ascidiidae); b, Chelyosoma production (Corellidae); c, Boltenia villosa (Pyuridae); d,
Cnemidocarpa finmarkiensis (Styelidae); st, proteins from the sperm of the ratfish (Hydrolagus colliei) used as a
marker standard [10]. B: Comparative electrophoretic analysis of PLasc (lane d) and the SNBPs of two species
from the genus Styela\ e, S. plicaia , f, S. montereyensis. The direction of electrophoresis is trom top (+) to bottom
(-). C: Schematic representation of the structural organization of the PLasc molecule; NT: amino-terminal domain
(2 arginine residues); CC, trypsin-resistant hydrophobic core (~75 amino acid residues); CT, carboxy-terminal
domain (70-75 amino acid residues).
also a trypsin-resistant peptide and an amino acid composition similar to that of PLasc (Table 1)
[35, 36], The protein Psty.2, of higher electrophoretic mobility, displays microheterogeneity and
consists of three distinct forms that can be resolved by HPLC [36] and that exhibit an almost
identical amino acid composition (Table 1). Table 1 also shows the enormous compositional
resemblance that exists between the C-terminal tail of PLasc and protein Psty.2. This fact is of a
special interest because it suggests a close relationship between these two molecules (see
discussion).
In summary, the sperm nuclei of the ascidiacean tunicates consist of a protein (PLasc)
which is different both from specific sperm histones of echinoderm and from the “typical
protamines” of the bony fish. This protein appears in all families studied and may represent the
504
M. CHIVA ETAL. : NUCLEAR PROTEINS OE CHORDATES
Table 1. — Amino acid composition (mol %) of the SNBPs from tunicates (a-g) and from the cephalochordate
Branchiostoma floridcie (h). As a comparison, the composition of a PL III protein (a SNBP from the bivalve
mollusc Crenomytilus grayanus [29]) is shown in lane (i); a, protein PLsty.l (PL of Styela plicata)] b, PLasc
(5. plicata ); c, trypsin resistant core of PLasc ( S . plicate)] d, C-terminal domain of PLasc (5. plicate ); e-g, each
one of the three components of Psty.2 protein (S. plicata).
Fig. 2. — A: Electrophoretic pattern of
Branchiostoma floridae SNBPs (lane c), shown
in comparison with the SNBPs from the tunicate
Styela plicate (lane b) and a chicken erythrocyte
histones standard (lane a). B: First nine amino
acid residues (N-terminus) of Pceph protein.
A a b c
B G/PRSRSRSAS
ancestral SNBP of ascidiacean tunicates. Nevertheless, in some particular groups, as in the genus
Styela, some variations to this general protein pattern have occurred; namely, the appearance of a
shorter and more basic molecule (Psty.2) in addition to molecules similar to PLasc (for example
PLsty.l). r
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
505
SNBPs from Cephalochordates
The phylum Cephalochordata consists of two genera (Branchio stoma and Epigonichtys )
[28], The SNBPs from Branchiostoma floridae have partially been characterized [40]. Their
electrophoretic pattern (shown in Fig. 2A) displays a main protein component (Pceph in Fig. 2A)
which is accompanied by small quantities of residual histones [40]. Pceph has an electrophoretic
mobility similar to that of tunicate protein Psty.2. Its size, estimated from the electrophoretic
behaviour is of about 120 amino acid residues, and its compositional analysis (Table 1) is very
simple. The protein consists of only six different types of amino acid residues, with arginine,
lysine, alanine and serine being the most abundant. It is interesting to note, with regard to the
amino acid composition and size, that Pceph is more similar to SNBPs from bivalve molluscs
than to other deuterostome SNBPs (Table 1). The N-terminal sequence (shown in Fig. 2B)
contains three alternating arginine-serine motives. Such an alternation of basic (R/K) and
phosphorylatable residues (S/T) has also been observed in the SNBPs from several gastropod and
bivalve molluscs [6, 13, 17], birds and mammals [30-31], but it is not present in the “typical
protamines” from bony fish (Fig. 5B).
In summary, the protein Pceph shares some characteristics with typical protamines such as
its low amino acid diversity, its basic composition and the presence of serine. Yet, Pceph and
“typical” protamines have several distinctive features. First, in Pceph lysine and arginine are
present in similar proportions (24.7% and 25.3% respectively) whereas protamines consist almost
exclusively of arginine. Secondly, Pceph displays an alanine rich amino acid composition
(21.7%) not found in protamines. Finally, Pceph has a much larger size than “typical protamines”
and contains the repetitive motifs arg-ser which is absent in bony fish protamines.
Table 2. — Amino acid composition (mol %) of the sperm histones of the lamprey Petromyzon marinus (PM) in
comparison to the somatic histones from calf thymus (CT) [26].
HI H2A H2B H3 H4
Source : MNHN , Paris
506
M. CHIVA ETAL. : NUCLEAR PROTEINS OE CHORDATES
SNBPs from Agnathans
The nuclear sperm proteins from Agnatha have only been studied in one species of lamprey,
Petromyzon marinas [40], Nuclei isolated from ripe sperm of P. marinas consist only of histones
(Fig. 3A-C). According to the chromatographic and electrophoretic behaviour as well as to the
compositional amino acid analyses (Table 2), the sperm histones of P. marinas do not exhibit any
specific characteristics that distinguish them from the somatic type. This is surprising considering
that agnathans have appeared relatively late in evolution and it contrasts with the strong tendency
of tunicates and cephalochordates to have specialized proteins in their sperm nuclei. It is necessary
to study the SNBPs of more agnathans in order to determine whether the absence of specialized
nuclear sperm proteins is just a specific characteristic of P. marinas or represents the general trend
in agnathans. Nevertheless, the absence of specific proteins and the presence of somatic-like
histones in the sperm nucleus also occurs in some groups of bony fish (see below).
HI
H2A
B
C
Fig. 3. — A: Light microscopy micrograph of ripe sperm used for the analysis of the nuclear sperm proteins of
Petromyzon marinus (x I 000). B, C: Two-dimensional electrophoretic protein patterns of SNBPs from
Petromyzon marinus (3C), and histones from chicken erythrocyte (3B) used as standard. The direction of the
electrophoresis was left (+) to right (-) (first dimension. AUT) and top (-) to bottom (+) (second dimension, SDS).
Source : MNHN , Paris
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
507
SNBPs from Chondrichthyes
The class Chondrichthyes consists of two lineages that diverged very early in its evolution:
Holocephali and Elasmobranchii [28]. The Elasmobranchii diverged later giving raise to the
Selachimorpha (sharks) and the Batidoidimorpha (skates). The electrophoretic patterns of the
SNBPs from eight selachians, one batoidean and one holocephalan species are shown in Fig. 4.
All contain several basic proteins in their sperm nuclei. One of these proteins (Pcon.Z3 in Fig. 4)
can be directly extracted with dilute acids. However, the other major proteins (Pcon-Zl, Z2)
require chemical reduction of the nuclei before they can be solubilized by acids. The need for
reduction prior to protein extraction is a characteristic of cysteine-rich proteins owing to the
formation of intermolecular S-S bridges in the ripe sperm nuclei. The SNBPs from the selachian
Scyliorhinus canicula have been characterized previously [18] and they are representative of the
SNBP pattern in all selachian species (see the similarity among selachian SNBPs in Fig. 4A). The
electrophoretic pattern of the SNBPs of one batoidean species ( Raja rhino) is shown in Fig. 4A.
Unfortunately, there is no information available on the amino acid composition of batoidean
SNBPs. The holocephalan Hydrolagus colliei contains several minor proteins and three major
proteins [37] (see also Fig. 4A). The amino acid compositions of the main H. colliei SNBPs are
shown in Table 3 in comparison with the corresponding proteins form S. canicula .
In Fig. 4B we compare the N-terminal sequence of Pcon-Z3 from H. colliei [37] with the
PCON.Z-1.2
PCON.Z-3
A
a
b
c
d e
f g h
j
(a) ARRRHSMKKKRKSVRRRKTRKNTRKRKNSLGR
B (b) ARSRSRRSYGRGRRRGGRRRRRRRRRRRGGR
Fig. 4. — A: Electrophoretic pattern of the SNBPs of eight selachian species (a-h). one batoidean (i) and one
chimaeriform (j). a, Galeus melastomus\ b, Scyliorhinus canicula (a, b. family Scyliorhinidae, Order Lamniformes);
c, Centroscymnus coelolepis; d, C. crepilater ; e. Centrophorus uyato ; f, C. squamosus\ g, Deania profundorum: ;
h, Etmopterus pusillus (c-h family Squalidae, Order Squaliformes); i. Raja rhina iRajidae, Rajiformes);
j, Hydrolagus colliei (Chimaeridae, Chimaeri formes). B: N-terminal sequence of the protein Pcon-Z3 from
Hydrolagus colliei (a) compared with the sequence of the protein Pcon-Z3 from Scyliorhinus canicula (b) (42].
508
M. CHIVA ETAL. : NUCLEAR PROTEINS OF CHORDATES
corresponding protein in S. canicula [42] (referred to as Z3 in the original works of GUSSE &
CHEVAILLIER [18]).
Judging from the information available, it appears that chondrichthyans share a common
SNBP pattern consisting of a very basic cysteine-lacking protein (Pcon-Z3) and a reduced set of
cysteine-containing proteins (Pcon-Zl, Z2). It also appears that the amino acid composition and
sequences of the SNBPs have undergone a remarkable divergence during the separation of
holocephalans and selachians (Table 3 and Fig. 4B; see [37] for a more extensive discussion).
Table 3. — Amino acid composition (mol %) of proteins Pcon.Zl, Z2 and Z3 from Hydrolagus colliei (HC), and
Scyliorhinus canicula (SC). Pcon.Zl. Z2 and Z3 compositions from S. canicula are from [8, 25. 42].
Res* = Total number of amino acid residues.
SNBPs from Osteichthyes
We have studied a number of bony fish species [35, 38-39. 41] and have exhaustively
reviewed the information on osteichthyan SNBPs [41]. The most striking feature that emerged
*r°™ these .analyses was the apparent lack of a unique protein pattern which could be considered
representative of all the SNBPs of this group. With a few exceptions [23, 24], the SNBPs of fish
belong to one of the five electrophoretic patterns shown in Fig. 5 [39]; namely a presence of
somatic histones and absence of any other sperm-specific nuclear protein (this is a similar
whh n°mi?kehHat a ieady deTbud in the a§nathan p- marinas)-, b, presence of somatic histones but
„,i r (ed increase in the histone HI content (in this case we will consider that the specific
nuclear piotein corresponds to the additional increase of the corresponding HI); c, presence of a
histone complement coexisting with one additional SNBP belonging to PL type- d presence of
?Z Z nmt PL completely replace histones in spSrm nuclei; and e presence of
d fl>m!rPP nmeS -h'Ch a S° replace comPIetely somatic histones. There appear to be important
difterences between typical protamines” and the SNBPs from groups b, c and d. The former
Source .
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
509
has
hbs
Cl C2
hds hes
->
*
A
t
B
a)
b>
c»
d)
el
f )
8
l
PRRRRQAS
PRRRREAS
PRRRRQAS
PRRRRRSS
PRRRRRSS
PRRRRSSS
RPVRRRRRT
RPVRRRRRY
RPVRRRRRY
RPVRRRRRY
RPVRRRRRY
RPVRRRRRA
20 28
i I
RRSTAERRRR
RRSTAARRRR
RRSTAARRRR
RRSTVARRRR
RRSTAARRRR
R V S R R R R
30 38
I I
R V V R R R R
R V V R R R R
R V V R R R R
R V V R R R R
R V V R R R R
RRGGRRRR
Fig. 5. — A: Several representative electrophoretic patterns of the main SNBPs from bony fish; a, somatic histones
(Trig la lucerna, Triglidae); b. somatic histones with increased quantities of HI ( Pagellus acarne , Sparidae);
c, somatic histones coexisting with an additional specific protein belonging to PL type (cl: Merluccius capensis,
Merlucciidae, c2: Cataetyx laticeps, Bythitidae); d. protein PL which replaces histones in sperm nuclei ( Mullus
surmuletus , Mullidae); e, “typical” fish protamine (Dicentrarchus labrax, Percichthyidae). Chicken erythrocyte
histones used as a standard (h) and commercial salmine (s) are shown for comparison in each electrophoresis.
B: Amino acid sequence of some bony fish “typical” protamines [31, 38]. a , Dicentrarchus labrax; b-c. Tuna
fish (Thunnus thynnus) b, fraction Yl. c, fraction Y2. d, fraction Zl. and e, fraction Z2; f, protamine 2b from
rainbow trout (Salmo irideus). C: Schematic representation of the structural organizaton of PL from
M. surmuletus ; NT: 20 aa residues; CC: ~75 aa residues; CT: 80-85 aa.
are very small molecules consisting of a few amino acid types (see Table 4-e and D. labrax
protamine sequence [38] in Fig. 5B). In contrast, the SNBP from the other groups are larger
molecules, and exhibit a more complex amino acid composition which may be considered similar
to that of the histones of the Hl/H5-family, but with a higher arginine content (type PL). These
510
M. CHIVA ETAL. : NUCLEAR PROTEINS OF CHORDATES
Table 4. — Amino acid composition (mol %) of the bony fish SBPs shown in figure 5; a) whole histones from T. lucerna
sperm nuclei; b) HI histone of P. acarne\ cl) specific sperm protein of M. capensis ; c2) SNBP of C. laticeps ; d)
main PL of M. surmuletus ; e) "typical" protamine of D. labrax.
t = trace amounts
features are common to both the PL proteins that coexist with histones in the sperm nuclei (lanes b
and c in Fig. 5A, and Table 4), and to those PL proteins that wholly replace histones, as occurs in
the family Mullidae (Fig. 5A lane d, and Table 4). The electrophoretic pattern of the SNBPs of
this family consists of two proteins of almost identical electrophoretic mobility and amino acid
composition. They could possibly have arisen from a unique ancestor by a mechanism of gene
duplication [38], The partial sequence of one of them [35] indicates that it consists of
approximately 180 amino acid residues organized in three structural domains (N-terminal, central
core and C-terminal), as in HI histones (Fig. 5C). The N-terminal region is 20 amino acid long
(50% of them being basic residues) and contains the repetitive motive Ser-Pro-basic-basic which
has been described in the N- and C-terminal regions of the sperm histones from echinoderms [33,
43]. The central core is trypsin-resistant and displays a high percentage of similarities with the
equivalent region of HI histones. The C-terminal zone is about 85 residues long and contains
most (-70%) of the basic amino acid residues of the molecule.
DISCUSSION
Characteristics and distribution of SNBP molecules
Taking into account the structural and compositional features of the deuterostome SNBPs
(other than histones), it is possible to group these molecules into two categories. I) proteins
similar to histones (mainly to the HI type), and II) highly specialized proteins.
The first group, referred to as PL (protamine-like) owing to its enhanced basicity, are
relatively large proteins, with a complex amino acid composition similar to that of histone HI. All
PL proteins studied to date share the same general molecular organization. All of them have a
trypsin-resistant core consisting mainly of neutral amino acid residues, flanked by very arginine
and/or lysine-rich N- and C-terminal domains. Depending on the species, PL proteins may be able
Source . MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
511
Fig. 6. — Different mechanisms of post-translational processing in SNBPs leading to specialized P proteins: A: Direct
translation from mRNA; e.g. ••typical” protamine of bony fish. [20]. B: Translation of a precursor which leads to
the appearance of a P protein by a single proteolytic cleavage; e.g. "protamines” of cephalopod molluscs [46],
C: Translation of a precursor which undergoes several progressive cleavages. Each cleavage introduces important
changes in the extent of chromatin condensation; e.g. "protamines" of neogastropod molluscs [4]; and the
mammal P2 protamine [7]. D: Translation of a large PL protein. The C-terminal domain is cleaved to generate a
very specialized P protein and a second PL protein; e.g. bivalve molluscs [5-6].
to replace somatic histones to different extents. Among the deuterostomes studied so far, PL
proteins appear in all ascidiacean tunicates (PLasc) as well as in some fishes ( Mullus , Merluccius ,
etc.). However, it is interesting to note that PL proteins are also present in protostomes such as in
the case of PL-I and PL-II of bivalve molluscs [3] and “protamines” of patellogastropods and
polyplacophorans [15, 16]. The origin of this type of protein is not well established. From the
sequences published [3] and the ongoing analysis of the sequences of PLasc and PL from Mullus
[35], it becomes apparent that the central core of these proteins shares an enormous similarity with
the globular core of the histones of the HI family. Therefore, PL proteins may be evolutionarily
related to this histone. In contrast to the central core, the PL N- and C-terminal tails exhibit an
enormous compositional variability. From the evolutionary point of view, there does not seem to
exist a direct link among the zoological groups which display PL proteins. In our opinion, the PL
sperm specific proteins may have appeared independently, several times during the evolution of
512
M. CHIVA ETAL. : NUCLEAR PROTEINS OF CHORDATES
the animal kingdom (bivalves, polyplacophors, patellogastropods and tunicates, and several times
among fish).
The proteins of the second type, which we refer to here as “very specialized proteins” (or
“P” proteins), exhibit the following characteristics: they are short molecules (from approximately
30 to 120 amino acid residues) and they exhibit a very simple amino acid composition (with only
a very few amino acid types). Arginine and/or lysine are present in a large amount (50%-80%)
and hydrophobic residues are scarce or absent. Among the deuterostomes studied here. P proteins
appear sporadically in tunicates (Psty.2 in genus Styela), in cephalochordates (Pceph), in
chondrichthyan fishes (Pcon.Z3) and in osteichthyes (typical protamines). However, as occurs
with PL proteins, P proteins are widely distributed over a broad spectrum of both
phylogenetically distant and closely related groups. Thus P proteins have been described in
bivalves [2], archaeogastropod molluscs [13-14, 16-17], neogastropods (“ripe” protamines) [4],
cephalopod molluscs [46], crustaceans [11], echinoderms (d>0 protein) [34] as well as in fish,
amphibians, birds and mammals [22, 31, 47], These P proteins are not always homologous and it
is reasonable to think that they have also appeared independently several times and from several
different cellular processes (Fig. 6) during the course of evolution. In the case of bivalve
molluscs, it has been demonstrated that some “specialized proteins” (PL-IV in the original works
of CARLOS et at. [5-6]) actually correspond to the C-terminal region of a larger protein precursor
belonging to the PL type (referred to as PL-I by some authors) (see also AUSIO, this volume).
They appear in the nuclei following a precise proteolytic cleavage (Fig. 6D). From this
perspective, the similarity between the amino acid composition of the Psty.2 protein and the C-
terminal part of PLasc (Table 1) is very suggestive of a possibly related cleavage post-translational
mechanism in the case of these proteins. It seems possible that some P proteins have arisen from a
simplification (genotypical or phenotypical) of other proteins of the PL type, although other
origins for particular P proteins (as is the case of Pcon.Zl, Z2) can not be disregarded.
General evolutionary > considerations
From the information presented in this chapter, it is quite evident that it is difficult to trace
an uninterrupted evolutionary line connecting the different SNBPs of deuterostomes. The
proposition ol an evolutionary link between histones and protamines which has been observed in
different phylogenetic groups (histone HI PL proteins -» very specialized P proteins; see also
AUSIO, this volume) has most likely occurred independently in each of them. Another important
consideration to be made arises from the fact that when a group consisting of a large number of
species is analyzed, the evolutionary trend within this group may be masked by the variability of
SNBPs present in the limited sample of species analyzed. This is for instance the case in bony
fish where the appearance of “protamines” seems to be sporadic. Two hypothesis have been put
forward to explain the apparent random distribution of SNBPs within this group. The first one
proposes a mechanism of horizontal evolution of the genes of the fish protamines. In the second
alternative, the phenomenon is explained by the loss of the expression of these genes during the
formation of some groups of fish [21, 41], It is important to stress the fact that fish protamines,
which for historical reasons have long been considered to be the “typical protamines”, in fact
represent only one of the many types of the highly specialized P proteins. Fish protamines do not
necessarily represent the linal goal of SNBP molecular evolution. Yet, they are of special interest
because ol the extent of similarity they share with the PI protamine ancestors of tetrapods [31].
AC KNO WLEDGMENTS
M. C„ N. S. and C. C. are very indebted to Professor J. A. Subirana for financial support and for providing lab
space. We arc also grateful to Drs. H. Kasinsky, D. Lloris, L. Z. Holland and N. D. Holland for helpful discussion and for
providing some of the valuable biological specimens whose SNBP protein compositions are discussed in this paper. This
work was supported by grant PB-93-1067 C1CYT (Spain) to J. A. Subirana and by NSERC grant OGP-0046-399 and NATO
grant CRG 930717 to J. Ausi6.
Source : MNHN. Paris
ADVANCES IN SPERM ATOZOAL PH YLOGENY AND TAXONOMY
513
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Source : MNHN. Paris
Heparin-Binding Proteins on Bull, Boar, Stallion,
and Human Spermatozoa
Juan Jose CALVETE * **, Libia SANZ * Markus REINERT *,
Zuzana DOSTALOVA * & Edda TOPFER-PETERSEN *
* Institut fur Reproduktionsmedizin,
Tierarztliche Hochschule Hannover, Bunteweg 15, 30559 Hannover- Kirchrode, Germany
** Instituto de Quimica-Fisica “Rocasolano” CSIC, Madrid, Spain
ABSTRACT
The seminal plasma from many mammalian species contains heparin-binding proteins which bind to the sperm surface
at ejaculation and appear to mediate sperm capacitation modulated by glycosaminoglycans (GAG) present in the
reproductive tract of the female. The molecular mechanisms underlying this process is only poorly understood, however.
Furthermore, seminal plasma heparin-binding proteins have been structurally characterized in only few mammalian
species. Here, we summarize our present knowledge on sperm surface-associated heparin-binding proteins in four different
mammals: boar, bull, stallion and man. The major conclusion of this comparative study is that the major heparin-binding
proteins from these species belong to only three different protein families. The relative abundance of proteins of different
families on spermatozoa from the different species, together with structural diversity between proteins of the same family
in different species, and species-specific topographical localization of homologous heparin-binding proteins on
spermatozoa, may contribute to the specific spermatozoon phenotype, which in turn may modulate species-specific
effects of GAGs on sperm capacitation.
RESUME
Les proteines liant l’heparine dans les spermatozoides du Taureau, du Verrat, du Cheval et de
l’Homme
Le plasma seminal de nombreuses especes de mammiferes contient des proteines liant Pheparine qui se lient & la surface
des spermatozoides h Peculation et semblent etre les mediateurs de la capacitation des spermatozoides, qui est modulee
par les glycoaminoglycanes (GAG) presents dans le tractus genital de la femelle. Toutefois, les mecanismes mofeculaires
sous-jacents & ce processus sont mal connus. De plus, la structure des proteines du plasma seminal liant PhSparine a ete
caracterisee seulement dans quelques esp&ces animales. Nous resumons ici l’etat actuel des connaissances sur les proteines
liees & Pheparine assocfees & la surface des spermatozoides chez quatre mammiferes: Verrat, Taureau, Cheval et Homme. La
conclusion principale de cette etude comparative est que les proteines majeures liant Pheparine de ces especes
appartiennent h seulement trois families differentes de proteines. L’abondance relative des profeines de differentes families
sur les spermatozoides des differentes especes, ainsi que la diversite structurale entre les profeines d'une meme famille dans
les differentes especes, et que la localisation topographique sp^cifique & Pespece des proteines homologues liant
Pheparine sur les spermatozoides, peuvent contribuer au phenotype specifique du spermatozoide, qui ensuite module les
effets specifiques des glycoaminoglycanes sur la capacitation des spermatozoides.
Calvete, J. J., Sanz, L., Reinert, M., DostAlovA, Z. & TOpfer-Petersen, E., 1995. — Heparin-binding proteins
on bull, boar, stallion, and human spermatozoa. In: Jamieson, B. G. M., Ausio, J., & Justine, J.-L. (eds). Advances in
Spermatozoal Phylogeny and Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 515-524. Paris ISBN : 2-85653-225-X.
516
J. J. CALVETE ETAL. : HEPARIN-BINDING PROTEINS ( MAMMALIA )
Following the differentiation of the haploid spermatozoon, the cell is released from the
epithelium of the seminiferous tubules passing from the testis to the epididymis. Mammalian
spermatozoa are highly differentiated by the time they leave the testis. Nonetheless, at this stage,
they do not have the ability to fertilize eggs. Spermatozoa gain this capability while passing
through the epididymis (epididymal maturation) and/or after residing in the female tract for some
period of time (capacitation) (reviewed in [35]). Though our knowledge of the molecular basis of
these processes is still in its infancy, one of the most prominent, and best documented,
physiological changes in the spermatozoa is a continous remodelling of their plasma membrane
components as spermatozoa travel through the various microenvironments within the male and the
female genital tracts [17, 35]. Both membrane-integrated and surface-adsorbed components
(lipids or proteins) either change their location in or on the plasma membrane, are altered,
masked, or replaced as a response to the constantly changing osmolarity and chemical
composition of the millieu surrounding the sperm. In particular, at the time of ejaculation,
spermatozoa from the distal (cauda) epididymis are mixed with secretions of the male accessory
sexual glands. Recent evidence suggests that components of seminal fluid upon attaching to the
sperm surface may regulate important sperm functions: some (glyco)proteins stabilize the plasma
membrane and may prevent premature acrosome reactions, and others are believed to mediate
interactions between spermatozoa and the zona pellucida. Here, we will restrict the discussion to
the structure and proposed biological role of heparin-binding proteins of the seminal plasma of
different mammals: boar, bull, stallion and man. Interestingly, the seminal plasma heparin¬
binding proteins of these four species belong to only three different protein families, which
include both capacitation factors and carbohydrate-recognition (putative zona pellucida-binding)
molecules. Differences between species in the relative abundance and topography of proteins from
different families on the sperm surface, may contribute to the species-specific physiology of
mammalian spermatozoa.
RESULTS AND DISCUSSION
Heparin-like GAGs and heparin-binding proteins in fertilization
Proteoglycans with heparin- and chondroitin sulphate-like glycosaminoglycan side chains
are secreted by the epithelium of the female reproductive tract, particularly at high concentration
during the follicular phase of the estrous cycle, and have been shown to specifically invoke sperm
capacitation in a number of mammalian species, as measured by the onset of agonist-inducible
acrosome reactions [32]. Thus, in vitro incubation of epididymal sperm from such different
mammalian species as bovine, hamster, human, rabbit, or equine, with glycosaminoglycans
significantly accelerated the development of the sperm capacitated state as judged by their
increased ability to fertilize homologous eggs. In addition, since exposure of bovine epididymal
spermatozoa to seminal plasma from the same species enhanced their ability to undergo the
acrosome reaction in the presence of heparin [25], it follows that the effects of heparin-like GAGs
appear to be mediated by seminal plasma heparin-binding proteins which coat the sperm surface at
ejaculation.
Seminal plasma heparin-binding proteins on bovine spermatozoa
Comparison of the heparin-binding protein composition of epididymal and ejaculated
spermatozoa showed the presence of a 30 kDa and several 15-17 kDa proteins in the ejaculated
spermatozoa [10]. The same group of proteins were adsorbed to the sperm surface following
exposure of epididymal sperm to seminal plasma [24]. The 30 kDa protein has not been
structurally characterized. The primary structures of the 15-17 kDa proteins, designated BSP-Aj,
BSP-A2 and BSP-A3, have been reported [16, 30], BSP-A] and BSP-A2 have the same
polypeptide backbone but the former contains a single O-glycosylated trisaccharide (NeuNAc a(2-
6)-Gal G(l-3)-GalNAc-) attached at threonine-1 1 [5], and together they are also known as Major
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
517
Protein (MP) or PDC-109 (Fig. 1A). Structurally, BSP-A1/2 and BSP- A3 are made up of two
tandemly-arraged homologous modules of around 40 residues that share the consensus sequence
of the collagen- and heparin-binding type-II modules of fibronectin [30] (Fig. 1A). Each of these
repeats contains the amino acid residues which have been identified by alanine scanning
mutagenesis to be critical for collagen binding [2], In addition, the isolated B-domain of PDC-109
retained affinity for immobilized collagen [2], Recently, a refined FP-NMR-solution structure of
the 45 -residue PDC-109 domain B has been reported [12]. However, the folding pattern of the
whole molecule and the heparin-binding site(s) remain to be established.
HSP-l : ‘DLQTTGADHSAT.VNP
C (2X) P F X Y ( 7X )
C < 8 — 1 OX ) W C ( 4X ) D Y (3-5X)W X Y C C
HSP-l :7S AjF P FlvJT] R
PDC-109 : 70|v F P F I Y G
BSP- A3 : 75 |V F P F I Y| E
BT
K
S
Y D 1r |C Tl T D|G S
y(e]t cdliCi g s
Y D T c II Ik I g s
L |7] R I [s]-
H WJm]- |sj-
t |f m| - n y
WCSlVTlPNYD
w c S L S|[p N Y D
W C S L S| Si N Y D
H H G A
k[d"r1 a
e Id gTT
( 2 X ) P F X Y ( 7 X ) C ( 8-10X )
W C ( 4X ) N Y (3-SX)
A : DLQTTGADH S ATVN P
A': DQQLIMTKHSATV —
B : TPENKCVFPFNYRGYRYYDCTRTDSFYR — WCSLTGTYSGIQV-RYCAA
B' : TDYAKCAFPFVYRGQTYDRCTTDGSLFRISWCSVTPNYDHHGAWKYC
HSP-1 - 1
PDC-109 - 1
BSP-R3
Fig. 1. — A: Comparison of the primary structure of the major heparin-binding proteins from stallion (HSP-l) and bovine
(PDC-109 and BSP- A3) seminal plasma. Identical residues in at least two proteins are shown in boxes. The
consensus amino acids of the fibronectin type II module and the spacing between them are shown below the
sequence alignment. T, O-glycosylated threonine residues. B: Amino acid sequence alignment of the A- and B-
type internal repeats of stallion heparin-binding protein HSP-l, and a scheme of the domain arrangement in HSP-l
and in bovine PDC-109 and BSP- A3. indicates the relative positions of O-glycosylation sites; S-S, disulphide
bridge.
Proteins cross-reacting with monospecific antibodies against either BSP- A 1/2, BSP- A3, or
BSP-30 kDa have been detected in the seminal fluids of human, porcine, hamster, mouse, and rat
[18]. However, only the gene expression of bovine BSP-A1/2 has been studied [29]. Southern
518
J. J. CALVETE ETAL. : HEPARIN-BINDING PROTEINS (MAMMALIA)
blot analysis of genomic DNA indicated that BSP-A1/A2 is coded for by a single gene per haploid
bovine genome [29]. The protein is secreted by the seminal vesicle epithelium at a concentration
of 16-25 mg/ml [5, 29].
The topographical localization, the nature of the sperm surface acceptor molecules of PDC-
109, and its physiological role are controversial. Thus, AUMULLER et al. [1] reported that PDC-
109 binds preferentially to the middle piece and neck region of bovine spermatozoa and identified
by immunoblotting analysis a 65-67 kDa protein duplet as a PDC-109 acceptor site on epididymal
spermatozoa. These authors postulated that binding of PDC-109 to its acceptor site might be
regarded as a physiological event related to the onset of hyperactivated sperm motility [1]. On the
other hand, Manjunath et al. have shown that isolated PDC-109 possesses the ability of binding a
number of ligands, such as apolipoprotein A-I (apoA-I) and apoA-I associated high-density
lipoproteins, different types of collagen (I, II, IV, and V), fibrinogen, heparin, calmodulin,
phospholipase A2 (PLA2), and phosphorylcholine-containing lipids [23]. Using
immunofluorescence, MANJUNATH et al. have reported that BSP-A1/2 is located on the entire
sperm head surface, “although the intensity was noticeably stronger at the midpiece region” [22],
These authors have proposed that BSP-A1/2 may play an important role in sperm lipid
modification that occurs during capacitation and the acrosome reaction. In their mechanistic
theory, binding of BSP-A1/2 to choline phospholipids on the sperm surface may block PLA2
from acting on these phospholipids and prevent sperm from undergoing a premature acrosome
reaction. In addition, acting as decapacitation factors sequestering cholesterol and choline
phospholipid, BSP-A1/2 may alter the fluidity and permeability of the spermatozoal membrane
and allow calcium to enter for activation of PLA2, which would then produce lysophospholipids
that are known to destabilize membranes and trigger membrane fusion (acrosome reaction).
In addition to the proposed role in membrane lipid remodelling events, MANJUNATH et al.
[22] have reported a specific interaction of BSP-A1/2, BSP-A3, and BSP-30 kDa with insulin-like
growth factor-II (IGF-II), and hypothesized that BSP-proteins bound to the cell surface could
modulate the IGF-II action by serving as carriers as well as cell surface binding site for the
hormone.
Our own data showing that, upon mixing of spermatozoa with seminal plasma at
ejaculation, 9 million molecules of PDC-109 on average become coated to the sperm surface, and
that this figure decreases only to 8 million molecules per spermatozoon after incubation for 24 h in
capacitation medium at 39° C [5], do not support a role for BSP-A1/2 as a decapacitation factor.
Furthermore, BSP-A1/2 eluted from a Sephadex G-200 gel filtration column as aggregated
molecules with an apparent molecular mass of 60-120 kDa [22], indicating that in addition to
binding to 65-67 kDa acceptor protein(s) and choline phospholipids, BSP-A1/2 becomes coated
to the sperm surface as a multimer. How the aggregation state of BSP-A1/2 affects the various
ligand-binding activities reported in in vitro systems deserves further investigation. Clearly, much
work is needed to establish the actual biological function(s) of, and the mechanisms used by, the
different members of the bovine seminal plasma (BSP) protein family on sperm physiology.
Another abundant heparin-binding component of bovine seminal plasma (~7 mg/ml) is
acidic seminal fluid protein (aSFP) [15]. Deduction of the primary structure of aSFP from a
complete cDNA clone [34] showed that aSFP shares 43% sequence identity with porcine
spermadhesins (see below) (Fig. 2). On average, 6 x 106 molecules of aSFP bind to a narrow
region on the apical part of the acrosome of an ejaculated spermatozoon, but this amount
decreases to undetectable levels in capacitated sperm [14], This indicates that aSFP might have a
role as a decapacitation factor, and shows that proteins from the same family (spermadhesin) play
different functions in the fertilization process of different mammalian species, i.e. in bull and boar
(see next section).
Source : MNHN, Paris
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
519
K P
K P
[d]p
H F
0S
nB
0H
Efcd
C G K E Y V E[LlLD g|p] P G S
C G K E Y V E V|q1 D G L P G [aJ
e]g[a|p G S
D [q]1rJ0 G [p]
D G L P G S
Eli
G0Y
K S0
d[n1 F L|K
G K
G K
G K
I C
0
0
T
I L
g]f0
S L M D
120
GPLPFPYF
AKA
121 130
ERQTI IATEKNIP
Fig. 2. — Alignment of the amino acid sequence of boar spermadhesins AWN, AQN-1, PSP-I, and AQN-3, and the bovine
seminal plasma polypeptide aSFP. Identical residues within at least two of these proteins are shown in boxes.
* , this residue is acetylated in AWN-1; °, AQN-3 also exists as a glycosylated form; N_, is the conserved
glycosylation point in PSP-I and the AQN-3 isoform. Two disulphide bridges between nearest-neighbour cysteine
residues are conserved within the boar spermadhesins.
The major boar sperm-associated heparin-binding proteins belong to the spermadhes in family
The boar seminal plasma and sperm-associated heparin-binding proteins have been isolated
using affinity chromatography on Sepharose-heparin [27]. A minor 18 kDa glycoprotein with an
N-terminal amino acid sequence DQHLPGRFLXPAITSDDKCVFPFIYKGNL... was
characterized [27]. This clearly showed that boar spermatozoa also possess a heparin-binding
protein of the same protein family as the bovine polypeptides. However, the major heparin¬
binding components, designated AQN-1, AQN-2 (PSP-I), AQN-3, AWN-1, and AWN-2 belong
to a novel protein family for which the term “spermadhesin” has been coined (reviewed in [6]).
The nomenclature used for boar spermadhesin was based on the first three amino acid residues of
their sequence, alanine(A)-glutamine(Q)-asparagine(N) and alanine(A)-tryptophan(W)-
asparagine(N); a number indicates the reverse-phase HPLC elution order of polypeptides
containing the same N-terminal sequence. These proteins are 111-133 amino acid long, contain
two conserved disulphide bridges between nearest-neigbor cysteine residues, and have 40-60%
sequence identity (Fig. 2). Posttranslational modifications contribute to the diversity of the family:
AWN-2 is identical as AWN-1 but has an acetylated N-terminal alanine residue; PSP-I is
constitutively glycosylated; AQN-3 and AWN (isoforms 1 and 2) are found as both non-
glycosylated and as N- and O-glycoforms. On the other hand, no glycosylated isoforms of AQN-
1 have been reported.
Spermadhesins were first identified by their carbohydrate-binding, and zona pellucida-
binding capabilities [6]. Subsequently, it was shown that, in addition to their lectin-like activity,
boar spermadhesins are multifunctional proteins which combine within the same molecule
heparin- (AQN-1, AQN-2, AQN-3, and AWN) and/or serine proteinase inhibitor- (AQN-1 and
AWN) binding abilities. It has been proposed that serine proteinase once bound to sperm surface
acceptor molecules, may stabilize or protect sperm surface membrane specific sites for sperm-egg
interaction [26]. Inhibitors are then released from the sperm surface during sperm residence in the
520
J. J. CALVETE ETAL. : HEPARIN-BINDING PROTEINS ( MAMMALIA )
female genital tract allowing zona pellucida-binding sites to become exposed. Therefore, the
spectrum of ligand-binding strongly suggests that boar spermadhesins may play a role in at least
two important aspects of fertilization, sperm capacitation and gamete interaction.
The major biological source of spermadhesins is the secretion of the seminal vesicle
epithelium, where the concentration of different spermadhesins ranges from 0.6-7. 2 mg/ml [13].
However, AWN-1 is also synthesized by the tubuli recti and the rete testis [31] and is the only
spermadhesin found on the surface of epididymal sperm. The amount of coated AWN-1 (5. 9-7. 5
x 106 molecules/epididymal spermatozoon) is sufficient to cover one-third of the entire surface of
the sperm head, i.e. the acrosomal cap, with a one-molecule-thick layer. We hypothesize that
AWN-1 may be one of the factors contributing to the fertilizing activity of epididymal
spermatozoa. Following ejaculation, 12-60 x 106 molecules of each AQN-1, AQN-2, AQN-3,
and extra 50 x 106 AWN molecules (isoforms 1 and 2) become adsorbed on the apical third of the
acrosomal cap of spermatozoa, the place where porcine sperm initiate binding to the zona
pellucida of the oocyte. However, approximately 60% of adsorbed spermadhesins AQN-1, AQN-
2, and AQN-3 are released after 3 h in vitro capacitation, the amount of AWN-1 decreases to the
level found on epididymal sperm, and the whole AWN-2 population is lost. This figure changes
only slightly upon 24 h capacitation.
Spermadhesins AWN-1 and AQN-3, as monomers, possess binding affinity for
phosphorylethanolamine (PE) matrices, and the PE-binding site is different from the
carbohydrate-recognition domain (unpublished results). Phosphorylethanolamine is a major
substituent of boar sperm membrane phospholipids [33]. This suggests that AWN-1 and AQN-3
may bind directly to sperm membrane lipids while the other spermadhesin moieties may coat on
top of them as aggregated molecules, and indicates that different subpopulations of spermadhesins
may play diverse roles as either decapacitation or acrosome stabilizing factors, positive
capacitation elements, and/or receptors for zona pellucida.
Glycosylated isoforms of spermadhesins AQN-3, PSP-I, and AWN bind heparin but fail to
bind zona pellucida glycoproteins and soybean trypsin inhibitor [7, 8, 9]. Therefore, attachment
of a glycosyl moiety may modulate the receptor function of spermadhesins isoforms, i.e.
switching the receptor function between a capacitation factor (heparin-binding) and a primary zona
pellucida-binding molecule. The functional inability of glycosylated spermadhesins has been
found to be due to steric blockade of the ligand-binding site which results from attachment of a
single oligosaccharide to each glycoform, either N-linked to Asn50 or O-linked to Ser52 or Thr95.
This indicates that the zona pellucida- and the inhibitor-binding sites may be located around the
glycosylated residues and are different from the heparin-binding site. AWN possesses a
consensus sequence for heparin-binding (3NRRSRS8) which, in a recently developed three-
dimensional model for spermadhesins [6], is located at the opposite side of the proposed zona
pellucida/inhibitor binding domain. These arrangement of binding domains would be in agreement
with the experimental results. Nevertheless, the exact three-dimensional structure and epitope
topography of spermadhesins, as well as the way in which the heparin-like glycosaminoglycan-
and zona pellucida glycoprotein-binding information is transduced, require further investigation.
Stallion spermatozoa possess heparin-binding proteins of both the BSP and the spermadhesin
family
The heparin-binding proteins on ejaculated stallion spermatozoa have recently been isolated
and structurally characterized [4], The major components, termed HSP-1 and HSP-2, belong to
the same protein family as bovine BSP- (A 1/2 and A3). We estimate that, together, HSP-1 and
HSP-2 may account for over 70% of the total sperm-associated heparin-binding proteins.
The primary structure of HSP-1 has been completed [3], Interestingly, it is a mosaic protein
which consists of 121 amino acid residues organized in two types of homologous repeats
arranged in the pattern AA'BB' (Fig. IB). Each of the N-terminal 13-15 residues long A-type
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
521
repeat (residues 1-15 and 16-28) contains two O-linked oligosaccharide chains each (threonine
residues 5, 12, 22, and 27) and they have 47% sequence identity. The B-type repeats span 44-47
amino acids each (residues 29-72 and 75-121), are not glycosylated, show 43% sequence
identity, possess two disulphide bridges (between cysteines, 34-58, 48-72, 80-106, and 94-121)
and have the consensus pattern of the fibronectin type-II module. The sequence of the A' domain
can only be aligned with the bovine proteins by introducing numerous gaps and the N-terminal A-
domain is absent in bovine BSP-A1/2 (PDC-109) and BSP-A3 (Fig. 1A). In addition, only the
glycosylation site at threonine 22 is conserved in BSP-A1/2. On the other hand, the B-domains are
highly conserved in bovine BSP-A1/2 and BSP-A3 proteins (Fig. 1A). However, the critical
residues for gelatin-binding are only conserved in the second (B') repeat. Altogether, it seems
reasonable to propose that the different combination of structural domains of the bovine and
equine BSP-proteins could confer species-specific properties on a common heparin-binding
polypeptide framework. Further characterization of the topographic localization, sperm surface
acceptor molecules and coating dynamics, may help to clarify this point.
Another abundant sperm-associated heparin-binding protein is a 16 kDa component which
may represent some 20% of the total stallion sperm-associated heparin-binding proteins. This 16
kDa protein is not glycosylated and is immunologically related to boar AWN spermadhesin [4]. In
addition, initial structural characterization of this stallion AWN-protein shows that it shares a high
degree of amino acid sequence with the boar protein (unpublished results). However, whereas the
boar AWN spermadhesin binds homogeneously to the whole acrosomal cap surface [28], an
indirect immunofluorescence study showed that the topographical localization of the 16 kDa
AWN-related protein is restricted to the equatorial segment in specimens of both epididymal and
ejaculated stallion spermatozoa [4], AWN is the only member of the equine spermadhesin family
found on either seminal plasma or spermatozoa.
In addition to proteins of the BSP and spermadhesin families, a minor component (25 kDa),
which probably represents less then 10% of the total heparin-binding proteins, with the N-
terminal amino acid sequence IIGGWEXEKHSKPWQVAVYHQGHFQXG..., has also found
associated with ejaculated stallion spermatozoa. This sequence shows a high degree of sequence
identity with serine proteinases of the kallikrein (EC 3.4.21.35) family, in particular with human
prostate-specific antigen, PSA (27 kDa) [20], and with the androgen-dependent arginine-esterase
(EC 3.4.21.34) mRNA product of canine prostate [11]. Human PSA is one of the enzymes
involved in cleaving structural proteins of the seminal coagulum [19]. Its function in stallion
sperm physiology has not been assessed, and deserves further study.
Heparin-binding proteins on human sperm: kallikrein and spennadhesins
Recently our laboratories have begun to characterize the human seminal plasma heparin¬
binding proteins which bind to the sperm surface. Using affinity chromatography, two major
components (80 kDa and 28 kDa) have been isolated. Each protein accounts for around 50% of
the total heparin-binding proteins in seminal plasma. The 28 kDa protein contains an amino acid
sequence identical with previously characterized human plasma (glandular) kallikrein. The
structure of the 80 kDa component(s) remains to be established. In addition, using indirect
immunofluorescence microscopy, we have localized the binding site of an AWN-
crossimmunoreacting protein to the equatorial segment of ejaculated human spermatozoa. Its
relative abundance on spermatozoa has not been studied, however. Although the biological
significance of these human molecules on sperm function remains obscure, we are currently
elucidating the primary structure of human AWN as a First step towards determining its function.
Conclusion
The mechanism by which heparin modulates capacitation and/or the acrosome reaction is
poorly understood. A major event in capacitation is believed to be the removal or alteration of a
522
J. J. CALVETE ETAL. : HEPARIN-BINDING PROTEINS ( MAMMALIA )
protective coat from the sperm plasma membrane. This structural alteration may correlate with
loss or reduction of plasma membrane cholesterol, uptake of extracellular calcium, and elevation
of the internal pH, all of which constitute important steps in the capacitation and early steps of the
acrosome reaction. The biological effects of heparin is not absolutely conserved in all mammalian
species. Since, in bull, boar, stallion, and man, the seminal plasma heparin-binding proteins
which become bound to the sperm surface belong to only three different protein families (Table
1), our working hypothesis is that species-specific modulation of sperm capacitation/acrosome
reaction exerted by heparin might be related to a combination of , at least, the following factors:
The chemical structure, amino acid sequence and posttranslational modifications, of
the heparin-binding proteins bound to the sperm surface;
The relative abundance of members of the different heparin-binding protein families in
the seminal plasma;
The topography and amount of the different heparin-binding proteins on the surface of
spermatozoa during capacitation.
Table 1. — Relative distribution of heparin-binding proteins of different protein families on mammalian spermatozoa.
NF, not found.
To dissect the relative biological relevance and synergy of these, and possibly other, factors
in heparin-mediated sperm capacitation is one of the main goals of our laboratories.
ACKNOWLEDGEMENTS
The work from the authors’ laboratories has been financed by grants To 114/3-1 (E.T-P.) from the Deutsche
Forschungsgemeinsam. 01KY9103 from Bundesministerium fur Forschung und Technologic (E.T-P.), Germany, and PB92-
0096 from the Direccidn General de Investigacion Cientffica y T6cnica (J.J.C., L.S.), Spain.
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Histone Gene Expression During Mammalian
Spermatogenesis: Structural and Functional Aspects
Detlef DOENECKE & Birgit DRAB ENT
Institut fur Biochemie und Molekulare Zellbiologie der Georg-August-Universitat,
Humboldtallee 23, D-37073 Gottingen, Germany
ABSTRACT
The chromatin of spermatogenic cells undergoes structural rearrangements upon differentiation from spermatogonia to
mature spermatozoa. During the haploid stages of mammalian spermatogenesis, histones are gradually replaced first by
transition proteins and then by protamines. The histone fraction in chromatin of spermatogenic cells is composed of
testis specific subtypes as well as such histone isoforms, which are also found in somatic tissues. The subtype patterns of
all histone classes except H4 change in a stage specific manner during mammalian spermatogenesis. This implies that
control mechanisms exist which regulate the cell type specific expression of the individual histone subtype genes. This
control may be exerted at the transcriptional level as exemplified by functional studies at the Hit promoter. Regulation
also may take place posttranslationally as demonstrated by the polyadenylation of part of the mRNA of spermatogenic
cells.
RESUME
Expression des genes des histones pendant la spermatogenese des Mammiferes: aspects
structuraux et fonctionnels
La chromatine des cellules spermatog£n6tiques subit des re-arrangements structuraux pendant la differenciation
progressant de la spermatogonie au spermatozoide mur. Pendant les stades haploi'des de la spermatogenese des
Mammiferes, les histones sont remplacees graduellement d’abord par des proteines de transition puis par des protamines.
La fraction des histones dans la chromatine des cellules spermatogenetiques est composee de sous-types specifiques du
testicule ainsi que d’ isoformes des histones qui sont aussi rencontrces dans les tissus somatiques. Les sous-types de toutes
les classes d’histones sauf H4 changent specifiquement en fonction des etapes de la spermatogenese des Mammiferes. Ceci
implique qu’il existe un mecanisme de controle, qui regule fexpression specifique a chaque type cellulaire des genes
individuels de chaque sous-type d’histone. Ce controle peut etre exerce au niveau transcriptionnel comme font montre les
etudes fonctionnelles sur le promoteur des Hit. La regulation peut aussi etre post-traductionnelle ainsi que le montre la
polyadenylation d'une partie des ARNm des cellules spermatogenetiques.
Histones are the basic chromosomal proteins of eukaryotic organisms. The histone protein
familiy is composed of five protein species which have been classified on the basis of size and
function. First, five different classes were defined by electrophoretic means and were termed HI,
Doenecke, D. & Drabent, B., 1995. — Histone gene expression during mammalian spermatogenesis: structural
and functional aspects. In: Jamieson, B. G. M., Ausio, J„ & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and
Taxonomy. Mem. Mus. natn. Hist, nat., 166 : 525-535. Paris ISBN : 2-85653-225-X.
526
D. DOENECKE & B. DRABENT : HISTONE GENE EXPRESSION {MAMMALIA)
H2A, H2B, H3 and H4 (for reviews, see [93, 95]). Two copies of each of the four histones
H2A, H2B, H3 and H4 form the nucleosomal core. Therefore, they are summarily described as
core histones in contrast to the HI class proteins, which interact with the linker DNA connecting
nucleosomal cores and are termed linker histones. Core and linker histones have been detected in
nearly all eukaryotes [95]. The yeast Saccharomyces cerevisiae is an exception in having no linker
histone [18], but its chromatin forms core nucleosomes and shows a subunit pattern with a
regular spacing [90].
Histone protein patterns have been monitored during spermatogenesis in a broad spectrum
of lower and higher eukaryotes [6, 7, 13, 16, 54, 62, 66, 67, 83, 85, 87]. Our group has
concentrated on mammalian systems and has studied the structure and expression of somatic and
testis-specific histone genes from man and mouse [2-4, 31, 32, 34-38]. In this contribution,
structural and functional features of testicularly expressed histone genes and gene products will be
discussed in relation to different stages of spermatogenesis (see Table 1).
Table 1. — Mammalian spermatogenic histone gene expression. Compilation of histone subtype proteins detectable at
specific stages of sperm differentiation before replacement by transition proteins and finally by protamines (data
from rat, mouse and man or from one or two only of these). Asterisks indicate expression data obtained using gene
probes.
Source .
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
527
OBSERVATIONS AND DISCUSSION
Testicular histone subtypes
The HI linker histone family of mammals comprises several variant isoforms. The most
detailed analysis of the H 1 complement has been done in human and murine chromatin in several
cell and tissue types. In humans, five main type HI histone genes have been described [3] in
addition to the gene encoding Hlo [31], which is a histone confined to highly differentiated cell
types, and the highly conserved Hit gene [34], which is only expressed in testicular cells. Hit
protein sequences are known from man [34], other primates [59], mouse [35], rat [22] and boar
[21] . The Hit protein is confined to male germ cells and is not a general meiosis specific variant
[65], As in humans, five different mouse HI proteins (or genes) [3, 33, 62, 88, 89, 97] plus Hlo
[4] and Hit [35] were described. As yet, only one rat main type HI gene [23, 36], the Hit gene
[22] and a partial Hl» cDNA sequence [17] have been described. The rat Hit gene and gene
promoter structures have been intensely studied [49-51] and will be discussed below. The Hit
fraction of the overall testicular HI complement amounts to about 25% [78], and somatic type
isoforms [62] and the Hlo fraction [84, 87] constitute the remaining part of the HI histone moiety
in testicular chromatin. In pachytene spermatocytes and later stages Hit may comprise a much
higher fraction [13, 67],
Core histone isoforms, which are restricted to testicular cells, have been described in several
species. A pair of testicularly expressed genes consisting of an H2A and an H2B gene was
described by Huh et al. [55], These genes appear to code for the previously described testicular
subtypes TH2A and TH2B, respectively [10, 58, 92, 98]. In addition to main type H2A and H2B
isoforms, two H2A subtypes, which are replication independent, i.e. H2A.Z and H2A.X [53,
64], have been described. H2A.X is enriched in testicular chromatin, whereas H2A.Z is
uniformly found in most somatic tissues [53]. A testicular subtype of H3 (TH3) has been isolated
from rat testis [91]. Its unique amino acid composition (including three cysteine residues)
indicates structural differences compared with all other known H3 subtypes, but as yet no TH3
gene has been identified in any mammalian genome. The replication independent H3 subtype
H3.3 is also expressed during spermatogenesis. For example, H3.3 has been observed in
spermatid stages of spermiogenesis [69], but it is also present at earlier stages of spermatogenesis
[67, 91]. H4 is the most conserved of all histone classes. Its 102 amino acid sequence is strictly
maintained in all mammalian species. This even applies to H4 genes which are differentially
expressed. For example, the human, rat or mouse Hit genes are located near testicularly
expressed H4 genes which code for the same H4 amino acid sequences as other H4 genes from
the same species.
Organization of mammalian histone gene clusters
The majority of histone genes in the murine and human genomes is clustered at specific
chromosomal sites. Except the Hl° gene, all known human HI genes and surrounding core
histone genes are located on chromosome 6 [3]. A minor portion of core histone genes maps to
chromosome 1 [46], and the solitary Hlo gene is located on the long arm of chromosome 22 [3].
The situation in the murine genome appears to be similar, since a major histone gene cluster
including the Hit gene has been mapped to chromosome 13 [26, 75], and the murine Hlo gene is
on chromosome 15 in a region which is syntenic with the region on the human chromosome 22,
where the human Hlo gene is located [3, 1 1].
The human Hit gene, which is expressed in pachytene spermatocytes (see below) forms
part of the major gene cluster on chromosome 6, which also contains the other HI genes [3].
Thus, the generation of H 1 histone patterns, which are characteristic for cells of specific stages of
spermatogenesis, must depend on a differential regulation of the genes within that major cluster.
In addition, the expression of the Hl° gene, which appears to be developmentally regulated
528
D. DOENECKE & B. DRABENT : HISTONE GENE EXPRESSION (MAMMALIA)
during differentiation of several cell types [99], must undergo tissue-specific control. It is
preferentially expressed during early stages of spermatogenesis [42] and in somatic cells it mainly
appears upon terminal differentiation [99].
The genes coding for the testicularly expressed histones TH2A, TH2B and TH3 have not
yet been mapped to specific chromosomal sites. They also may form part of the major histone
gene cluster. GRIMES et al. [49] have shown that an H4 gene, which is located near the rat Hit
gene, is testicularly expressed. It has the same primary structure as other mammalian H4 proteins.
On the basis of its variant nucleotide sequence and testicular expression, this gene may be termed
H4t [49, 94]. Its association with the Hit gene, which is located within the major cluster of
somatically expressed histone genes, implies that it is not a solitary gene. In contrast to the Hit
gene, expression of this neighbouring H4 (H4t) gene is not confined to spermatogenic cells, but
its mRNA also has been detected in a rat myeloma cell line.
Cell type specific histone patterns at different stages of spermatogenesis
The HI patterns of different somatic cell types or germ cells are not uniform, but vary in
their HI subtype composition. In several mammalian species, five main type HI protein species
(termed HI a-Hle) were described [62], In rat testes, the subtypes Hla-Hle can be detected (Hla
and Hlc predominating) during all stages of spermatogenesis until the primary spermatocyte stage
[12, 62]. Similarly, the subtypes Hla and Hlc predominate in mouse germ cells until the meiotic
prophase [62]. Immunocytochemical analysis of murine tubuli seminiferi showed the greatest
level of reactivity in primary spermatocyte nuclei using antibodies against Hla [79].
Developmental studies showed that the first expression of the Hla gene occurs in 7 day old mice
at a stage when intermediate and B type spermatogonia appear [79], In situ hybridizations with
human testis detected the mRNA coding for human Hl.l (equals Hla according to [76]) until the
stage of round spermatids [14, 15]. Thus, the subtype Hla appears to be a major constituent in
the chromatin of mammalian germ cells [13, 67]. In addition, the subtypes Hlb, c and d
contribute to the germ cell chromatin [62, 67, 78].
The HI subtype Hl° has been described in unfractionated mammalian testis cell
preparations [83, 85, 86], The predominant expression of the Hl° gene in spermatogonia was
suggested by promoter studies of GARCIA-IGLESIAS et al. [42], In that work, the Hlo promoter
was ligated upstream of a B-galactosidase gene and the expression of this construct was monitored
in transgenic mice. The analysis showed that the promoter was used in several tissues, such as
specific cell types in kidney, brain and testis. Testicular mRNA synthesis was mostly confined to
spermatogonia, but immunofluorescence studies with Hlo antibodies indicated expression in
Sertoli cells, too. Thus, expression of the Hl° gene may be confined to early stages of
spermatogenesis, but somatic cells in the testis also express the Hlo gene.
The Hit protein is absent from spermatogonia and is first detected in pachytene
spermatocytes [35, 60, 69], This has been demonstrated at the protein level in chromatin from
mouse and rat cells fractionated by elutriation centrifugation [44], After the cloning of the genes
coding for the human, murine and rat Hit proteins [22, 34, 35], Northern blot and in situ
hybridization analysis has confirmed these protein data showing that the mRNA is only found in
pachytene spermatocytes [60] whereas the proteins are preserved in the subsequent stages until
histone replacement by transition proteins [69],
The major change in chromatin structure during the meiotic prophase is also evident in the
H2A/H2B class of histones. The subtypes TH2A and TH2B both become first detectable in
pachytene spermatocytes of rat and mouse [20, 67, 78, 92], The subtype H2A.X, which, like
Hlo, is a non replication-dependent histone [72, 96], has been detected in type A spermatogonia
[64], Expression of a modified H2B protein has been found during mouse spermiogenesis [70].
In a cDNA library constructed from spermatid RNA, an H2B cDNA sequence was observed
which was extremely similar to other mouse H2B gene sequences, but the C-terminus coded for
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ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
529
12 additional amino acids, 7 of which were hydrophobic. Northern blots with RNA from other
tissues indicated that this transcript was testis-specific [70]. Similarly, a polyadenylated H2A
mRNA was detected in mouse round spermatids [71].
The histone to protamine transition during human spermiogenesis does not result in a
complete removal of all histones [43, 52], About 15% of the human sperm DNA appears to
remain associated with core histones, i.e. mainly with H2A (H2A.X and trace amounts of
H2A.Z), several H2B isotypes, H3.1, H3.3 and highly acetylated H4 [43]. In contrast to these
remaining core histones, no association of any H 1 subtype with mature sperm chromatin has been
observed.
The gradual changes of the core and linker histone moieties during the development of male
germ cells and the changes in chromatin morphology and gene activity suggest a functional role
for the individual histone subtypes. However, correlations between specific structural features of
histones and functional differences have not yet been established in any somatic or germ cell
system. Hl», which appears to be confined to early stages of spermatogenesis, is correlated with
terminal differentiation in several cell types (for review, see [99]). Its avian countertpart H5 is
confined to the condensed, transcriptionally inactive nuclei of avian red blood cells [5]. The high
arginine content [30, 3 1] of the H5 histone is considered as one of the reasons for its condensing
capacity. Compared with the other HI subtypes, Hit is also enriched in arginine, but DE LUCIA et
al. [29] have shown by circular dichroism analysis that Hit has a lower condensing capacity than
the other HI subtypes. Thus, Hit may even contribute to activating effects in the chromatin of
developing germ cells rather than repressing nuclear activity. It may thus help to decondense the
chromatin structure for the specific needs of the meiotic and haploid stages of germ cell
development.
Postsynthetic histone modifications
Posttranslational modifications of histone proteins have been primarily observed at
spermatid stages of spermatogenic cell development. Recently, the phosphorylation of Hit in
elongating spermatids has been described [69]. In the same study, which used vitamin A as a
means to synchronize rat seminiferous epithelia into few stages of spermiogenesis, additional
bands of H2A.1, H2A.2 and TH2A were observed and were interpreted as postsynthetic
modifications. A complex pattern of phosphorylation of the H2A.X subtype has been observed in
murine testicular cells [45]. Another type of histone modification is the conjugation with
ubiquitin. This has been described for H2A histones during rooster spermatogenesis [ 1 ].
The most impressive modification of histones during spermatogenesis is their
hyperacetylation. This modification of the H4 histone structure is correlated with a broad
spectrum of cellular processes, including transcriptional control and chromatin assembly (for
review, see [95]). H4 hyperacetylation occurs in elongating spermatids [47, 48, 68]. This is the
stage when displacement of histones by transition proteins begins [69, 73]. Thus, the association
of highly acetylated H4 with the stage of histone displacement in rat spermatids is in agreement
with the idea that reducing the positive charge of specific lysine residues may help to displace
histones from chromatin during spermiogenesis.
Regulation of testicular histone gene expression
The location of spermatogenesis related histone genes within clusters of somatic histone
subtype genes implies that control steps discriminate between the different member genes of the
gene cluster. This control may take place at the transcriptional level, but also posttranscriptionally,
i.e. during processing of the primary transcript or by influencing the stability of specific histone
mRNAs (for review, see [74]). At the transcriptional level, promoter structures of specific histone
genes may contain sequence motifs where interaction with germ cell-specific transcription factors
controls the specific expression of the respective genes.
530
D. DOENECKE & B. DRABENT : HISTONE GENE EXPRESSION (MAMMALIA)
The mechanism of HI histone gene regulation in somatic cells is not yet fully understood.
Sequence analysis of HI gene promoters in all vertebrate systems studied revealed that the
heptanucleotide A A AC AC A is conserved at a position 100 nucleotides upstream of the
transcription start site [25, 27, 281. Functional studies indicated the involvement of this HI -box in
the S-phase-dependent expression of HI genes [27, 28, 61], but variants of this sequence motif
have been observed [38]. A second sequence element, which is involved in the regulation of HI
genes, is the CCAAT box, which is the binding site for an Hl-specific regulatory factor [41], The
sequence analysis of the rat, human and murine Hit promoters revealed that their sequences
contained all main features of S-phase-dependent HI genes: TATA-box, GC-rich element,
CCAAT-motif and Hl-box [22, 34, 35, 50, 51]. Thus, the known regulatory elements within
Hit promoter structures apparently do not reflect the fact that the Hit gene is not transcribed
during DNA replication, but at the pachytene stage of the meiotic prophase. GRIMES and
coworkers [50, 51] searched for a testis-specific element and defined the palindromic
hexanucleotide CCTAGG, which is located between the GC-rich element and the CCAAT-box of
the rat Hit promoter as the testis-specific promoter element [50, 51]. This element was identified
as the site of interaction of testis-specific DNA-binding proteins at the promoter in pachytene
spermatocytes [51]. Further support for a functional role of this sequence element may be derived
from the human Hit promoter, where this sequence motif is conserved at the same site [32, 34],
The palindromic arrangement, however, may not be mandatory, since the mouse Hit promoter
shows a varied element, CCTGGG, at the same location [35].
The rat TH2A and TH2B genes are grouped together, and they are divergently transcribed
from a joint promoter region of about 240 nucleotides in between the two genes [56-58], In both
directions, TATA- and CCAAT-boxes are located upstream of the two genes. In addition, the
TH2B gene promoter contains the OctI element ATTTGCAT, which is a characteristic regulatory
element in all H2B gene promoters [40] but also in control regions of several other genes. For
example, variant Oct factors binding to such elements have been detected during mouse
embryogenesis and are specifically expressed in germline cells [81]. In conclusion, the promoter
arrangement of the TH2B gene does not vary from consensus H2B promoter structures and it
does not reflect the replication-independent, testis-specific expression of this histone gene.
Functional studies with the TH2B promoter in fibroblast cells revealed that the CCAAT- and
octamer elements of this promoter are involved in the S-phase dependent expression of the TH2B
gene when transfected into these somatic cells [56-58]. Subsequent studies showed that
differential methylation at specific sites of the TH2B promoter contributes to the tissue-specific
transcription of this TH2B gene [20] and that a repressor protein specific for the rat TH2B gene
was present during early stages of spermatogenesis [63].
The gene coding for TH3, which has been described as a testis-specific H3 subtype in rat
spermatogonia [91], has not yet been detected. Thus, no data on cell specific regulation of
testicular H3 histones exist. As mentioned above, GRIMES et al. [49] have shown that an H4 gene
is closely associated with the rat Hit gene. SI nuclease analysis has shown that this particular H4
gene is transcribed in the testis predominantly during the pachytene stage, but it is also expressed
in a tumor cell line. This is in contrast to the neighbouring Hit gene, which is solely transcribed
in pachytene spermatocytes [50, 94],
The control of histone gene expression is not restricted to transcriptional regulation (for
review, see [74]). Processing of the primary transcript and mRNA stability of replication
dependent histone gene products depend on the presence of a dyad symmetry element at the 3’
end of the mRNA, which is not polyadenylated [8, 82], The only exceptions from this rule are the
S phase-independent replacement histone variants, such as Hlo, H3.3, H2A.Z or H2A X which
are all encoded by polyadenylated histone mRNAs. Remarkably, the Hit genes of rat, mouse and
m£xAhow the same dyad symmetry elements as replication dependent histone genes and the
mRNAs are non polyadenylated.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
531
Posttranscriptional regulation of testicular histone gene expression
As mentioned above, main type, S phase-dependent histone mRNAs in somatic cells are
poly(A)- in contrast to the mRNAs encoding the replacement histone subtype mRNAs. In addition
to these specific subtype mRNAs, polyadenylated testicular histone mRNAs have been described
[39], These are at least in part derived from genes which are transcribed to poly(A)--mRNA in
somatic cells. A poly(A)+ histone H2B mRNA with an extended reading frame and a consensus
AAUAAA polyadenylation signal has been detected in mouse spermatids [70]. Recently, a
polyadenylated H2A gene transcript was found in murine round spermatids. In this case, the
poly(A) tail was not preceded by the somatic AAUAAA signal sequence [71]. In a detailed
analysis of histone mRNAs in chicken spermatids, CHALLONER et al. [19] detected an H2B
mRNA subpopulation, which was polyadenylated despite the fact that the histone mRNA was
derived from a gene which is transcribed as a poly(A)--mRNA in somatic cells. The transcript
from this same gene was elongated by 26 or 28 nucleotides beyond the histone mRNA consensus
termination site, and a poly(A) tail was added to this elongated mRNA.
A major step in histone gene regulation is the control of mRNA degradation [74], This has
not been specifically studied in testicular histone gene expression, but the addition of poly(A) tails
to part of the histone mRNA population during spermatogenesis suggests that it is a means to
increase the stability of this mRNA, which is either synthesized at post-meiotic stages or is
synthesized at early spermatogenesis and is preserved for later stages of development, when a
certain pool of mRNAs for histone replacement may be needed.
Conclusions
Modulation of the chromatin structure during spermatogenesis requires changed patterns of
histone proteins and histone modifications which contribute to restructuring of chromatin and to
the transition towards the inactivation of the genome in generating the condensed genome of
mature sperm. Specific histone subtypes, which differ from their somatic counterparts have been
described for all histone classes except H4. The most drastic changes in histone gene expression
and chromatin restructuring occur during the meiotic prophase, when specific subtypes of HI,
H2A and H2B, i.e. Hit, TH2A and TH2B, are synthesized. These testis specific isoforms remain
associated with the chromatin of cells during the haploid stages of sperm cell differentiation. At
this spermiogenesis period, remodelling of chromatin before the final deposition of protamines
may require a specific chromatin structure which is accessible for regulatory factors such as non¬
histone proteins and for transition proteins replacing the histone moiety. This specific chromatin
structure may be established by specific subsets of core and linker histones and by their
posttranslational modification.
ACKNOWLEDGEMENTS
Work performed in the laboratory of the authors was supported by a grant from the Deutsche
Forschungsgemeinschaft.
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early stages of spermatogenesis. Journal of Biological Chemistry, 267: 15271-15273.
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76. Parseghian, M. H., Henschen, A. H., Krieglstein, K. G. & Hamkalo, B. A., 1994. — A proposal for a coherent
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Biochemical and immunocytochemical analysis of a histone HI variant from the mouse testis. Journal of Cell
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80. Rasheed, B. K. A., Whisenant, E. C. & Bhatnagar, Y. M., 1989. — Physical mapping of mouse histone gene
clusters. Biochimica et Biophysica Acta, 1048: 110-112.
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187-191.
83. Seyedin, S. M. & KlSTLER, W. S., 1980. — HI histone subfractions of mammalian testis. Biochemistry, 18: 1371-
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85. Seyedin, S. M. & Kistler, W. S., 1981. — HI histones from mammalian testes. Hit is associated with
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Proceedings of the National Academy of Sciences USA, 72: 2714-2718.
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TH3, a germ cell specific variant of histone 3 in the rat testis. Journal of Biological Chemistry , 259: 8769-
8776.
92. Trostle-Weige, P. K., Meistrich, M. L., Brock, W. A., Nishioka, K. & Bremer, J. W., 1982. — Isolation and
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1260-1268.
Source . MNHN. Paris
Source : MNHN . Paris
Sequence, Evolution and Transcriptional Regulation
of Avian-Mammalian PI Type Protamines
Rafael OLIVA
Human Genome and Molecular Genetics Research Group,
Faculty of Medicine, University of Barcelona, Diagonal 643, 08028 Barcelona, Spain
ABSTRACT
Methodological approaches to protamine PI sequence determination have evolved from the initial protein sequencing
methods to the robust cloning and PCR-based techniques. Twenty-seven different mammalian-avian PI type protamine
genes and 32 different PI amino-acid sequences are now available and allow detailed phylogenetic analysis and the study of
transcriptional control mechanisms. All mammalian-avian PI type protamines contain a well conserved N-terminus with
the consensus "ARYR" followed by alternating "S/T-S-S” phosphorylatable residues. Eutherian mammalian Pis contain
cysteine residues, whereas birds, prototherian and metatherian protamines lack cysteine. Thus cysteine appeared after the
divergence of marsupials, monotremes and placental lineages. Overall detailed phylogenetic analysis of the gene
sequences indicates that the evolution of PI genes is in agreement with the expected species evolution supporting that
these genes have evolved vertically.
RESUME
Sequence, evolution et regulation transcriptionnelle des protamines de type PI des Oiseaux et
Mammiferes
Les approches methodologiques dc determination de sequence des protamines PI ont evolue depuis les premieres
methodes de s^quen^age de proteines jusqu’aux techniques fiables basees sur le clonage et la reaction d'amplification en
chaTne. Vingt-sept genes differents de protamines dc type PI des Oiseaux et Mammiferes et trente-deux sequences
differentes d’acides amines sont maintenant disponibles et permettent une analyse phylogenique et une etude des
mecanisme de controle de la transcription. Toutes les protamines dc type PI des Oiseaux et Mammiferes contiennent une
extremity N-terminale bien conservee avec la sequence consensus “ARYR” suivie par la s6quence alternee de residus
phosphorylables “S/T-S-S". Les protamines PI des Mammiferes Eutheriens contiennent des residus de cysteine, alors que
les Oiseaux, les Protheriens et les M6thath6riens n’en ont pas. La cysteine est done apparue apres la divergence des lignees
des Marsupiaux, des Monotremes et des Placentaires. Une analyse phylogenique generale et detaillee des sequences de
genes indique que Involution des genes des PI est cn accord avec Revolution attendue des especes, ce qui indique que ces
genes ont 6volue verticalement.
Protamines are small (30-60 amino acids) and very positively charged proteins (40-70%
arginine) which appear at the late stages of spermatogenesis in many but not all animal, and some
plant, species [6, 11, 16, 21-23, 25, 43, 50, 52, 60, 69-73]. In those species in which they
occur, such as in all mammals [6, 52], birds [13, 52, 53,], some teleost fish [11, 16], some
reptiles [25, 70] and amphibians [25] they replace most of the histones during spermiogenesis and
Oliva, R., 1995. — Sequence, evolution and transcriptional regulation of avian-mammalian PI type protamines.
In: Jamieson, B. G. M.. Ausio, J., & Justine, J.-L. (eds). Advances in Spermatozoal Phylogeny and Taxonomy. Mem.
Mus. natn . Hist, nat., 166 : 537-548. Paris ISBN : 2-85653-225-X.
538
R. OLIVA : AVIAN-MAMMALIAN PI TYPE PROTAMINES
become the major sperm nuclear protein [47, 48, 52, 54], There are two basic groups of
protamines in mammals; the PI protamines which have been found in all mammalian species that
have been analyzed and the P2 protamines which have been found in humans [5, 8, 42, 79, 80]
and a limited number of other mammals such as mouse, guinea pig and stallion [10, 59, 66],
However, pro-P2 protamine genes have been sequenced from eight species of primates [66].
Both types of protamines contain cysteine which can form disulphide bonds and contribute to the
stability of the condensed sperm nucleus. Bird protamines lack cysteine [13, 45, 49, 52] although
they are clearly related to mammalian PI protamines as several identical amino acid sequence
motifs are present in both cases. Because of the high variability of protamines and protamine
genes it is very difficult at present to explain the evolution of these proteins within an entire
phylum. In many cases the limited number of sequences available precludes their connection into
a coherent evolutive pathway. Thus the focus in this review has been placed in the avian
mammalian-mammalian PI type protamine for which a considerable amount of information is
now available. Other papers and reviews cover other vertebrate or invertebrate groups ([4, 12, 13,
16, 25, 43, 49, 52, 60, 70, 71, 73], see also CHIVA, SAPERAS, CaCERES & AUSIO, this
volume, and PRATS & CORNUDELLA, this volume). Protamine genes are a clear example of
highly tissue-specific genes. However the mechanisms that direct their specific expression in the
testis are not fully understood [18, 22, 50, 52, 80]. Thus the last section of this review covers the
progress made in the understanding of the transcriptional control of the PI genes.
RESULTS AND DISCUSSION
Methodological approaches to protamine PI sequence determination:
The methods initially available to sequence protamines were based on end group analysis,
proteolytic digestion, isolation, sequencing, and overlapping of the protamine peptides. The
presence of several arginine tracts in each protamine with very similar sequences made this
approach technically difficult. The first reported avian-mammalian PI type complete sequences
using these methods corresponded to bull [15; Table 1], Gallus domesticus [45] and boar [76].
Some discrepancies in the initial reported sequences were found when the corresponding
protamines were re-examined by automated micro-sequencing or by cloning of the protamine
cDNAs and genes [37, 49, 40]. Subsequently the use of automated protein micro-sequencing led
to the determination of the sequences of human PI [41], stallion PI [2, 9], ram [71] and rabbit,
goat and rat [3], Partial sequences corresponding to a few N-terminal residues have also been
reported for many mammalian protamines [6] (Table 1).
Simultaneously to the onset of the use of automated micro-sequencing, the methods of the
cDNA synthesis, donning and sequencing were also developed and applied to protamine genes
(Table 1). The first mammalian protamine cDNA sequence corresponded to mouse PI [29]. Since
no probes were initially available to screen the cDNA library, this initial sequence was obtained by
characterization of selected clones preferentially expressed in spermatids [28]. Subsequently, the
use of the mouse PI cDNA clone as a probe led to the determination of the sequence of bovine
protamine PI cDNA [35], Simultaneously, the bovine protamine PI cDNA sequence was also
independently obtained using oligonucleotides designed from the previously known amino acid
sequence [31], The mouse PI cDNA also led to the isolation of the boar protamine 1 cDNA [37]
and rat PI cDNA [30], The bovine probe led to the isolation and sequencing of the human
protamine PI cDNA [36]. However the mammalian probes would not recognize the avian
protamine genes because of marked divergence in the nucleotide sequences between these species.
Thus the cDNA sequence corresponding to rooster protamine was obtained by random
sequencing of 210 clones from a rooster testis cDNA library until the sequence of one clone
predicted an amino acid sequence similar to the previously reported at the protein level for galline
[55]. The availability of a cDNA probe from galline led to the rapid isolation and sequencing of
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
539
1. — Chronological list of reports on protamine PI sequences with indication of the species and methods used for
sequencing.
540
R. OLIVA : AVIAN-MAMMALIAN PI TYPE PROTAMINES
another avian protamine, the quail ( Coturnix japonica ) [53], as well as to the isolation and
sequencing of the chicken genomic clones [49]. The same approach using cDNA as probes to
screen genomic libraries was also followed to obtain the genomic sequences corresponding to bull
PI [32], mouse PI [24], human PI [18] and boar PI [26] (Table 1). The availability of probes
for all these genes also led to the determination of their copy number which proved to be one copy
per haploid genome for PI protamines in mammals, two identical genes (coding region) per
haploid genome in Gallus domesticus, and one copy of P2 per haploid genome in mammals. This
indicated that the numerous basic protamine type molecules present in the sperm nucleus [PI, P2,
P3, P4 and others] of mammals corresponded only to two types of protamines [PI and P2]. The
PI genes are located adjacent to other spermatid-specific genes in the mammalian genome [19, 34,
46], As some mammalian protamine genes were sequenced by independent laboratories, some
minor discrepancies in the reported sequences also emerged, such as in the bull genes [31, 35]
and between the human PI sequence initially reported [18] with that subsequently redetermined in
several independent individuals [62],
The availability of the PI genomic sequences from human, bull, mouse and boar allowed
their comparison in a search for conserved flanking sequences from which to design consensus
oligonucleotides. This approach proved to be extremely efficient leading to the isolation and
sequencing of the Saguinus imperator PI protamine [63], Orcinus orca PI [1], and subsequently
several primates (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, gibbon,
Cercopithecus patas, Alouatta seniculus ) [68], several human individuals (Mediterranean,
Korean, Sudanese, American Indian) [62], additional eutherian mammals (rat, guinea pig, cat
bear, elephant, horse, camel, deer, elk moose, gazelle) [61] and the monotremes, platypus and
echidna [67]. The determination of a Wallaby partial amino acid sequence [7] by protein micro¬
sequencing led to the sequencing of the opossum protamine PI [77, 78]. A similar PCR-based
approach was followed to amplify and sequence the promoter region of the rat, guinea pig,
gorilla, orangutan, anubis baboon and red monkey [64].
Although PCR from genomic DNA is a very valuable tool in the amplification and
sequencing of new protamine genes it does not provide information on whether the sequenced
genes are expressed or not (for instance, if a sequenced gene is a pseudogene). In the case of PI
genes, the fact that all mammalian species where the sperm nuclear protein content has been
analyzed contain protamine PI together with the single copy number of the PI genes in mammals,
suggests that most (if not all) of the mammalian species whose PI sequence has been determined
by PCR also express the sequenced gene. However this could be a limitation in the prediction of
functional properties based on the derived amino acid sequence in the case of those proteins (such
as P2 protamine) which are not ubiquitously expressed in mammals [66].
The availability, at present, of a large number of PI sequences should allow design of new
primers with which to amplify and sequence the PI genes corresponding to species which have
remained so far elusive. In the case of the P 1 genes the PCR approach has been successful in the
amplification of sequences corresponding to members of the class from which the oligos were
predicted (e.g. mammals), but failed in the amplification of the PI genes corresponding to other
classes (e.g. reptiles or amphibians). Thus determination of the sequences of protamine genes in
other vertebrate classes (or in other phyla) will probably require laborious groundwork based on
either protein micro-sequencing (followed by oligonucleotide design and PCR) or cDNA based
approaches [29, 55]. However once one or a few nucleotide sequences became available in the
additional phyla and classes [4, see PRATS & CORNUDELLA, this volume], the same PCR-based
approach successlully used in mammalian-avian Pis should also work in the determination of
protamine sequences corresponding to most of the members of other classes.
Source :
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
541
Bull
Goat
Ram
Orca
Boar
Horse
Mouse
Rat
Rabbit
Pygmy chimp
Common chimp
Gorilla
Human
Orangutan
Red monkey
Gibbon
Red howler
Marmoset
Opossum
Echidna
Platypus
Chicken
Quail
10 20 40 40 50 60
ARYRCCLTH- -SGSRCRRRRRRRCRRRRRR-FG - RRRRRR - VCCRR - YTVIRCTRQ
ARYRCCLTH- -SRSRCRRRRRRRCRRRRRR-FG - RRRRRR - VCCRR - YTWRCTRQ
ARYRCCLTH- - SRSRCRRRRRRRCRRRRRR- FG - RRRRRR - VCCRR - YTWRCTRQ
ARNRC-RSP — SQSRCRRPRRR-CRR-RIR-CC - RRQ-RR - VCCRR - YTTTRCARQ
ARYRCCRSH- - SRSRCRPRRRR-CRRRRRR-CC - PRR-RRA- - VCCRR - YTVIRCRRC
ARYRCCRSQ— SQSRCRRRRRRRCRRRRRR-SV - R-Q-RR- - -VSCRR- - - YTVLRCRRRR
ARYRCCRSK- -SRSRCRRRRR-RCRRRRRR-CC - R-RRRR - RCCRRRRSYT- IRCKKY
ARYRCCRSK— SRSRCRRRRR-RCRRRRRR-CC - R-RRRR— -RCCRRRRSYT -FRCKRY
VRYRCCRSQ-- SRSRCRRRRR-RCRRRRRR-CC - QRRRVR - KCCRR — TYT-LRCRRY
ARYRCCRSQ — SRSRCYRQRR — SRRRKRQ- SC - QTQRRAM- - RCCRRR — SR- LRRRRH
ARYRCCRSQ— SRSRCYRQRQ-RSRRRKRQ-SC - QTQRRAM- -RCCRRR- -SR-MRRRRH
ARYRCCRSQ — SRSRCYRQRQ-TSRRRRRR-SC - QTQRRAM- -RCCRRR — NR-LRRRKH
ARYRCCRSQ— SRSRYYRQRQ-RSRRRRRR-SC - QTRRRAM — RCCRPR — YR- PRCRRH
ARYRCCRSQ— SQSRCCRRRQ-RCHRRRRR-CC - QTRRRAM— RCCRRR— YR-LRCRRH
ARYRCCRSQ— SRSRCCRQRR-RCRRRRRR-RC - RARRRAM — KCCRRR — YR-LRCRRY
ARYRCCRSQ - - SRSRCYRRGQ - RSRRRRRR- SC - QTRRRAM- -RCCRPR- -YR- LRRRRH
ARYRCCRSRSLSRSRCYRQRP- RCRRRRRR- SC - RRP-RAS- -RCCRRR— YR-LRRRRY
ARYRCCRSQ - - SRSRCYRQRR- RGRRRRRR-TC - RRR-RAS- -RCCRRR- -YK-LTCRRY
ARYRR- RSRSRSRSRYGRRRRR- SRSRRRR- SRRRRRRRG - RRG- - RGYHRRS PHRRRRRRRR
ARFRPSRSR — SRSLYRRRRR — SRRQRSRRGGRQTGPRKITRRGRGRGKSRRRRGRRSMRSSRRRRRRRRN
ARFRRSRSR — SRSLYRRRRR — SRR - GGRQTRSRKLSR- SRRRGRSRRRKGRRSRRSSRRS — RRRN
ARYRRSRTR- - SRS PRSRRRRRRSGRRR - SPRRRRRYGSARRSRRSVGGRRRR- YGSRRRRRRRY
ARYRRTRTR — SRSR - RRRRSRRRR - SSRR-RRYGRSRRSYRSVGRRRRR-YGRRRRRRRRY
Fig. 1. — Alignment of the reported avian-mammalian PI amino acid sequences (see Table 1).
Implications for protamine PI gene evolution
The coincidence of the four C-terminal amino acids between mammalian Pis and galline
(the protamine from Gallus domesticus) has been known for nearly two decades [45]. However,
because of the lack of cysteine residues in bird protamines and the presence of these amino acids
in mammalian protamines, both types of protamines had been classically classified as belonging to
different types. According to Bloch’s classification, galline was type 1 (or a true protamine)
whereas mammalian protamines were type 2 (stable or keratinous protamines). The similarity
between mammalian and rooster protamine became stronger when the sequence predicted from the
genome (and in accordance with the re-sequence obtained for the N-terminus of the protein) [49];
(Fig. 1) was used instead of the initial sequence reported [45]. For instance, the single threonine
residue present in galline occupies exactly the same position as that present in ram and bull [52]
(Fig. 1). The determination of the quail protamine sequence [53] also revealed the presence of the
N-terminal ARYR motif and a size (56 residues) closer to mammalian Pis (50 residues) than
those with galline (61 residues; Fig. 1). An alternating triple phosphorylatable site (T/S-S-S) is
also found at positions 9-15 in all avian-mammalian PI protamines (Figs 1, 2) [6, 20, 52, 57,
58]. The size among bird protamines appears consistently similar to that of mammals (Fig. 1) [13,
14]. Altogether the data strongly suggest the existence of an avian-mammalian protamine gene line
during evolution (Fig. 2).
A new insight has come form the recent determination of the sequence corresponding to the
prototherian (the monotremes platypus and echidna) [67] and the metatherian protamines (the
marsupials) [7, 69, 77, 78]. All these sequences lack cysteine and the corresponding genes
contain one intron. Thus these species are closer to birds according to their lack of cysteine but
closer to eutherian mammals according to the presence of the single intron. Detailed phylogenetic
analysis indicates that these sequences are half way between eutherian mammals and birds [7, 49,
67, 77, 78]. Based on the limited (but significant) similarity in the introns between prototherian
542
R. OLIVA : AVIAN-MAMMALIAN PI TYPE PROTAMINES
Fig. 2. — Possible pathway of mammalian-avian protamine PI gene evolution.
and eutherian mammals it was concluded that the introns in the protamine PI genes of
monotremes, marsupials and eutherian mammals were derived from a single intron that was
probably inserted into the ancestral gene prior to the divergence of the theria and prototheria 150
to 170 million years ago [67] ( Fig. 2).
The comparison of all protein and DNA sequences further strengthens the idea that
protamines are amongst the most rapidly diverging proteins studied [68]. This variation may
allow discrimination of closely related species or even individuals in some cases. For instance, a
sequence polymorphism has been found in the human PI gene [62], Molecular analysis of the PI
genes from nine primates revealed that within primates the rate of evolutionary change is much
higher than that within other mammalian orders [68]. Interestingly, the primate PI data confirm
that human-gorilla-chimpanzee PI protamines are indeed very similar but, unlike the slightly
closer association between chimpanzee and human derived from analysis of other genes [66], the
human-gorilla relationship is slightly favoured in the case of the PI genes [68],
Overall phylogenetic analysis of all PI sequences (Table 1) indicates that the molecular
evolution of PI genes is in agreement with the expected species evolution supporting that these
genes have evolved vertically [61, 83] (Fig. 2).
Source . MNHN . Pans
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
543
-150
*140
-130
-110
-100
CCCTCCCT- - - -CACCAAGCACCTCCCACATGC XAIATATGG
C • CTCCCT - CACAAGGC - CCTCCCACAIGC ICATATAIGG
TACACTCGGGGGC -CTGCCCGCCTCTCAAATGC XATATATGG
TACACTCGGGGGC- CTGCCCTCCCCTCACATGC XAIATATGG
TACACTCAGGGGC-CTGCCCACCCCTCACATGC XATATATGG
TACACTCGGGGGC-CTACCCACCCCTCACATGC XATAIATGG
TACTICTAGGGGC- CTTCCCGCCCCTCACAIGC ICATAIAIGG
GA - CAAAG-CCCTGCCCACCCCTCTCATGC XATATTTGG
GA- TGCCAAAG- CCCTGCCCA- -CCTCTCATGC XATATTTGQ
ICATGATTTGGGCAG-CTCTGACCCT
iCACGA I GCAGGCCGACT C T GGCCCT
tCATGAT GCAGGCC - AC - CTGGC - AT
ICATGATGTAGGCC - AC- CTGGC - AT
ICA T GA T GCAGGCC • AC - CT GGC - AT
ICAT GATGCAGGCC - AC - CTGGCCAT
tCATGAT GCAGGCC • AC - C T GGC - A T
tCATGGTACAGGTCCTCACTGGCCAT
ICATGGTACAGGTCCTCACTGGACAT
AAGTGG . CTCTTGCCAT
b
ProtIC
TATA
-70
CTGGG-TCTCT
CTAGG-CCTCT±i
CC-AG-CCCCTT r
CC-AG-CCCCT
CC-AG-CCCCTT r
CCCAG-CCCCTTT
CCCAG-CCCCT
CCTGGTCCTCTTT
CCTGGTCCTCTTr
CCGGG
GACCTCACAAT
GACCTCACAAT
GCCCTCACAAT
GCCCTCACAATt,
GCCCTCACAAT
GCCCTCACAAT;
t»gccctcacaat;
GACTTCATAATT
GACTTCATAATT
TCACCTCACAAT 1
-50 -40
iACCAGGACCCTGCCCGGGTC
iACCAGGGCCCTCCCCGCGTC
iACC AACGGCCCCC T GGC A T C
iACCAACGGCCTCCTGGCATCT
jACCAACGGCCCCCTGGCATCr.
lACCAACAGCCTCCTGGCATCT
j ACCAACGGCCCCC T GGCG T C T
CCTAGGGGCCA-CTAGTAT
CCCAGGGGCCA-CTAGTAT
3TCCTGGGMGTCCTGGGTT
AT
ATAAfc.
AT.
lAGGCCG
AGGCCC
AGGCCG
iAACCTG
AGGCCG
iAGGCTG
iAGGCCG
AGGAAG
iAGGAAG
iAGGCCA
ttggtcctggtcacctcacaa
-10 *1 *10 *20 *30 *40
GGAAGTCGGC-CCCTG--TCACAGCCCACAAA-TTCCACCTGCTCACAGGTTGGCTGGCTCAACCAAG
AGCAGTCAGC- -CCCTGGCACACAGCCTCCAAAGTTCCACCTGCTCACAGGTTGGCTGGCTCAACCAAG
CAGAGCTGGC--CCCTGACTCACAGCCCACAGAGTTCCACCTGCTCACAGGTTGGCTGGCTCAGCCAAG
CAGAGCTGGA- • CCCTGAC T CACAGCCCACAGAGT TCCACC TGCTCACAGG T T GGCTGGCTCAGCCAAG
CAGAGCTGG---CCCTGACTCACAGCCCACAGAGTTCCACCTGCTCACAGGTTGGCTGGCTTAGCCAAG
CACAGCTGGC- ■ CCC T GAC T CACAGCCCACAGAGT T CCACCTGC T CACAGGT T GGCTGGCTCAGCCAAG
CAGAGCTGG-- CCCTGACTCACAGCCCACAGAGTTCCACCTGCTCACAGGTTGGCTGGCTCAGC-AAG
AGGG T GC T GGC T CCCAGGC - CACAGCCCACAAAAT T CCACC TGC T CACAGGT T GGC TGGC T CGACCCAG
AGGG T GCTGGCTCTCCAGC-CACAGCCCACAAAATT CCACCTGC T CACAGGT T GGC TGGCTCGACCC AG
-AGAGCTCGG- *CCC TGGC TCACAGCCAACAAAGTTCCACCTCCTCACAGGTTGGCTGGCTCAGCTGAA
• • • ••**••• •• • •••••••• ■ •
•60 *70 *80 *90 f
GCGGTATCCCCTGCTCTGAGCAT--CC-AGGCCGAATCCACCCAGCACCATGGCCAG BULL
GCGGTAT CCCCTGCTCTGAGCAT - - CA - AGACT GAGTCCA T CCA TCACCAT GGCCAG BOAR
GTGGTG- -CCCTGCT CTGAGCAT - - TC- AG-CCAAGCCCATCCTGCACCATGGCCAG HUHAN
GTGGTG- -CCCTGCTCTGAGCAT- - TC-AG-CCAAGCCCATCCTGCACCATGGCCAG REO HONKET
GTGGTG*- CCC TGC TC T GAGCAT - • T C - A G GCCGAGCC C A TCC T GCACC A T GGCCAG GORILLA
GTGGTG- -CCCTGCTCTGAGCAT- -TC-AGGCCAAGCCCATCCCGCACCATGGCCAG BA6COW
GTGGTG- - • TCTGCTCT GAGCAT - -TC-AGGCCAAGTC- • A TCT GCACC AT GGCCAG ORANGUTAN
GTGGTGTCCCCTGCTCTGAGCCA- -GC- - • T CCCGGCC AAGCCAGCACCA T GGCCAG HC«JSE
GTGGTGCCCCCTGCTCTGAGCCA--GC---TCCCGGCCAAGCTAGCACCATGGCCAG RAT
GTGGTGCTCTCTGTTCTGAGCCAAGTCTAGGCCAAGTTCATCTAGTGCCATGGCCAG GUINEA PIG
• ••• • •• ••••••• •
SR* ProtIC TATA ATG
Fig. 3. — a: Alignments of all available mammalian PI gene promoter sequences. An asterisk indicates that a position is
conserved in all species. The nt positions are referenced relative to the tsp [+1]. The conserved SRE, “TGTGAGG”,
ProtIC and the TATA box are boxed. The arrow over the ProtIC indicates the sequence which is palindromic with
the “TGTGAGG” sequence (also arrowed) The start codon (ATG) is indicated by a downward arrow. After [64].
b: Position of the ProtIC , SRE and “TGTGAGG" sequences present in the PI genes and position of ProtlC-like
sequences present in other testis-expressed genes. The putative tsp is indicated [+1]. The numbered open boxes
indicate the position of sequences identical or similar to ProtIC \ The number in the open box indicates the number
of matches to the 12mer ProtIC [thus, 12 indicates a perfect match]. The beginning of the coding region is shown
by the unnumbered solid boxes 3’ to the tsp. A broken line indicates that the sequence is not available. The SRE is
indicated by the two connected arrows facing each other. The “TGTGAGG” sequence is showed by a shaded box and
the position and presence of a TATA box is indicated by the ellipse. After [64].
Transcriptional regulation of protamine PI genes
Transcription of the avian-mammalian PI type genes occurs in the post-meiotic, haploid
stages of spermatogenesis as determined by Northern blot analysis of RNA from testis at different
stages of development or from sorted cells [17, 35, 52, 55], and by in situ hybridization [35, 38,
44, 55]. Run-off assays on isolated mouse nuclei indicate that the mouse PI gene is activated at
the round spermatid stage [39], What are the mechanisms leading to this specific activation? The
availability of the nucleotide sequences from mouse, human and bull led to the prediction of some
potential regulatory elements. For instance a TATA box is present in all of them [18, 24, 32], a
544
R. OLIVA : AVIAN-MAMMALIAN PI TYPE PROTAMINES
CRE-like element [50], CAT box, CG boxes and additional sequences [18, 24, 32, 50, 52], A
different approach in the prediction of potentially important sequences has been the comparison of
homologous or heterologous protamine genes in the search for the conserved regions with the
assumption that important regulatory sequences would have been conserved in evolution [50].
Thus the following comparisons were reported: mouse PI and P2 genes [24]; human, bull and
mouse PI genes [33]; human PI and P2 genes, mouse PI, bull PI and human PI [18]; porcine
genes [26]; and chicken, bull PI, mouse PI and trout protamine [50]. A common problem in all
the studies comparing protamine gene PI sequences was that the homology between the different
PI genes available for analysis was relatively high so that discrimination between conserved
regulatory sites and sites conserved simply because of a close origin in evolution was not possible
in many cases. This problem was solved when the promoter region of additional PI genes was
sequenced thus increasing the total number of sequences available for comparison [64] (Fig. 3).
Four highly conserved sites were detected in the 5 'region (-160 to -1) of the protamine
genes [64]. The first one (-29 to -35) corresponds to the already previously described TATA box,
but with the novelty of being preceded invariably in all species by the di-nucleotide TC (Fig. 3).
The second conserved region (-55 to -66) was named Protamine 1 Consensus (ProtlC) which is a
CRE-like sequence (but always differently). The relevance of this conserved element in the
expression of the P 1 genes is strongly supported by the demonstration of a mouse testis trans¬
acting factor ([75], Tet-1) which binds and matches in the mouse the first 11 bp of the
corresponding ProtlC sequence. Independent experiments using different oligonucleotides
corresponding to the mouse PI 5' region [82] showed that the region -35 to -70 led to the
appearance of three different specific bands in gel retardation assays upon incubation with nuclear
extracts from different tissues. Only one of those bands was testis-specific. Similar results have
been obtained with the rat PI sequence using rat nuclear extracts [65], The third highly conserved
region detected in the 5 'of all PI genes corresponds to the sequence TGTGAGG (-88 to -82).
This sequence is a palindrome of the seven central nt of ProtlC (binding sequence for factor Tetl
in the mouse) and is exclusively present in this region in all PI genes whose sequence is available
suggesting an important function in the differential expression of the protamine PI genes. This
region corresponds to box E in the mouse promoter [24]. The fourth highly conserved region
corresponds to MATGCCCATATWTGGRCAYG and has the typical structure of serum response
elements (SRE) [64]. This region demonstrates specific interaction with a factor present in rat
nuclear extracts from different tissues with a distinctive shifted band appearing in the testis
extracts [65]. Also the equivalent region in the mouse PI gene (described as Box O) demonstrates
specific binding with a factor present in mouse nuclear extracts. Thus two highly conserved
sequences, the ProtlC (also referred as to CRE-like in all PI sequences or Tetl binding site in the
mouse), and the SRE appear to bind a testis specific factor or a testis-specific combination of
factors [65]. Both of these sequences lie within the minimal region (from bp -150 to bp -37)
described to direct spermatid-specific expression with a heterologous promoter from a human
growth hormone reporter gene [56, 81, 82].
ACKNOWLEDGEMENTS :
This work was supported with grants from the “Fondo de Investigaciones Sanitarias” (FIS93/0670) from Spain, and
from the Direccion General de Investigation Cientffica y Tecnica (DGICYT PB92-0810).
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SYSTEMATIC INDEX
A
Acanthobothrium 59
Acanthobothrium filicolle benedenii 89
Acanthobothrium filicolle filicolle 89
Acanlhochitona pygmaea 132
Acanthochitona viridis 132
Acanthochitonida 146
Acanthochitonidae 132
Acanthocotyle 58
Acanthodasys 108
AcanthopleUra granulata 132, 134
Acanthopleuridae 146
Ac ant hums monroviae 57
Acarus siro 223
Acipenser 346
Acipenser sturio 346
Acmaeidae 171
Acoela 38, 39
Acomys 409, 410, 417, 418
Acotylea 41
Acrosterigma reeveanum 157
Actinodactylella 47
Actinodactylella blanchardi 46
Actinodactylellidae 41, 46
Aculifera 145
Ade lotus brevis 322
Adlerocystis 224
Aegla 252
Agama adramitana 367
Agama agama 367
Agama stellio 367
Agamidae 367
Agelaius phoeniceus 438, 440, 441
Agnathans 506
Alaria 58
A Ices alces 539
Algyroides alleni 364
Aliaporcellana 258
Aliaporce liana suluensis 254
Allogalathea 258
Allogalathea elegans 253
Alopias pelagicus 314, 316, 317
Alopiidac 316
Alouatta seniculus 539, 540
Alytes 328, 330, 346, 352
Alytes obstetricians 322
Amblychelipas javanicens 169
Amblychilepas 172
Am by stoma macula turn 336
Ambystomidae 336
Amniotes 343, 344, 355
Amphibdella 58
Amphibia 321, 333
Amphilina 59, 73
Amphilinidea 69, 73
Amphisbaena darwinii 360
Amphiscolops 39
Amphiuma tridactylum 336
Amphiumidae 336
Anas platyrhynchos 346
Ancylostoma caninum 122
Angiostrongylus cantonensis 122
Anguimorpha 380
Annulipalpia 306
Anolidae 367
Anolis carolinensis 367
Anomalopus verreauxii 360
Anomura 253, 254, 256
Anopheles maculipennis 306
Anoplodiscus 58
Anoplodiscus cirrusspiralis 48
Anoplura 304
Anthocidaris crassispina 16, 17
Anthopharynx sacculipenis 43
Anlhopleura 18
Anthopleura midori 16, 17
Anura 328, 336, 337
Apatemon 58
Apatemon graciliformis 55, 57, 61
Aphalloides 58. 63, 69
Aphalloides coelomicola 55, 57, 61
Aphelasterias japonica 477, 484
Aphelenchoides blastophihorus 122
Apis mellifera 464
Aplacophora 137
Aporina 59
Aporina delafondi 88. 89, 90
Aporocotyle 58, 63
Aporocotyle spinosicanalis 55, 57, 62
Aprasia repens 372
Arbacia lixula 484
Arbacia punctulata All, 484
Archaeobrachyura 276
Archaeogastropoda 180
Archeognatha 304
Arcticoidea 160
Aristaeomorpha foliacea 232, 234, 236, 237, 239, 243,
244, 246, 248. 249
Aristeidae 232, 234
A l istens antennatus 232, 234, 236, 237, 239, 243, 244,
248, 249
Artemesia 233
Arthroleptella 337
Arthroleptella lightfooti 334, 336, 337
Ascaphus 328, 334, 340, 346, 352, 377
Ascaphus truei 322, 464
Ascaris megalocephala 122,124
Ascaris suum 1 22
A sc id i a 17, 466
A sc id i a callosa 503
Ascidia sydneiensis seniea 16, 17
Asphondylia ruebsaameni 306
Aspiculuris tetraptera 122
550
SYSTEMATIC INDEX
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
551
Carassins auratus 450, 464
Carcharhinidae 316
Carcharhiniformes 316, 319
Carcharhinus plumbeus 316, 317
Carcharias taurus 314,316,317
Carcinoscorpius 204, 210, 216
Carcinoscorpius rotundicauda 2 1 6
Cardinalis cardinalis 438, 440, 441
Cardioidea 160
Cardita niuricaici 158
Carditidae 162
Carditoidea 162
Cardium edule 1 58
Carlia 361, 362, 375, 379, 380
Ccirlici pectoralis 361, 362, 372, 374, 376
Cataetyx laticeps 464, 465, 467, 509, 510
Catagapelus nigrcms 306
Catenotaenia 59
Catenotaenia pusilla 89
Catenulida 38
Caudata 466
Cavia porcellus 539
Cecidomyiidae 306
Cellana capensis 170
Cellana testudinaria 170
Cellana toreiuna 1 70
Cemocotyle 58
Centrophorus squamosus 507
Cenlrophorus uyato 507
Centroscy minis coelolepis 507
Centroscymnus crepilater 507
Ceniroscymnus owstoni 316, 317, 319
Centurus carolinus 440
Cephalobus quinilinealus 122
Ccphalochordates 505
Cephalodasys 1 1 6
Cephalodasys maximus 106, 112, 116
Cephaloscy Ilium umbratile 316, 317, 318
Cerastoderma edule 1 80, 1 82
Ceratitis capitata 285, 306
Cercomeridea 56, 69
Cercopithecus patas 539, 540
Cerris puludum 286
Cervus elaphus 539
Chaetoderma 145
Chaetoderma argenleum 132, 138, 140, 144
Chaetoderma canadense 132, 138, 140
Chaetodermomorpha 132, 140, 145, 146
Chaetonotida 106, 112, 116
Chaetonotidae 106
Chaetonotus maximus 106, 108
Chaetopleura apiculata 132, 137
Chaetopleuridae 132, 146
Chaelopteryx gessneri 306
Chalcides 359, 379
Chalcides ocellatus 360, 370, 376
Chalcides ocellatus tiligugu 360, 362, 362
Chama macerophylla 158
Chamaeleonidae 368
Chamoidea 160
Chara corallina 469
Chartoscirta cineta 286
Cheiridium museorum 207
Chelicerata 203
Chelonia 355, 376
Chelyosoma production 454, 458, 503
Cherax destructor 47
Childia groenlandica 39
Chimaera phantasma 315, 316, 317
Chimaeridae 316
Chimaeriformes 316
Chiromantis 337, 340
Chiromantis xerampelina 333, 336, 337, 338, 341
Chironomidae 306
Chironomus 306
Chiroptera 421
Chirostylidae 254, 258
Chiton luberculatus 132, 137
Chitonidae 132, 146
Chlamydia trachomatis 463, 464, 470
Chlamydomonas 15, 16, 17, 18, 20, 21, 28
Chlamydomonas reinhardti 16, 17
Chlamydoselachidae 316
Chlamydoselachus anguineus 313, 314, 316, 317
Chlorostoma argyrostoma lischkei 169
Chondrichthyes 507
Choricotyle 58
Chrysomya megacephala 285
Chthonerpton ind is tine turn 336
Cichlidogyrus 58
Circe cf plicatina 157, 160
Cleistogamia longicirrus 46
Cleithrarticus 55, 57, 58, 67, 68
Clibanarius 256
Clibanarius corallinus 253
Clibanarius erythropus 253
Clibanarius longi tarsus 253
Clibanarius misanthropus 253
Clibanarius nathi 253
Clibanarius taeniatus 253, 268, 270, 271
Clibanarius virescens 253
Clitellio 194, 196, 198
Clitellio arenarius 192, 196
Clonorchis 58
Cnemidocarpa finmarkiensis 503
Cnemidophorus 364, 370, 375, 379, 380
Cnemidophorus lemniscatus lemniscatus 364
Cnemidophorus sexlineatus 347, 362, 366, 368, 370,
374, 376
Codakia punctata 156, 157, 160
Coelogynoporidae 41
Coenobita 256
Coenobita brevimanus 254
Coenobita clypeatus 253
Coenobita perlatus 254, 258
Coenobita purpureus 254
Coenobita rugosus 253
Coenobita spinosus 253
Coenobita variabilis 253
Cocnobitidae 253, 254, 256
Coleoptera 297, 304
Collyricloides 58
552
SYSTEMATIC INDEX
Coluber constrictor 372
Coluber viridiflavius viridiflavius 372
Colubridae 376
Concinnbcotyla 58
Constrictor 372
Constrictor constrictor 372
Coralliodrilus 190, 196, 197. 198
Coral liodrilus rugosus 189, 190. 191, 192, 195
Corbicula 158, 162, 163
Corbicula fluminea 158, 162
Corbicula sandai 158
Corbiculoidea 160
Coronella austriaca 372
Corrigia 58
Cosmopolites sordidus 297, 298, 299
Cotugnia 59
Cotugnia polyacantha 89
Coturnix coin mix 346
Coturnix japonica 539. 540
Cotylea 41
Cotylophorum 58
Craspedella 46
Crassatellidae 162
Crassostrea 17
Crassostrea angulata 180, 182, 184
Crassostrea gigas 16, 17
Crenilabrus cinereus 57
Crenomytilus grayanus 504
Cricetomyinae 414
Cricelulus migratorius 539
Crocodilia 355
Crocodylus 349
Crocodvlus johnstoni 344, 346, 347, 348, 349, 350,
352, 355
C rota l us adamant e us 372
Crustacea 231, 243, 251, 265
Crypthecodinium cohnii 463, 464, 470
Cryptoblepharus virgatus 361, 362, 372, 374
Cryptobranchidae 336
Cryptobranchus 341
Cryptobranchus alleganiensis 336
Cryptocellus 216
Cryptocellus boned 207, 208, 211, 214
Cryptochiton stelleri 129, 130, 132, 142
Cryptocotyle 58
Ctenotus 362, 379, 380
Ctenotus robustus 360, 362, 376
Ctenotus taeniolatus 360, 362
Culex pipiens 304, 306
Culicidae 306
Cupriguanus 367
Cupriguanus scapulatus 366
Curculio elephas 299
Cyclodorippoidea 276, 277
Cylindrotaenia 59
Cylindrotaenia hickmani 89
Cymonomus 268, 273, 277
Cynopterus brachyotis 422
Cyrnus trimaculatus 306
Cyta latirostris 2 1 8
D
Dagnaudus ( =Paramo/a) petterdi 268, 276
Dahlias licha 313, 315, 316. 317
Dalyelliida 41, 44, 74
Dalyelliidae 41, 44
Dardanus 254, 256
Dardanus arrosor 253, 258
Dardanus crassimanus 253
Dardanus lagopodes 254
Dardanus megistos 253
Dardanus scute Hat us 254
Deania 3 1 8
Deania calcea 316,317
Deania historicosa 316, 317
Deania profundorum 507
Decadidvmus gulosus 46. 49
Decapoda 231, 243, 251, 265
Delma tincta 372
Dendrobranchiata 231, 243
Dendrobranchiate 233, 236
Dendrofissurella 172
Dendrofissurella scutellum 169
Dendromurinae 414
Dendromus 409
Dendromus mesomelas 410, 414
Dendromus mystacalis 410. 414, 416
Deomys 409, 417, 418
Deomys ferrugineus 410, 414, 416
Deontosloma californicum 122
Dermaptera 304
Dermestes frischii 299
Dicentrarchus labrax 509, 510
Diceratocephala boschmai 46
Diclidophora 58, 73
Dicrocoelium 58
Dictyostelium 23, 24, 28, 32, 34
Dictyostelium discoideum 23, 24, 26, 27, 28, 29,30, 31,
32, 33, 34
Dictyostelium giganteum 26, 31
Dictyostelium mucoroides 26
Dictyostelium purpureum 26, 3 1
Dicyema misakiensis 21
Didelphis marsupialis 539
Didymorchidae 41, 47
Didymorchis 47
Didymozoon 58, 69, 70
Diemictylus 341
Digenea 59, 69, 70, 73
Dioctophyma renale 122
Diodora aspera 1 69
Diogenes 256, 260
Diogenes custos 253
Diogenes miles 253
Diogenes pallescens 254
Diogenidae 253, 254, 256
Dionchus 58
Dipetalonema dessetae 122
Dipelalonema setariosum 122
Dipetalonema viteae 122, 124
Diphyllobothrium 59
ADVANCES IN SPERM ATOZOAL PHYLOGENY AND TAXONOMY
553
Diphyllobothrium latum 89
Diplectanum 58, 73
Diplodasys ankeli 106, 108, 110, 112
Diplozoon 58, 63, 64, 71, 72, 74
Diplozoon gracile 55, 57, 64
Diplozoon nipponicum 64
Diplura 304
Diptcra 301, 302, 304, 308
Dirofilaria immitis 122
Discoglossus 327, 328, 330, 346. 352
Discoglossus pictus 322
Divales bipustulatus 299
Divariscintilla 155, 156, 160, 163
Divariscintilla troglodytes 158
Divariscintilla yoyo 158
Dixa 306
Dixidae 306
Donacidae 160
Donax 163
Donax deltoides 157, 160. 163
Donax madagascariensis 158, 163
Donax serra 158, 163
Donax sordidus 158, 163
Donax trunculus 158, 163, 180, 182, 455
Doliopharyngiophora 74
Draculiciteria 108
Dreissena polyniorpha 158
Dreissenoidea 160
Dromaius novae ho llandiae 346
Dromiacea 272
Dromidia antillensis 281
Dromidiopsis 272, 274, 276, 277
Dromidiopsis edwardsi 268, 270, 271. 272
Dromiidae 272
Drosophila 102
Drosophila melanogaster 285, 306
Drosophilidae 306
Drymarchon corais 372
Duthiersia 59
Duthiersia fmibriata 89
Dynomene 272, 274
Dvnomene aff. devaneyi 265, 266, 267, 268, 2'
276, 278
Dynomenidae 274
Dysdera 210
E
Echeneibothrium 55, 57, 59, 68
Echeneibothrium beauchampi 89
Echinobothrium 59
Echinobothrium affine 89
Echinobothrium brachysoma 89
Echinobothrium harfordi 89
Echinobothrium typus 89
Echinococcus 59
Echinococcus granulosus 89
Echinoderms 475, 476, 491
Echinoida 466
Echinostoma 58, 97, 102
Echinostoma caproni 98, 100
Echinostoma liei 98, 101
Echinostoma togoensis 55, 57, 61
Echymipera kalubae 398
Echymipera rufescens 398
Ekphymatodera thomasoni 122
Elapidae 376
Eledone 45 1
Elenchus japonicus 291, 292
Elenchus tenuicornis 291, 292
Elephas 539
Emballonura furax All
Emberizidae 440
Embioptera 304
Emerita 260
Emerita analoga 253
Emerita asiaiica 253
Emerita talpoida 253
Empididae 306
Emydura 349
Emydura kreffti 344
Encotyllabe 58
Enoplus anisospiculus 122
Enoplus demani 122
Ensis ensis 158, 455
Ensis minor 45 1
Entobdella 58
Eonycteris spelaea 422
Ephemeroptera 304
Epicrius mollis 218
Epigonichtys 505
Epimenia 138, 140
Epimenia australis 132, 138, 143
Ec/uus caballus 539
Erpocotyle 58
Escherichia coli 470, 493
Etisus 280
Etmopterus 313, 316, 318
Etmopterus brachyurus 316, 317
Etmopterus molleri 316, 317
Etmopterus pusillus 316, 317, 507
i, 272, Eucarida 236
Euccstoda 69, 72, 73
Eucidaris 476
Eucidaris crassa 480
Eucidaris esculentus 479, 480
Eucidaris tribuloides 476, 477, 484
Eucrassatella cumingii 158
Eucrassatella kingicola 158
Eudromia elegans 346
Euglena gracilis 470
Eukalyptorhynchia 41, 43
Eumeces laticeps 360, 362
Eumunida 258
Eumunida sternomaculata 254
Eupagurus angulatus 253
Eupagurus bernhardus 253
Eupagurus prideauxii 253
Euphausia 236
Eurytrema 58
Eusimonia mirabilis 214
Source . MNHN. Paris
554
SYSTEMATIC INDEX
Eutheria 466
Euzetrema 58
Exechia seriatci 303, 304, 306
F
Fasciola 58
Fecampiida 41, 47
Fecampiidae 41
Felis catus 539
Fibricola 58
Filistata 2 1 1
Filistata insidiatrix 208, 21 1
Fissure l la 172
Fissurella mutabilis 169
Fissurella natalensis 169
Fissurellidae 169, 172
Fissurelloidea 169
Forcipulata 466
Fra gum hemicardium 157
Fragum unedo 157, 160
Fulvia tenuicostata 158
Furnestinia 58, 68
Furnestinia echeneis 55, 57. 66
G
Galathea squamifera 253
Galathea strigosa 253
Galatheidae 253, 258
Galatheoidea 253, 254
Galeocerdo cuvier 316, 317
Galeommatoidea 160
Galeus 3 1 8
Galeus eastmani 316, 317
Galeus melastomus 507
Galeus nipponensis 315, 316, 317
Gallus domeslicus 346, 442, 453, 538, 539, 540. 541
Ganeo 58
Gasterophilidae 306
Gasterophilus intestinalis 306
Gasterosteus 465, 466
Gastrocotyle 58, 73
Gastromermis 122, 124, 125
Gastropoda 180
Gastrotricha 105
Gazella dorca 539
Gebia littoral is 253
Gekkonidae 370
Gekkota 380
Geocentrophora 42
Geocentrophora intersticialis 42
Geocentrophora wag ini 42
Geocentrophora wasiliewi 42
Geopelia striata 344, 346, 347, 348, 349, 350
Gerbillinae 412
Gerbillurus 409
Gerbillurus paeba 410, 412, 413, 417
Gibbula cineraria 1 80, 1 82
Gibbula divaricata 453
Gibbula lumida 169
Gibbula umbilicalis 169, 180, 182
Gieysztoria 44, 46
Glaridacris 59
Glciridacris catostomi 89, 90
Glauconome 157, 160
Glossosomatidae 306
Glyphotaelius pellucidus 306
Gnathostoma 122
Gobio gobio 57
Gobi us micropus 57
Goeridae 306
Gonapodasmius 58, 69
Gonoplasius 58, 73
Gorgodera 58, 97, 98, 99
Gorilla gorilla 539
Gotocotyla 58, 63, 71
Go toco ty la acanthura 65
Graffilla buccinicola 46
Graffillidae 41, 46
Grus vipio 346
Gymnophiona 336
Gynaecotyle 58
Gyratrix 43
Gyrocotyle 59, 73
Gyrocotylidca 69, 73
Gyrodactylus 58
H
Haematoloechus 55, 57, 58, 61, 63, 69, 97, 98, 99
Haemonchus contortus 124
Halichondria 18
Halichondria japonica 16, 17
Halictophagus chilensis 291, 292
Haliotidae 169, 172
Haliotis discus 169
Haliotis diversicolor aquatilis 169
Haliotis midae 169
Haliotis rufescens 169
Haliotoidea 169
Hanleyella asiatica 137
Haplobothrium 59
Haplobothrium globuliforme 89, 90
Helcion dunkeri 170
Helcion pectunculus 170
Helcion pellucidus 170, 180, 182
Helcion pruinosus 170
Heleophryne 340, 341
Heligmosomoides poly gyrus 119, 120, 122, 123, 124
Heligmosomoides poly gyrus bakeri 120
Hemicentrotus 1 7
Hemicentrotus pulcherrimus 16, 17, 18
Hemidactylus frenatus 370
Hemidactylus mabouia 370
Hemiptera 285, 304
Hemisotidae 336
Hemisus marmoratus 336, 337
Hemitriakis japanica 316, 317, 319
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
555
Heptathela 210, 212, 213
Hepiathela kimurai 210, 211, 212. 213, 218
Heterakis gallinarum 122, 124
Heteraxine 58
Heteraxinoides 55, 57, 58, 63. 64
Heterocon>le 58
Heterodera ( Globodera ) rostochiensis 122
Heterodera (Globodera) virginiae 122
Heterodera ( Heterodera ) avenae 122
Heterodera ( Heterodera ) schachtii 122
Heterodonta 180
Heterodontidae 316
Heterodontiformes 316, 319
Heterodontus japonicus 314, 316, 317
Heterodrilus 189, 190, 198
Heterodrilus minisetosus 190, 191. 192
Heterodrilus pentcheffi 190, 191, 192, 195
Heteronotia 370, 380
Heteronotia binoei 346, 347, 368, 369, 370, 374, 376
Heterorluibditis bacteriophora 122
Heterotremata 277
Heteroxenotrichula 108
Heteroxenotrichula squamosa 106, 108, 112, 115
Hexanchiformes 316, 319
Hexostoma 58
Hippa 260
Hippa pacifica 254, 258
Hippidae 253, 254, 258
Hippoidea 253, 254
Hirundinidae 439
Holothuria 466
Holothuria tubulosa All, 484
Holothuria tubulosa 491, 492, 493, 494, 495, 496, 498
Homo sapiens 539
Homola ranunculus 268, 276
Homolidae 276
Homolodromia 265, 266, 272, 274. 277, 281
Homolodromia kai 265, 266, 267, 268, 270, 272, 274,
276, 278
Homolodromiidae 212
Homologenus 276
Homolomannia sibogae 276
Homoptera 304
Hydrolagus colliei 503, 507, 508
Hydrolagus mitsukurii 316, 317
Hydropsyche pellucidula 306
Hydropsychidae 306
Hydroptila aegyptia 306
Hydroptila angulata 306
Hydroptila forcipata 306
Hydroptila tineoides 306
Hydroptilidae 306
Hyla 321, 327, 328
Hyla japonica 322, 324, 327, 328
Hyla meridionalis 322
Hyla rubra 334
Hylobates lar 539
Hymenochirus 467
Hymenolepis 59
Hymenolepis diminuta 89
Hymenolepis microstoma 89
Hymenolepis nana 88, 89, 90
Hymenoptera 304
Hyperoliidae 336
Hyperolius horstocki 336
Hypoconcha 272, 281
Hypoconcha arcuata 281
I
Ichthyopiidae 336
Integripaipia 306
Ichthyopis glutinosus 336
Icteridae 440
Iguania 379
Iguanidae 366, 376
lliacantha subglobosa 211
Inanidrilus 198
Inanidrilus bulbosus 191, 192
Inanidrilus leukodermalus 191, 192
Inermicapsifer 59
Inermicapsifer guineensis 89
Inermicapsifer madagascariensis 89
Insecta 285, 291, 297, 301
Isancistrum 58
Ischnochiton albus 132
Ischnochitonidae 132, 146
Isoodon macrourus 398, 405
Isoodon obesulus 398
Isoptera 304
Isurus oxyrinchus 314, 315, 316, 317, 319
J
Jacana jacana 346
K
Kalyptorhynehia 41, 43
Katharina lunicata 132
Keroplatus reaumuri 303, 306
Kronborgia 40, 49, 75
Kronborgia isopodicola 47, 48
Kuhnia 58
L
Lacerta laevis 364
Lacerta lepida 364
Lacerta lepida lepida 364
Lacerta sicula campestris 364
Lacerta taurica 364
Lacerta viridis 364
Lacerta vivipara 347, 364
Lacenidac 364, 376
Lacistorhynchus 59
Lacistorhynchus tenuis 89, 90
Lagarocotyle 58
Source . MNHN. Paris
556
SYSTEMATIC INDEX
Lamellodiscus 58
Lamnidae 316
Lamniformes 316, 319
Lampropeltis ge lulus 372, 374
Lampropholis 362, 379, 380
Lampropholis delicata 344, 349, 361, 362, 367. 372,
374, 376
Lasaea 155, 156, 163
Lasaea subviridis 158
Latimeria 352, 354
Latimeria chalumnae 346
Latreillia 265, 266, 268, 271, 276, 277. 281
Latreilliidac 277
Laireillopsis gracilipes 268. 271, 276
Lecithoepitheliata 41, 42
Lepetodrilidae 169, 172
Lepetodriloidea 169
Lepetodrilus fucensis 168, 169, 172
Lepidochitona 131
Lepidochitona dentiens 132, 134, 137
Lepidochitona fernaldi 132. 137
Lepidochitonidae 146
Lepidodasyidae 106
Lepidodermella squamata 106, 108
Lepidopleuridae 132, 146
Lepidoptera 304
Lepidozona retiporosa 132, 134
Leptoceridae 306
Leptocerus tineiformis 306
Leptochiton 140
Leptochiion asellus 129, 130, 132, 136, 137, 141, 142
Leptocoris trivittatus 286
Lcptodrusus budtzi 306
Leplopelis flavomaculatus 336, 337
Lialis 359, 375, 380
Lialis burtonis 347, 367, 370, 371, 372, 376. 380
Lilium longiflorum 469, 470
Limnephilidae 306
Limnephilus bipunctatus 306
Limnephilus rhombicus 306
Limnodriloides gurwitschi 199
Limnodriloidinae 191, 192
Limnodynasies 327
Limnodynastes peronii 322
Limn l us polyphemus 205, 213, 214, 216. 223, 450
Liolaemus 367
Liolaemus austromendocinus 366
Liolaemus weigmanii 360
Liophis miliaris 372, 374
Liotella parvirota 169
Lit bodes 260
Lit bodes maja 253
Lithodidae 253
Litoria 327, 465. 466
Litoria caerulea 322
Litoria fallax 322
Litoria gracilenta 322
Litoria lesueuri 322
Litoria peronii 322
Litoria rubella 322
Litosomoides carinii 122
Littorina littorea 180, 181, 182
Littorina saxatilis 180, 181, 182
Littorinidae 466
Loa !oa 122
Lobatostoma 58, 71
Loimosina 58
Loligo 449
Loligo pealeii 453
Lomidae 254, 258
Lomis 252, 260
Lomis birta 254, 258
Lomoidea 254
Lopburomys 410, 417
Lottia digitalis 170
Lottia pelta 170
Lottia strigatella 170
Loitiidae 170, 466
Lucinoidea 160
Lunella cinerea 169
Lunella granulata 1 69
Luriculus australiensis 46
Luridae 41 , 46
Lygodactylus 380
Lygodactylus picturatus 370, 376
Lyreidus 277
Lyreidus brevifrons 268, 273. 276
Lytechinus pictus 470, 477, 480, 484
Lytechinus variegatus All
M
Macaco mulatto 539
Macoma nasuta 45 1
Macrodasyida 106, 110, 112
Macrodasyidae 106
Macrodasys 106. 108. 116
Macroglossinae 422
Macroglossus minimus 422, 425
Macro gyrodactylus 58, 73
Macropbtbalmus 280
Macrophtbalmus crassipes 268, 278
Macroschisma sinense 169
Macrostomida 39
Macrostomum 39, 49
Macrostomum tubum 39
Macrolis 398
Macrotis lagotis 397, 398, 399, 400, 401, 402, 404,
405, 406
Mactroidea 160
Maehrenthalia 43
Majids 268
Malacotbrix 409
Malacotbrix typica 410, 414, 416, 417
Mallophaga 304
Mammalia 356, 397, 409, 421, 515, 525, 537
Mantodea 304
Marc bant ia polymorpba 469
Maricola 41
Maritrema 58
Marsupalia 466
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
557
Masticophis flagellum flagellum 372
Mastigoproctus giganteus 207, 213
Mathevotaenia 59
Mathevotaenia herpestis 88, 89
Meara 38
Mccoptera 304
Mecynostomum 39
Mecynostomum auriium 39
Megachiroptera 422
Megalocotyle 58, 73
Megaloptera 304
Meiogymnophallus 58
Melampophylax me lamp us 306
Melanerpes carol inus 346
Meleagris gallopavo 346
Meloidodera floridensis 122
Meloidogyne acronea 122
Meloidogyne arenaria 122
Meloidogyne graminicola 122
Meloidogyne hapla 122
Meloidogyne incognita 1 22
Meloidogyne oryzae 122
Menaethius monoceros 275
Meretrix 20
Meretrix lusoria 16, 17
Merluccius 5 1 1
Merluccius cape ns is 509, 510
Merluccius merluccius 57
Mesacanthion hirsutum 122
Mesodasys laticaudatus 106, 111, 112
Metagonimus 58
Metamicrocotyla 58
Metapeneopsis 233
Metastrongylus apri 122
Metazoa 17, 18
Me tore his 58
Metridium senile 454, 458, 467
Microcarina surge rea 169
Microchiroptera 422
Microcotyle 58
Microdalyellia 44
Microphallus 58
Micropterna sequax 306
Microstomum 39
Micrurus fulvius 372
Mictyris 280
Mictyris longicarpus 268, 278
Miniopterinae 422
Miniopterus 428
Miniopterus schreibersii 422, 425, 426, 427, 428
Mixophyes fasciolar us 322
Mollusca 129, 155, 167, 179, 451
Molothrus ater 438. 440
Moniezia 55, 59, 68
Moniezia benedeni 89, 90
Moniezia expansa 57, 89, 90
Monobothrium 59
Monobothrium wageneri 89
Monocelididae 41
Monocotyle 58
Monodonta australis 169
Monodonta labio labio 169
Monodonta turbinata 169
Monoecocestus 59
Monoecocestus americanus 89
Monogenea 63, 71, 72, 73
Monopisthocotylea 68, 72, 73
Monopylephorus 198, 199
Monopylephorus limosus 192
Montfortula 172
Montfortula conoidea 169, 172
Mopalia 466
Mopalia ciliata 132, 134, 136
Mopalia lignosa 132
Mopalia muscosa 132, 134
Mopaliidae 132, 146
Mull us 511
Mullus barbatus 454
Mul I us surmuletus 458, 509, 510
Multicotyle 58, 71, 73
Multipeniata 42
Muni da 254, 258
Munida rugosa 253
Munidopsis 254, 258
Murina leucogaster 422, 425
Murininae 422
Mus muse ulus 16, 17, 539
Mustelus 3 1 8
Mustelus canis 316, 317
Mustelus griseus 315, 316, 317
Mustelus manazo 316, 317
Mycetophilidae 306
Mycrohylidae 336
Myiarchus crinitus 440
Myliobatididae 316
Myliobatiformes 316
Myliobatis tobijei 316, 317
Myloncliulus nainitalensis 122
Myocidaris 476
Myotini 422
My ot is 421, 422, 424, 425, 427
Myotis adversus 422
Myotis formosus 422
Myotis hosonoi 422, 425
Myotis lucifugus 422, 427
Myotis macrodactylus 421, 422, 424, 425, 427
Myotis nattereri 421, 422, 424, 425, 426, 427
My sella 155, 156, 163
Mysella tumida 158
Mystacides azure a 306
Mystromyinae 412
Mystromys 409, 410
Mystromys alba 410, 411, 412, 417
Mytilidae 466
Mytiloidea 180
My til us 449, 451, 458, 485
Mytilus californianus 451, 452, 484
Mytilus edulis 16. 17, 180, 182, 184. 455. 464, 465
Myxinidocotyle 58, 73
558
SYSTEMATIC INDEX
N
Nacella delesserti 170
Nacellidae 170
Nangura 349
Nangura spirosa 360, 362, 374
Natrix natrix 372
Natrix t esse lata tesselata 372
Neanthes 18
Neanthes diversicolor 16, 17
Necturus maculosus 336
Nemastoma 216
Nemastoma lugubre 206
Nematocera 306
Nematoda 119
Nematodirus battus 1 22
Nematoplanidae 41
Nematospiroides dubius 120
Nematotaenia 59
Nematotaenia chantalae 88, 89
Nemertoderma 38, 50, 129, 130, 149
Nemertodermatida 38
Neoaplectana intermedia 122
Neobatrachus pelobatiodes 322
Neocarus 207
Neoceratodus 343, 346, 354, 355
Neoceratodus forsteri 344, 354
Neochasmus 58
Neodasyidae 106
Neodermata 75
Neodasys 106, 108, 116
Neodorippe 265, 266, 278. 280
Neodorippe astuta 268, 275, 278
Neodorippe callida 275, 278
Neomeniomorpha 132, 138, 140, 146
Neopolystoma 58
Nepa rubra 286
Nerita picea 138
Nerodia sipedon 372, 374
Nicoletiella lutea 205
Nippostrongylus 124
Nippostrongylus brasiliensis 122, 124
Noctiluca 470
Notonecta glauca 286
Notoplana 464
Notoplana japonica 42
Nucella lapillus 180, 181, 182
Nucellidae 466
Numida meleagris 346
Nuttalina flexa 132
Nyc talus 421, 424, 427
Nyctalus noctula 422
Nymphon 204, 205, 208
Nymphon gracile 216
Nymphon rubrum 216
O
Octomacrum 58
Ocyphaps lophotes 349
Ocypode 280
Ocypode ceratophthalma 268, 278
Odocoileus virginianus 539
Odonata 304
Odontaspididae 316
Odontoceridae 306
Odontocerum albicorne 306
Odontophrynus 327
Odontophrynus cul tripes 322
Odontorhynchus 43
Oecetis furva 306
Olavius planus 192
Oligochaeta 189
Omphalus pfeifferi 169
Onchobothrium 59
Onchobothrium uncinatum 89
Oncorhyncus keta 453
Onithochiton quercinus 132
Oochoristica 59
Oochoristica agamae 89
Opilioacarus 207
Orcinus orca 539, 540
Orectolobidae 316
Orectolobi formes 316, 319
Orectolobus japonicus 314, 315, 316, 317
Orfelia 302, 303, 306
Ornithodorus tholozani 205
Ornithorincus anatinus 539
Orthoptera 304
Orthotrichia costalis 306
Oryctolagus cuniculus 539
Osieichthyes 508
Ostrea edulis 180, 182, 184
Ostreoidea 180
Otomyinae 416
Otomys 409, 417
Otomys irroratus 410, 416
Otoplanidac 41
Ovis aries 539
Oxy stele impervia 169
Oxystele sinensis 169
Oxy stele tabularis 169
Ox)'stele tigrina 169
Oxystele variegata 169
Oxyuranus 376
Oxyuranus microlepidotus 372
P
P achy medusa 327
P achy medusa dacnicolor 322
Page 1 1 us acarne 509, 510
Paguridae 253, 254, 256
Paguristes maculatus 253
Paguristes oculatus 253
Paguroidea 253, 254
Pagurus 252, 256, 260
Pagurus bernhardus 253, 268, 270
Pagurus chevreuxi 254, 258
Pagurus excavatus 253
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
Pagurus hirtimanus 254
Pagurus prideaux 253
Pagurus punctulatus 253
Pagurus striatus 253
Paleognathae 466
Paludicola 41
Pan paniscus 539
Pan troglodites 539
Panagrellus silusiae 1 22
Pantodon 349, 352
Panulirus 234, 239
Paphia 157
Papio doguera 539
Paradynomene 272, 274, 277
Paradynomene tuberculata 268, 270, 271, 272
Paragonimus 58
Paramacrostomum tricladoides 39
Paramecium 15, 16, 17, 18
Paramecium caudatum 16,17
Paramo la bathyalis 276
Parapaguridae 254, 256
Parapeneus 233
Parapeneus longirostris 232, 233, 234, 236, 239. 249
Paras it us berlesei 2 1 8
Paravortex 46, 47
Paravorte x cardii 46
Paravortex karlingi 46
Paravortex tapetis 46
Parechinus angulosus 477, 480
Paridae 439
Paromolopsis boasi 276
Parus bicolor 439
Passer domesticus 438, 439, 441, 442
Passeridae 439
Passeriformes 437
Patella ap banes 1 70
Patella argenvillei 170
Patella aspera 1 70
Patella barbara 170
Patella caerulea 1 70
Patella candei 1 70
Patella canescens 170
Patella cf miniata 170
Patella chapmani 170
Patella cochlear 1 70
Patella compressa 170
Patella concolor 170
Patella depressa 170
Patella ferruginea 1 70
Patella flexuosa 170
Patella granatina 1 70
Patella granularis 1 70
Patella laticostata 170
Patella longicosta 170
Patella miliaris 170
Patella miniata 1 70
Patella obtecta 170
Patella oculus 1 70
Patella peronii 170
Patella rustica 170, 180, 182
Patella sqfiana 1 70
Patella labularis 1 70
Patella vulgata 170
Patellidae 168, 170
Patellogastropoda 168, 174, 180
Patelloida 174
Patelloida profunda albonotata 170, 171
Patelloida saccharina lanx 170, 171
Patiria miniata All , 484
Pec ten maximus 455
Pectinodrilus 190, 197
Pectinodrilus molestus 189, 190, 192, 195
Pelusios adansoni 57
Penaeidae 232, 233
Penaeus 233, 234, 239
Penaeus aztecus 232, 233
Penaeus japonicus 232, 233, 234
Penaeus kerathurus 232, 233, 234
Penaeus setiferus 232, 233
Penaeus vannamei 232, 233
Peneopsis 233
Peneopsis serrata 232, 233, 239
Perameles gunni 398
Perameles nasuta 398
Peromyscus maniculatus 120
Perorcytes longicauda 398
Perotrochus quoyanus 169. 171
Perotrochus westralis 169, 171
Petrolisthes 258, 260
Petrolisthes armalus 254
Petrolisthes lamarckii 253
Petromyzon marinus 450, 467, 505, 506, 508
Phaenocora 48
Phaenocora anomalocoela 43
Phallodrilinae 190, 191, 192
Pharyngostomoides 58
Phasianellidae 169, 172
Phasmida 304
Philopotamidae 306
Philopotamus 308
Philopotamus ludificatus 306
Philopotamus montanus 306
Phlcbobranchiata 466
Phodopus sungorus 539
Pholcus 208, 216
P/tolcus phalangioides 207, 208, 210, 211, 216
Phyllobothrium 59
Phyllobothrium gracile 89
Phymaturus 366
Phymaturus palluma 366
Physaloptera 122
Phyloptus avellanae 223
Pilodius 280
Pilodius areolatus 268, 275
Pip a 467
Pipidae 336
Pipistrellini 422
Pipistrel! us 421, 424, 427
Pipistrellus abramus 421, 422, 424, 425, 426, 427
Pipistrellus abramus abramus 427
Pipistrellus abramus javanicus 427
Pipistrellus avii 422
560
SYSTEMATIC INDEX
Pipistrel Ins endoi 422, 424, 425
Pipistrellus javanicus 422, 424, 425
Pipistrellus javanicus javanicus 427
Pipistrellus pipistrellus 427
Piranga rubra 439
Pisidia 258
Pisidia longicornis 253
Placamen calophyllum 157
Planipennia 304
Planoceros 18
Planoceros reticulata 16, 17
Platyhelminthes 37, 55, 69, 74, 87. 97, 149
Plecoptera 304
Plecotini 422
Plecotus 421, 424
Plecotus auritus 422, 425
Plectanocotyle 58
Plectrocnemia geniculata 306
Plethodon albagula 336, 352
Plethodontidae 336
Pleurodeles 327
Pleurodeles waltlii 336
Pleuroncodes planipes 253
Pleurotomaria africana 1 7 1
Pleurotomariidae 169, 171
Pleurotomarioidea 169
Podarcis 364
Podocotyle 58
Podotremata 270
Pogona 359, 360, 368, 375, 379, 380
Pogpna barbata 363, 367, 368, 370, 376
Polistes dominilus 292
Polycentropodidae 306
Polycentropus irroratus 306
Polycentropus mortoni 306
Polycladida 41, 42
Polycystis naegelii 43, 44
Polydelphis 122
Polylabroides 58
Poly onyx 258
Polyonyx transversus 254, 258
Polyopisthocotylea 63, 71, 73
Polyplacophora 131, 136
Polysphondylium pallidum 26
Poly stoma 58
Polystomoides 58
Pomadasys incisus 57
Pongo pygmaeus 539
Porcellanidae 253, 258
Porcellanopagurus 254, 256, 260
Portunus pelagicus 268, 275, 280
Postharmostomum 58
Potamonautes 280
Potamonautes perlalus 268
Potamophylax cingulatus 306
Pricea 58
Primates 466
Prionace glauca 314, 316, 317
Prionomys 409
Prionomys batesi 410, 414, 416, 417
Pristiophoriformes 319
Proctoeces 58, 63, 69
Proctoeces maculatus 55, 57, 61
Prokoenenia wheeleri 206, 212, 213
Prolecithophora 41, 42, 56
Promacrostomum gieysztori 39
Promesostomidae 41
Prorhynchidae 41
Prorhynchus 42
Proseriata 41, 42
Prosorchis 58, 59. 63
Prosorchis ghanensis 55, 57, 60
Proteidae 336
Proteocephalus 59
Proteocephalus longicollis 89
Protodrilus 129, 130, 138
Protomicrocotyle 58, 73
Protostrongylus rufescens 122
Protozoa 17
Protura 304
Provortex balticus 46
Provorlicidae 41, 46
Psammechinus miliaris 480
Pseudanthobothrium 59
Pseudanthobothrium hanseni 89
Pseudocentrotus depress us 1 6
Pseudodactylogyrus 58, 73
Pseudodiplorchis 58, 73
Pseudograffilla arenicola 46
Pseudomazocraes 58
Pseudomonas aeruginosa 470
Pseudopythina 163
Pseudopythina rugifera 158
Pseudostomella etrusca 106, 108, 110, 112, 113, 116
Pseudoterranova decipiens 122
Psocoptera 304
Psudocentrotus depressus 17
Pterastericola 46, 75
Pterastericola asamushii 48
Pterastericola astropectinis 46
Pterastericolidae 41, 46
Pterinotrema 58
Pteriomorphia 180
Pteropodidae 422
Pteropodinae 422
Pteropus 424
Pteropus dasymallus 422, 424, 425, 426
Pungitius pungitius 464
Pycnogonum littorale 204
Pycnoporus 58
Pygopodidae 370
Pygopus lepidopus 372
Pyragraphorus 58
Python sebae 372
Pyxicephalus adspersus 336, 339
Q
Quinqueserialis 58
Quiscalus quiscula 438, 440. 441
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
561
R
Raillietina (Raillietina) tunetensis 88. 89. 90
Raillietina 59, 89
Raja eglanteria 314. 316, 317, 319
Raja miraletus 57
Raja rhina 507
Rajidae 316
Raj i formes 316
Ramphomyia 306
Ramphotyphlops 385. 386, 387, 390, 393, 394, 395
Ramphotyphlops australis 386, 387, 388, 390, 392, 393,
395
Ramphotyphlops endoterus 386, 387, 388, 390, 392,
393, 395
Ramphotyphlops waitii 386, 387, 388, 390, 392, 393,
394, 395
Rana 57, 98, 327. 328, 448. 465, 466
Rana calesbeiana 455
Rana clamitans 322
Rana dybowskii 322, 324, 327, 328
Rana fuscigula 331, 338
Rana nigromaculata 322, 324, 327, 328
Rana pipiens 322
Rana ridibunda 464
Rana rugosa 322, 324, 328
Ranidae 336
Ranina 211
Ranina ranina 268, 273, 274, 276
Raninoidea 276
Raninoides 268, 273, 276, 277
Raphidioptera 304
Rat t us non'egicus 539
Reduviidae 285
Reptilia 359
Retinometra 59
Retinometra serrata 89, 90
Retronectes atypica 38
Retronectes sterreri 38
Rhabditis pellio 122
Rhabdocoela 43
Rhacophoridae 336
Rhacophorus 328, 340
Rhacophorus arboreus 322, 337
Rhacophorus schlegelii 322
Rhea 348
Rhigonema madecassum 122
Rhinobatidae 316
Rhinobatiformes 316
Rhinobalos schlegelii 314, 315, 316, 317
Rhinochimaera pacifica 316, 317
Rhinochimaeridae 316
Rhinolophidae 422
Rhinolophinae 422
Rhinolophus 424, 427
Rhinolophus cornutus 422, 424, 425
Rhinolophus ferrumequinum 422. 424, 425, 427
Rhinolophus imaizumii 422, 425
Rhinolophus luctus 422, 425
Rhinolophus megaphylla 344, 347, 348
Rhinolophus monoceros 422, 425
Rhizodrilus 192, 198
Rhizodrilus russus 192
Rhyacodrilinac 190, 192
Rhyacodrilus 192, 197
Rhyacodrilus arthingtonae 192
Rhyacophila (Hyporhyacophila) tristis 306
Rhyacophila < Pararhyacophila) italica 306
Rhyacophila f Rhyacophila ) dorsalis 306
Rhyacophila (Rhyacophila) foliacea 306
Rhyacophilidae 306
Rudi tapes decussatus 1 58
Rugogaster 58, 71, 73
S
Saccharomyces cerevisiae 526
Saccostomus 409, 410, 418
Saccostomus campestris 410, 414, 417
Saguinus imperator 539, 540
Salamandridae 336
Saldula sanatoria 286
Salmo irideus 509
Salmoninae 466
Sandonella 59
Sandonella sandoni 89, 90
Sanguinicolidae 63
Sauria 378
Scatophaga 304, 306
Scatophagidae 306
Schistosoma 55, 56, 57, 58, 62, 70, 71, 74
Schistosoma bovis 57
Schistosoma curassoni 57
Schistosomatium 71
Schizomus 210, 213, 216, 220
Schizomus palaciosi 206, 208, 210, 213
Schizorhynchia 41, 44
Sciara 306
Sciaridae 306
Scincidae 360
Scincomorpha 378
Scintilla 155, 156, 158, 160, 163
Scissurellidae 169, 172
Scissurelloidea 169
Scrobicularia 162. 163
Scrobicularia plana 158, 163, 180, 182
Scrobiculariidae 160
Scuiariellidae 41, 47
Scutus 172
Scut us antipodes 169, 172
Scutus sinensis 169
Scutus unguis 169
Scyliorhinidae 316, 466
Scyliorhinus 464
Scvliorhinus canicula 464, 507, 508
Sea cucumber 493, 494
Sege stria senoculata 208, 210. 214
Sejus togatus 218
Semnodactylus wealii 336, 337, 338
Sepia officinalis 466
Serges tes arcticus 232, 233, 234, 236, 237
Source . MNHN. Paris
562
SYSTEMATIC INDEX
Sergestidae 232, 236
Sericostomci italicum 306
Sericosioma pedemontanum 306
Sericostomatidae 306
Seritia stichopi 46
Serpentes 372. 380
Sicyonia 233, 234
Sicyonia brevirostris 232, 234
Sicyonia carinata 232, 234
Sicyonia ingentis 232, 234, 249
Sicyonidae 232, 234
Silo mediterraneus saturniae 306
Silurana 463, 466, 467
Silurana epitropicalis 466, 467
Sinezona 169, 172
Siphonaptera 304
Siphonops annulatus 336
Siro 208, 210, 211, 212, 224
Siro duricorius 205, 206, 207, 214
Siro rubens 206
Skeneidae 169, 173
Smithsonidrilus 190, 194, 198
Smithsonidrilus hummelincki 189, 190, 191, 192, 195
Solen cape ns is 158
Solen cylindraceus 158
Solenocera membranacea 232, 233, 234, 236, 239
Solenoceridae 232. 236
Solenoidca 160
Solenopharyngidae 41
Sparidae 466
Spams aurata 57
Sphaerechinus granulosus 480
Sphaerodactylus cinereus 370
Sphaerolaimus hirsutus 122, 124
Sphenodon 346, 347, 348, 349, 350, 352, 355, 370,
376, 377
Sphenodon punctatus 344, 376
Sphenodontida 355
Sphyranura 58
Sphyrna lewini 314, 315, 316, 317
Sphyrnidae 316
Spirorchis 55, 57, 58, 62. 63
Spisula 453
Spisula solidissima 158. 180, 182, 452, 454. 458, 464
Spisula trigonella 157, 160
Squalidae 316
Squali formes 316, 319
Squalus 3 1 8
Squat us acanihias 466
Squalus brevirostris 316, 317
Squalus japonicus 3 1 6, 3 1 7
Squamata 356, 359, 376. 385, 466
Squatina japonica 313, 314, 316, 317
Squatinidae 316
Squatiniformes 316, 319
Stactobia caspers i 306
Stallion 520
Steatomys 409
Steatomys parvus 410, 414, 416. 417
Stegonotus cucullatus 372, 374
Stenodactylus selvini 367
Stenophylax permistus 306
Stenoplax conspicua 132, 134, 137
Sienopus hispidus 236, 237, 239
Slilesia 59
Stilesia globipunctata 89, 90
Stitndromia (=Petalomera) lateralis 268, 270, 272
Stimdromia 271. 272, 274, 276
Stophilus multistriatus 299
Stophilus oryzae 299
Strepsiptera 291, 304
Strongylocentrotus intermedins 484
Strongylocentrotus nudus 480
Strongylocentrotus purpuratus 476, All, 478, 480, 484
Strongylopus 339
Strongylopus grayii 336, 339
Struthio camelus 346
Sturnidae 440
Stum us vulgaris 438, 440
Styela 465, 502, 503, 504, 512
Stye I a monte reyens is 502, 503
Styela plicata 451, 502, 503, 504
Slyelidae 466
Stylops 292, 295
Sus scrofa 539
Sympagurus 254, 256, 260
Symsagittifera 97
Symsagittifera schultzei 98, 100
Syndesmis echinorum 46
Syndisyrinx punicea 46, 48
T
Tacliycineta thalassina 439, 440
Tachyglossus acid eat us 539
Tac hyp lens 204, 210, 216
Tachypleus gigas 205, 208, 216
Tachypleus tridentatus 216
Taenia 59
Taenia hydatigena 89
Talabrica aurora 1 58
Tapes 20
Tapes japonica 16, 17
Tapes rhomboides 158
Tarantula 213
Tarantula marginemaculata 213
Tarentola mauritanica 370
Tarentola mauritanica mauritanica 370
Taricha granulosa 344, 352, 354
Tarnania 303
Tarsipes rostratus 397, 398, 405, 406
Tatera 409
Tat era leucogasler 410, 412, 413, 417
Tectura scutum 170
Tectus pyramis 169
Teiidae 364
Tellina 160, 162
Tellina rostrata 156, 157, 160, 163
Tellinidae 160
Tellinoidea 160
Telmatobius 321, 327, 328
ADVANCES IN SPERMATOZOAL PHYLOGENY AND TAXONOMY
563
Telmatobius hauthali 322
Temnocephala 46
Temnocephala dendyi 46
Temnocephala minor 47
Temnocephalida 41, 46
Temnocephalidae 41, 46
Tephritidae 306
Terricola 41
Tetrabothrius 59
Tetragnatha 207, 208, 216
Tetragnatha montana 21 1
Tetrahymena 15, 16, 17, 18, 20
Teirahymena pyriformis 16, 17
Tetrahymena thermophila 470
Tetranchyroderma 106, 108, 110
Tetranchyroderma papii 106. 110
Tetranychus urticae 206, 210, 223
Tetraonchoides 58
Tetraonchus 58
Tetrapods 343, 354, 355
Teulhodea 466
Thalassina 258, 260
Thalassina squamifera 252, 254
Thalassinidae 254
Thalassinidea 252, 253, 254
Thalassinoidea 254
Thalassodrilides 198, 199
Thalassodrilides inert 192
Thalassodrilus 197, 198, 199
Thalassodrilus prostatus 192
Thaumastoderma 108, 116
Thaumastodermatidae 106, 110
Thelastoma periplaneticola 464
Thoracotremata 277
Thraupidae 439
Thunnus thynnus 509
Thy one 25
Thyone briareus 484
Thysaniezia 59
Thysaniezia ovilla 88, 89, 90
Thysanoptera 304
Tiliqua 362, 370, 379, 380
Tiliqua scincoides scincoides 360, 368, 374, 376
Tipula 304, 306
Tipulidae 306
Tivela polita 158
Tomopterna delalandii 336
Tonicella lineata 132, 134, 137
Tonicella marmorea 132
Tonicellidae 132, 146
Torpcdinidae 316
Torpediniformes 316
Torpedo tokionis 314, 316, 317
Trachypeneus similis 232, 233
Trapezium sublaevigatum 157, 160
Trepaxonemaia 39, 40, 41, 56, 102
Triakidac 316
Triatoma infestans 285, 286, 288
Trichinella nativa 122
Trichinella pseudospiralis 122
Trichinella spiralis 122, 124
Trichocera hiemalis 306
Trichoceridae 306
Trichoptera 301, 303, 304, 308
Trichuris muris 1 22
Tricladida 41, 43
Tricolia capensis 169
Tridacna maxima 157
Tridacnidae 466
Trig la lucerna 509, 510
Trigonostomidae 41
Trilocularia 59
Trilocularia acanthiaevulgaris 89
Tri turns viridescens 336
Trizopagurus strigimanus 254
Trochidac 169, 172,466
Trochoidea 169
Trochopus 58
Trochus nigropunctatus 169
Troglocaridicola 47
Tropiduridae 367
Tropidurus torquatus 367
Tubifex 196, 198, 199
Tubifex tubifex 192
Tubificinae 191, 192
Tubificidae 198
Tubificoides 190, 196, 198, 199
Tubificoides amplivasatus 189, 190, 191, 192
Tubiluchus corallicola 1 1 6
Tunicates 502
Turbanella 1 1 6
Turbanella ambronensis 106, 108, 112, 114
Turbanella cornuta 106, 108
Turbanellidae 106, 112
Turbinidae 169, 172
Turbo cidaris natalensis 169
Turbo coronal us 169
Turbo sarmaticus 169
Turdus migratorius 439
Turidae 439
Tylenchulus semipenetrans 122
Tymolus 268, 273, 277
Typhlonectes natans 335, 336
Typhlonectidae 336
Typhlopidae 385
Typhloplana virida 43
Typhloplanida 41, 43, 74
Typhloplanidae 41
Tyrannidac 439, 440
Tyrannus vertical is 439
Tyrophagus putrescentiae 223
U
Uca 280
Uca dussumieri 268, 278
Udonella 75
Udonellidae 74
Umagillidae 41, 46
Upogebia pusilla 253
Upogebiidae 253
Source . MNHN . Paris
564
SYSTEMATIC INDEX
Jraeotyphlus narayani 336
Uranomys 409, 410, 417, 418
Urastoma 41. 42, 48, 49
Urasionia cyprinae 42
Z
Zalipais laseroni 169, 173, 175
Zygentoma 304
Urodela 336
Urodcles 337
Urolophidae 316
Urolophus aurantiacus 316, 317
Uromastyx philbyi 367
Uroptychus 254, 258
Jrsus americanus 539
V
Varanidae 368
Varanus 359, 368, 379, 380
Varanus gouldii flavirufus 347, 367, 368, 369, 370, 376,
380
Varroa jacobsoni 214, 223
Veneroida 180
Veneroidea 158, 160
Venerupis aureci 158
Venerupis corrugata 158
Verutus volvingentis 122
Vespertilio 421, 424, 427
Vespertilio supercins 422
Vcspcrtilionidae 422
Vespertilioninac 422
Vetigastropoda 171, 174
Viper a aspis asp is 372
Vireo olivaceus 439
Vireonidac 439
W
Wormaldia copiosa 306, 308
Wormaldia occipitalis 306, 308
X
Xeinostoma 277
Xeinostoma richeri 268, 273
Xenopus 327, 337, 339, 340, 341, 463, 466. 467
Xenopus laevis 322, 334, 337, 338, 455, 467
Xenopus tropicalis 466, 467
Xenos 294
Xenos moutoni 291, 292, 294
Xenos ve spar urn 291, 292, 294, 295
Xenotrichula 108
Xenotrichula intermedia 106, 112
Xenotrichula punctata 106, 108, 112
Xenoirichulidae 106, 112
Xiphinema diversicaudatum 122
Xiphinema theresiae 122
Xylopagurus 254. 256, 260
Remerciements aux rapporteurs / Acknowledgements to referees
La Redaction lient a remcrcier les experts exterieurs au Museum national d'Histoire naturelle dont les noms suivcnt, d’avoir bien voulu
contribuer, avec les rapporteurs de FEtablissement, h revaluation des manuscrits (1986-1995) :
The Editorial Board acknowledges with thanks the following referees who, with Museum referees, have reviewed papers submitted to the
Memories du Museum (1986-1995):
Source : MNHN , Paris
Seventh International Symposium on Spermatology:
Plenary Papers
reprinted from Reproduction , Fertility and Development 7(4), 1995.
Published December 1995 by CSIRO Australia. 310 pages. Price: $US66 plus $6 for postage and handling.
Contents
Free radicals, lipid peroxidation and sperm function. R. John Aitken
Genetic control of mitosis, meiosis and cellular differentiation during mammalian spermatogenesis.
Debra J. Wolgemuth, Kunsoo Rhee, Shuang Wu and Stuart E. Ravnik
The azoospermia factor (AZF) of the human Y chromosome in Yql 1: function and analysis in spermatogenesis.
P. H. Vogt. A. Edelmann, P. Hirschmann and M. R. Kohler
'Chauvinist genes' of male germ cells: gene expression during mouse spermatogenesis. E. M. Eddy
A model for understanding gene regulation during spermatogenesis: the mouse testis Pdha-2 promoter.
Rocco C. Iannello. Julia Young. Sony Sumarsono. Martin Tymms and Ismail Kola
The molecular biology of SRY and its role in sex determination in mammals. Peter Koopman
Interleukin- 1, interleukin-6 and the germ cell-Sertoli cell cross-talk. B Jegou. C. Cudicini, E. Gomez and J. P. Stephan
The epididymis as protector of maturing spermatozoa. Barry T. Hinton. Michael A. Palladino, Daniel Rudolph and Jacquelyn C. Labus
The sperm centrosome during fertilization in mammals: implications for fertility and reproduction.
Christopher S. Navara. Calvin Simerly. Sara Zoran and Gerald Schatten
Sperm competition: evolutionary causes and consequences. T R Birkhead
Developmental expression and possible role of perinuclear theca proteins of mammalian spermatozoa. Richard J. Oko
Three-probe fluorescence in situ hybridization to assess chromosome X. Y. and 8 aneuploidy in sperm of 14 men from two healthy
groups: evidence for a paternal age effect on sperm aneuploidy.
W. A. Robbins. J. E. Baulch. D. Moore II. H.-U. Weier. D. Blakey and A. J. Wyrobek
Spermatology for understanding, managing and conserving rare species.
David E. Wildt. Budhan Pukazhenthi. Janine Brown. Steven Monfort. JoGayle Howard and Terri Roth
Sperm antigens and immunocontraception. Lorraine E. Kerr
Micro-assisted fertilization. Dianna Payne
The use of epididymal sperm for assisted reproduction in men with acquired, irreparable obstructive azoospermia.
P. Patrizio , T. Ord. J. P. Balmaceda and R. H. Asch
Regulation of sperm motility at the axonemal level. Claude Gagnon
Derivation and reliability of kinematic measures of sperm motion. Russell O. Davis and Rebecca J. Siemers
Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function.
P. F. Watson
Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review.
Lawrence A. Johnson
Ionic control of sperm function. Lynn R. Fraser
Interactions between gametes leading to fertilization: the sperm’s eye view. Bayard T. Storey
Synchronous multispecific spawning on coral reefs: potential for hybridization and roles of gamete recognition. Russ Babcock
Workshop report: clinical CASA — the quest for consensus. D. Mortimer, R. J. Aitken, S. T. Mortimer and A. A. Pacey
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From 1993 (Volume 155), the Memoires du Museum are published without serial titles.
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Pucadelphys andinus (Marsupialia, Mammalia) from the early Paleocene of Bolivia 168 on
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This volume is divided into three sections: Invertebrate and General, Vertebrates, and Proteins. It is
essential reading for those interested in spermatozoal ultrastructure and its use in taxonomy and phylogeny
of invertebrates and vertebrates, the practical application of computerized cladistic procedures, the nature,
evolution and molecular biology, including sequencing and transcription, of sperm nuclear proteins and
their gene expression, calmodulin-mediated events in sperm of slime-moulds and Metazoa, the fine
structure of sperm components, including patterns of subunits in axonemal microtubules, molecular
structure of dynein arms, demonstration of cytoskeletal elements using immunofluorescence and silver
staining, recording and interpreting patterns of sperm motility in terms of fertilization biology and other
themes representing major recent advances in comparative spermatology.
The text includes 41 contributions from 73 authors and should, indeed, prove useful to all students
of reproductive biology and phylogeny of groups such as the Platyhelminthes, Gastrotricha, Nematoda,
Mollusca, Annelida, Chelicerata, Crustacea, Insecta, Pisces, Amphibia, Reptilia, Mammalia, and Aves.
Barrie G. M. Jamieson is a Professor of Zoology in the University of Queensland (Brisbane,
Australia). In addition to his work as an oligochaete taxonomist, he has published many papers on
spermatozoal ultrastructure and phylogeny of several invertebrate and vertebrate groups, and two books
on Insect and Fish sperm.
Juan Ausio is Professor of Biochemistry at the University of Victoria (Canada) and was previously
a member of the Spanish Research Council (C.S.I.C.) in Barcelona (Spain). He is one of the world
specialists of sperm nuclear proteins and chromatin structure.
Jean-Lou Justine is Professor at the Museum national d’Histoire naturelle (Paris, France), and was
previously at the University of Dakar (Senegal). He has worked on spermatozoal ultrastructure and
phylogeny mainly on two groups of parasitic worms : the Platyhelminthes and the Nematoda.
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