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AITES

INTERNATIONAL JOURNAL OF BATRACHOLOGY

October 1997 Volume 15, N° 2

Alytes, 1997, 15 (2): 49-71. 4

The genus Chiasmocleis Méhely, 1904

(Anura, Microhylidae)

in the Atlantic Rain Forest of Brazil,

with description of three new species

Carlos Alberto Gonçalves DA CRUZ *,

Ulisses CARAMASCHI ** & Eugênio IZECKSOHN *

* Departamento de Biologia Animal, Universidade Federal Rural do Rio de Janeiro,

23851-970 Seropédica, RJ, Brazil

** Departamento de Vertebrados, Museu Nacional do Rio de Janeiro, Quinta da Boa Vista,

20940-040 Rio de Janeiro, RJ, Brazil

A taxonomic study of the species of the genus Chiasmocleis occurring

in the Atlantic Rain Forest of eastern Brazil is presented. Chiasmocleis bice-

goi Miranda-Ribeiro, 1920 is synonymized with C. albopunctata (Boettger,

1885), and rendered extralimital for the present work. The tion

of C. urbanae Bokermann, 1952 with C. leucosticta (Boulenger, 1888) is

reaffirmed. Chiasmocleis leucosticta and C. schubarti Bokermann, 1952

are redescribed, and three new species are described.

INTRODUCTION

The genus Chiasmocleis, proposed by MÉHELY (1904), currently contains 12 species,

distributed from Panama to southern South America, north and east of the Andes (FROST,

1985). The validity of the species included in this genus has not been assessed by a modern

review. Materials from the Atlantic Forests of Brazil indicate such an assessment is needed.

Herein, we present a review of the species of Chiasmocleis inhabiting the Atlantic Rain Forest

of eastern Brazil. This systematic review includes: (1) re-examination of previously described

taxa; (2) redescription of the valid species; and (3) description of three new species.

MATERIALS AND METHODS

Abbreviations of the collections housing specimens are: BMNH (Natural History

Museum, London, United Kingdom); CFBH (Célio F. B. HApDAD Collection, Universidade

Bibliothèque Centrale Muséum

y NM.

k 001

50 ALYTES 15 (2)

Estadual Paulista, Rio Claro, SP, Brazil); EI (Eugênio IZECKsoHN Collection, Universidade

Federal Rural do Rio de Janeiro, RJ, Brazil); MNRJ (Museu Nacional do Rio de Janeiro, RJ,

Brazil); MZUSP (Museu de Zoologia, Universidade de Säo Paulo, SP, Brazil); SPCS (Sérgio

POTSCH DE CARVALHO E SiLvA Collection, Universidade Federal do Rio de Janeiro, RJ,

Brazil); UFV (Museu de Histéria Natural, Universidade Federal de Viçosa, MG, Brazil).

Specimens examined are listed in app. 1.

Measurements, in millimeters, are: SVL (snout-vent length); HL (head length); HW

(head width); IND (internarial distance); END (eye to nostril distance); ED (eye diameter);

UEW (upper eyelid width); IOD (interorbital distance); THL (thigh length); TL (tibia

length); FL (foot length).

NOMENCLATURAL HISTORY

Each of the five names proposed for a member of the genus Chiasmocleis occurring in the

Atlantic Rain Forest is discussed in chronological order.

ENGYSTOMA LEUCOSTICTUM BOULENGER, 1888

This species was described on the basis of a female specimen (type not stated; BMNH

88.4.23.3 registered as holotype, according to FRosT, 1985) collected at “Sierra do Catharina,

Province Santa Catharina, Brazil” (BOULENGER, 1888). Currently this locality is in the

Catarina Mountain Range, in the state of Santa Catarina, southern Brazil. PARKER (1934)

transferred the species to the genus Chiasmocleis, under the combination Chiasmocleis

leucosticta.

CHiasmocLEIs BiCEGOI MIRANDA-RIBEIRO, 1920

The species was described based on a single specimen from “Os Perus, S. Paulo”

(MiRANDA-RIBEIRO, 1920). Currently, this locality is a suburb within the western side of the

city of Säo Paulo, state of Säo Paulo, Brazil. The holotype (MZUSP 924, formerly MZUSP

595), a juvenile (15.0 mm SVL) of undetermined sex, is in very poor condition, totally

discoloured, the ventral and pectoral regions dilacerated, and arms and legs almost totally

destroyed. Direct comparison of the holotype of C. bicegoi with a recently collected young

specimen of Chiasmocleis albopunctata Boettger, 1885, obtained at Iper6, state of Säo Paulo

(MNRY 17324, 13.9 mm SVL), indicates that the former is a junior synonym of the latter.

Chiasmocleis albopunctata is an open area inhabitant, occurring in the “cerrados” of Brazil in

the states of Säo Paulo, Minas Gerais, Goiäs, Mato Grosso, Mato Grosso do Sul, and Distrito

Federal, and in Paraguay and Bolivia. Iperé is about 90 km from the type locality of C.

bicegoï, and it is perfectly acceptable that the species occurred near the city of Säo Paulo at the

end of the last century, when the holotype was collected by BiCEGo. The shape of the snout,

although with some deformation in the holotype of C. bicegoi, is coincident in both speci-

mens; vestiges of the white blotches on the snout and dorsolateral region of the body

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 51

characteristic of C. albopunctata are still observed in the holotype of C. bicegoi. Thus,

synonymization of C. bicegoi with C. albopunctata renders it extralimital for the present work.

The citation of C. bicegoi for Itaguai, state of Rio de Janeiro, by BOGART & NELSON (1976)

applies to a new species described in the account below.

NECTODACTYLUS SPINULOSUS MIRANDA-RIBEIRO, 1924

The genus Nectodactylus was proposed by MIRANDA-RIBEIRO (1924) to accomodate a

new species, named N. spinulosus (syntypes MNRJ 525 and 5582), from “Humboldt — Sta.

Catharina - Brasilia” (currently Corupä, state of Santa Catarina, Brazil). The genus was

synonymized with Chiasmocleis by PARKER (1934), resulting in the combination Chiasmocleis

spinulosa. Later, CARVALHO (1954) synonymized Nectodactylus spinulosus with Engystoma

leucostictum, thus recognizing Chiasmocleis leucosticta as the valid name for the species.

CHIASMOCLEIS SCHUBARTI BOKERMANN, 1952

The species was described based on a young specimen (holotype MZUSP 2309) from

“Cérrego Juncado, Linhares”, state of Espirito Santo, Brazil (BOKERMANN, 1952). This is a

perfectly valid and recognizable species.

CHIASMOCLEIS URBANAE BOKERMANN, 1952

The species was described on the basis of a purported male specimen (holotype MZUSP

9033) from “Ilha de Säo Sebastiäo”, on the coast of the state of Säo Paulo, Brazil (BOKER-

MANN, 1952). Chiasmocleis urbanae was synonymized with C. leucosticta by BOKERMANN

(1966); this action was not recognized in FRosT (1985), where C. urbanae was treated as a valid

species. Examination of the holotype of C. urbanae showed it to be a female specimen and

identical to specimens of C. leucosticta from the state of Santa Catarina and the southern

portion of the state of Säo Paulo. Consequently, we agree with BOKERMANN (1966) and

consider C. urbanae to be a synonym of C. leucosticta.

SPECIES ACCOUNTS

Chiasmocleis leucosticta (Boulenger, 1888)

Engystoma leucostictum Boulenger, 1888.

Chiasmocleis leucosticta: PARKER, 1934.

Nectodactylus spinulosus Miranda-Ribeiro, 1924.

Chiasmocleis spinulosa: PARKER, 1934.

Chiasmocleis urbanae Bokermann, 1952.

Diagnosis. - À medium sized species of Chiasmocleis diagnosed by the following combination

of characters: (1) SVL 19.0-22.8 mm in males, 21.8-25.5 mm in females; (2) body trunk ovoid;

Source : MNHN, Paris

52 ALYTES 15 (2)

Fig. 1.- Dorsal and ventral views of Chiasmocleis leucosticta (Boulenger, 1888) (MNRJ 17901).

(3) snout short, tip slightly truncate in dorsal view, and slightly protruding in lateral profile;

(à) hand and foot of male webbed, of female half webbed; (5) fingers and toes lacking disks,

fringed on free parts in females; (6) fingers and toes with few, small lateral dermal spines only

in males; (7) dorsal surfaces with small dermal spines; (8) in preservative, color on dorsum

dark brown with small white dots irregularly distributed, and a light longitudinal mid-dorsal

line; (9) posterior sides of legs with a light longitudinal line; (10) venter roughly marbled in

dark brown and pale cream.

Description. — Size medium for the genus; body trunk ovoid (fig. 1); head short, as broad as

long; nostrils at the tip of snout, not protuberant, and directed laterally; snout short, tip

slightly truncate in dorsal view (fig. 2 A), and slightly protruding in lateral profile (fig. 2 B);

internarial distance smaller than eye to nostril distance, and only slightly smaller than eye

diameter; eye diameter less than eye to nostril distance; canthus rostralis only slightly defined;

loreal region oblique, flat; lips not flared; eyes small, only slightly protruding; upper eyelid

width one third of the interorbital space; interorbital area flat; cranial crests and occipital fold

absent; postorbital fold present; tympanum absent; upper jaw projecting beyond lower;

mandible with truncate, trilobed anterior margin; tongue large, ovoid; choanae small, round-

ed, widely separated; a small, subgular vocal sac present.

Arms slender, lacking tubercles and crests on forearm. Hand of male (fig. 2 C) webbed,

of female (fig. 3 A) half webbed; fingers lacking disks, fringed on free parts in females, with

lateral dermal spines only in males; finger length 1 < II < IV < II; subarticular tubercles well

developed, rounded; supernumerary tubercles absent; palmar tubercle large, divided in two

parts; thenar tubercle large, rounded, at the base of finger I.

Legs short, robust; knee and heel lacking tubercles; tibial and tarsal ridges absent. Foot

of male (fig. 2 D) webbed, of female (fig. 3 B) half webbed; toes lacking disks and fringed on

free parts; toe length I < II < V < III < IV; toes with few, small lateral dermal spines in males,

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 53

Fig. 2. - Chiasmocleis leucosticta (Boulenger, 1888), male (MNRJ 17901). Dorsal (A) and ventral (B)

views of head. Hand (C). Foot (D).

Source : MNHN, Paris

ALYTES 15 (2)

Fig. 3. - Chiasmocleis leucosticta (Boulenger, 1888), female (MNRJ 17902). Hand (A). Foot (B).

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 55

# C: leucosticta

& C:carvalhoi

X C: schubarti + C.capixaba

% C.atlantica + C:carvalhoi

Ü 100 200 300KN

Fig. 4. - Geographical distribution of the species of Chiasmocleis in the Atlantic Rain Forest of Brazil

(states of southeastern and southern regions of Brazil: MG, Minas Gerais; ES, Espirito Santo; RJ,

Rio de Janeiro; SP, Säo Paulo; PR; Paranä; SC, Santa Catarina; RS, Rio Grande do Sul).

lacking in females; subarticular tubercles well developed, rounded; supernumerary tubercles

absent; an oval inner, but no outer, metatarsal tubercle. Thigh length slightly less than tibia

length; knee and elbow widely separated with limbs adpressed to sides of body; combined

thigh and tibia length approximately 83 % of snout-vent length in males, 85 % in females;

heels slightly superposed when flexed legs held at right angles to body; foot length approxi-

mately 66 % of snout-vent length in males, 69 % in females.

Source : MNHN, Paris

56 ALYTES 15 (2)

Skin smooth above and beneath; dorsal and ventral surfaces of body and limbs with

small, uniformly distributed dermal spines, more numerous in males. Anal opening not

modified, lacking para-anal tubercles and glands around anus.

In preservative (70 % ETOH), color on dorsum dark brown with small white dots

irregularly distributed; a light longitudinal mid-dorsal line meeting, above the anus, another

light longitudinal line present on the posterior sides of legs. Venter roughly marbled in dark

brown and pale cream; male throat infuscated.

Variation. — Variation in measurements is presented in tab. 1. Females are larger than males,

with interdigital webbing less developed. In some females, a light line is present on ventral

surface of arms and pectoral region, where it assumes a V-shape, crossed by a perpendicular

light longitudinal mid-ventral line.

Geographical distribution. - Known from the states of Säo Paulo and Santa Catarina, in

southeastern and southern Brazil (fig. 4). The occurrence of the species in the state of Paranä

is predictable.

Remarks. - Specimens of C. leucosticta were collected in temporary ponds inside the forest,

from about 30 to 800 m above sea level. The reproduction is explosive, when numerous male

and female specimens congregate at the reproductive sites. KASAHARA & HADDAD (1997)

reported a population of C. leucosticta from Ribeiräo Branco, state of Säo Paulo, to be

tetraploid (4 n = 48).

Chiasmocleis schubarti Bokermann, 1952

Chiasmocleis schubarti Bokermann, 1952.

Diagnosis. - À medium sized species of Chiasmocleis diagnosed by the following combination

of characters: (1) SVL 19.2-26.3 mm in males, 20.4-34.5 mm in females; (2) body trunk ovoid;

(3) snout short, tip rounded in dorsal and lateral profile; (4) hand not webbed, foot only

slightly webbed at the base of toes in both sexes; (5) fingers and toes lacking disks, slightly

fringed; (6) fingers and toes of males with small, lateral dermal spines, absent in females; (7)

dorsal and ventral surfaces of body and limbs with small, uniformly distributed dermal spines

in males, absent in females except in anal region; (8) in preservative, color on dorsum

uniformly dark brown; (9) posterior sides of thighs with a light longitudinal line; (10) venter

roughly marbled in dark brown and pale cream.

Description. — Size medium for the genus; body trunk ovoid (fig. 5); head short, broader than

long; nostrils near the tip of snout, not protuberant, and directed laterally; snout short, tip

rounded in dorsal and lateral profile (fig. 6 A-B); internarial distance smaller than eye to

nostril distance, and slightly larger than eye diameter; eye diameter less than eye to nostril

distance; canthus rostralis only slightly defined; loreal region oblique, flat; lips not flared; eyes

small, only slightly protruding; upper eyelid width one third of the interorbital space;

interorbital area flat; cranial crests and occipital fold absent; postorbital fold present; tympa-

num absent; upper jaw projecting beyond lower; mandible with truncate, trilobed anterior

margin; tongue large, ovoid; choanae small, rounded, widely separated; a small, subgular

vocal sac present.

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 57

Fig. 5. - Dorsal and ventral views of Chiasmocleis schubarti Bokermann, 1952 (MNRIJ 17538).

Arms slender, lacking tubercles and crests on forearm. Hand (fig. 6 C) not webbed in

both sexes; fingers lacking disks, slightly fringed; fingers with small, lateral dermal spines in

males, absent in females; finger length I < II < IV < III; subarticular tubercles well developed,

rounded; supernumerary tubercles absent; palmar tubercle large, divided in two parts; thenar

tubercle small, rounded, at the base of finger I.

Legs short, robust; knee and heel lacking tubercles; tibial and tarsal ridges absent. Foot

(fig. 6 D) only slightly webbed at the base of toes in both sexes; toes lacking disks, slightly

fringed; toe length I < II < V < III < IV; toes with small, lateral dermal spines in males, absent

in females; subarticular tubercles well developed, rounded; supernumerary tubercles absent; a

small, rounded inner, but no outer, metatarsal tubercle. Thigh length slightly less than tibia

length; knee and elbow widely separated with limbs adpressed to sides of body; combined

thigh and tibia length approximately 85 % of snout-vent length in males, 82 % in females;

heels slightly superposed when flexed legs held at right angles to body; foot length approxi-

mately 70 % of snout-vent length in males, 64 % in females.

Skin smooth above and beneath; dorsal and ventral surfaces of body and limbs with

small, uniformly distributed dermal spines in males, absent in females except in anal region.

Anal opening not modified, lacking para-anal tubercles and glands around anus.

In preservative (70 % ETOH), color on dorsum dark brown; irregular white blotches on

outer surfaces of arms and forearms; dorsal surfaces of hands and feet with small white dots

Source : MNHN, Paris

58 ALYTES 15 (2)

Fig. 6. - Chiasmocleis schubarti Bokermann, 1952 (MNRJ 17538). Dorsal (A) and ventral (B) views of

head. Hand (C). Foot (D).

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 59

Fig. 7. - Dorsal and ventral views of Chiasmocleis atlantica sp. nov. (MNRIJ 17550, holotype).

irregularly distributed; a light longitudinal line on the posterior sides of thighs. Venter

roughly marbled in dark brown and pale cream; male throat infuscated.

Variation. — Variation in measurements is presented in tab. 1. In some specimens, a light

longitudinal mid-dorsal line is present.

Geographical distribution. - Known from the states of Espirito Santo and Minas Gerais in

southeastern Brazil (fig. 4).

Remarks. - Scattered specimens of C. schubarti were collected in pitfall traps installed in low

and highland forests, 40 and 800 m above sea level. Explosive reproduction, when numerous

male and female specimens congregate, was observed in temporary ponds inside the forest.

KASAHARA & HADDAD (1997) reported a diploid number of 24 chromosomes for this species.

Chiasmocleis atlantica sp. nov.

Holotype. - MNRJ 17550, adult male, collected at Tinguä (22°36'S, 43°26'W,; ca. 40 m

altitude), municipality of Nova Iguaçu, state of Rio de Janeiro, Brazil, on 27 December 1972,

by C. A. G. DA CRUZ, S. D. L. RAIMUNDO, J. G. SILVA and E. IZECKSOHN.

Paratopotypes. - MNRIJ 17551-17554 and EI 8940-8954, collected with the holotype; MNRJ

17549, on 30 January 1971, by J. Jim, V. C. JESUS and E. IZECKSOHN.

Diagnosis. - À medium sized species of Chiasmocleis diagnosed by the following combination

of characters: (1) SVL 22.0-25.0 mm in males, 30.0-31.8 mm in females; (2) body trunk ovoid;

(3) snout short, tip truncate in dorsal view, and slightly protruding in lateral profile; (4) hand

and foot not webbed; (5) fingers and toes lacking disks, slightly fringed; (6) fingers and toes of

Source : MNHN, Paris

60 ALYTES 15 (2)

males with lateral dermal spines, absent in females; (7) dorsal and ventral surfaces of body

and limbs with uniformly distributed dermal spines in males, absent in females except in anal

region; (8) in preservative, color on dorsum uniformly brown; (9) posterior sides of thighs

with a light longitudinal line; (10) venter roughly marbled in brown and pale cream.

Description. — Size medium for the genus; body trunk ovoid (fig. 7); head short, wider than

long; nostrils at the tip of snout, not protuberant, and directed laterally; snout short, tip

truncate in dorsal view (fig. 8 A), and slightly protruding in lateral profile (fig. 8 B); internarial

distance smaller than eye to nostril distance, and larger than eye diameter; eye diameter less

than eye to nostril distance; canthus rostralis slightly defined; loreal region almost vertical,

slightly concave; lips not flared; eyes small, only slightly protruding; upper eyelid width one

third of the interorbital space; interorbital area flat; cranial crests and occipital fold absent;

postorbital fold well developed; tympanum absent; upper jaw slightly projecting beyond

lower; mandible with truncate, trilobed anterior margin; tongue large, ovoid; choanae small,

rounded, widely separated; a small, subgular vocal sac present.

Arms slender, lacking tubercles and crests on forearm. Hand (fig. 8 C) not webbed in

both sexes; fingers lacking disks, slightly fringed; fingers with lateral dermal spines in males,

absent in females; finger length I < II < IV < III; subarticular tubercles well developed,

rounded; supernumerary tubercles absent; palmar tubercle large, divided in two parts; thenar

tubercle small, rounded, at the base of finger I.

Legs short, robust; knee and heel lacking tubercles; tibial and tarsal ridges absent. Foot

(fig. 8 D) only slightly webbed at the base of toes in both sexes; toes lacking disks, slightly

fringed; toe length I < II < V < III < IV; toes with lateral dermal spines in males, absent in

females; subarticular tubercles well developed, rounded; supernumerary tubercles absent; a

small, oval inner, but no outer, metatarsal tubercle. Thigh length slightly less than tibia length;

knee and elbow widely separated with limbs adpressed to sides of body; combined thigh and

tibia length approximately 87 % of snout-vent length in males, 78 % in females; heels slightly

superposed when flexed legs held at right angles to body; foot length approximately 70 % of

snout-vent length in males, 67 % in females.

Skin poorly rugose above and smooth beneath; dorsal and ventral surfaces of body

and limbs with uniformly distributed dermal spines in males, absent in females except in

anal region. Anal opening not modified, lacking para-anal tubercles and glands around

anus.

In preservative (70 % ETOH), color on dorsum uniformly brown; dorsal surfaces of

hands and feet with small white dots irregularly distributed; a light longitudinal line on the

posterior sides of thighs. Venter roughly marbled in brown and pale cream; male throat

infuscated.

Measurements of holotype. - SVL 22.8; HL 6.9; HW 8.0; IND 2.0; END 2.0; ED 2.0; UEW

1.0; IOD 2.7; THL 10.4; TL 10.7; FL 16.7.

Variation. - Variation in measurements is presented in tab. 1. In some specimens, a light

longitudinal mid-dorsal line is present.

Etymology.-The specific name, a Latin adjective, refers to the Atlantic Rain Forest of eastern

Brazil.

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 61

Fig. 8. — Chiasmocleis atlantica sp. nov. (MNRIJ 17550, holotype). Dorsal (A) and ventral (B) views of

head. Hand (C). Foot (D).

Source : MNHN, Paris

62 ALYTES 15 (2)

Geographical distribution. - Known from the states of Rio de Janeiro and Säo Paulo in

southeastern Brazil (fig. 4).

Remarks.-Specimens of C. atlantica were collected in lowland forests, at 40 m above sea level.

Explosive reproduction, when numerous male and female specimens congregate, was obser-

ved in temporary ponds inside the forest.

Chiasmocleis capixaba sp. nov.

Holotype. - MNRIJ 17514, adult male, collected at the municipality of Aracruz (19°59’S,

40°12’W,; ca. 60 m altitude), state of Espirito Santo, Brazil, on 29 November 1995, by J. L.

GASPARINI.

Paratopotypes. - MNRIJ 17515-17529, collected with the holotype; MNRJ 17532-17534, on

2-5 November 1994, by R. P. Basros and J. L. GaspaRINI; MNRJ 17891-17895, on 29

November - 1 December 1995, by J. L. GasPaRINI; MNRIJ 17535-17537, on 15-17 January

1996, by J. P. POMBAL Jr., C. F. B. HADDAD and J. L. GasPaRINI; EI 8955-8956, on 20-25

November 1994, by R. P. Basros and J. L. GaspARINI;, CFBH 2668-2669, on 20-25 November

1994, by R. P. Basros and J. L. GAsPaRINI;, CFBH 2685, on 23-27 January 1995, by J. P.

POMBaAL Jr. J. L. GaspaRiNI and C. F. B. HaDDAD; CFBH 2693-2695, on 27 November 1995,

by J. L. GasPaRINI;, CFBH 2701-2702, on 30 November 1995, by J. L. GASPARINI; CFBH 2714,

on 20 December 1995, by J. L. GASPARINI.

Diagnosis. — À small sized species of Chiasmocleis diagnosed by the following combination of

characters: (1) SVL 14.7-16.5 mm in males, 17.9-21.7 mm in females; (2) body trunk elongate

ovoid; (3) snout short, tip rounded in dorsal and lateral views; (4) hand not webbed, and foot

webbed in both sexes; (5) fingers and toes lacking disks, extensively fringed on free parts; (6)

fingers and toes of males with lateral dermal spines, absent in females; (7) dorsal and ventral

surfaces of body and limbs with small, uniformly distributed dermal spines in males, absent in

females except in anal region; (8) in preservative, color on dorsum grayish brown with gray

blotches irregularly distributed; (9) posterior sides of legs with or without a light longitudinal

line; (10) venter finely marbled in brown and pale cream.

Description. — Size small for the genus; body trunk elongate ovoid (fig. 9); head short,

approximately as broad as long; nostrils at the tip of snout, not protuberant, and directed

anterolaterally; snout short, tip rounded in dorsal and lateral views (fig. 10 A-B); internarial

distance equal eye diameter, smaller than eye to nostril distance; canthus rostralis rounded;

loreal region oblique, slightly concave; lips not flared; eyes small, only slightly protruding;

upper eyelid width smaller than half of the interorbital space; interorbital area flat; cranial

crests and occipital fold absent; postorbital fold present; tympanum absent; upper jaw slightly

projecting beyond lower; mandible with truncate, trilobed anterior margin; tongue large,

ovoid; choanae small, rounded, widely separated; a small, subgular vocal sac present.

Arms slender, lacking tubercles and crests on forearm. Hand (fig. 10 C) not webbed in

both sexes; fingers lacking disks, extensively fringed; fingers of males with lateral dermal

spines, absent in females; finger length I < II < IV < III; subarticular tubercles well developed,

rounded; supernumerary tubercles absent; palmar tubercle large, divided in two parts; thenar

tubercle large, rounded, at the base of finger I.

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 63

Fig. 9. - Dorsal and ventral views of Chiasmocleis capixaba sp. nov. (MNRIJ 17514, holotype).

Legs short, robust; knee and heel lacking tubercles; tibial and tarsal ridges absent. Foot

of male (fig. 10 D) extensively webbed, of female webbed only at the base of toes; toes lacking

disks and fringed on free parts; toe length I < II < V < III < IV; toes with lateral dermal spines

in males, absent in females; subarticular tubercles well developed, rounded; supernumerary

tubercles absent; an oval inner, but no outer, metatarsal tubercle. Thigh length slightly longer

than tibia length in males, and slightly less in females; knee and elbow widely separated with

limbs adpressed to sides of body; combined thigh and tibia length approximately 79 % of

snout-vent length in males, 74 % in females; heels slightly superposed when flexed legs held at

right angles to body; foot length approximately 66 % of snout-vent length in males, 59 % in

females.

Skin smooth above and beneath; dorsal and ventral surfaces of body and limbs with

small, uniformly distributed dermal spines in males, absent in females except in anal region.

Anal opening not modified, lacking para-anal tubercles and glands around anus.

In preservative (70 % ETOH), color on dorsum grayish brown with gray blotches

irregularly distributed; loreal region dark gray; a light longitudinal mid-dorsal line on body

and a similar line on the posterior sides of thighs present or absent. Ventral surfaces of body

and limbs finely marbled in brown and pale cream; male throat infuscated.

Measurements of holotype. - SVL 15.5; HL 5.0; HW 5.2; IND 1.0; END 1.3; ED 1.4; UEW

1.0; IOD 1.8; THL 6.4; TL 6.6; FL 10.3.

Variation. - Variation in measurements is presented in tab. 1. Females are larger than males,

With interdigital webbing less developed. The light mid-dorsal longitudinal line on body and

the similar line on posterior surfaces of thighs are observed in approximately half of the

examined specimens. These lines appear associated with a light line on ventral surfaces of

arms and pectoral region, where it assumes a V-shape.

Source : MNHN, Paris

64 ALYTES 15 (2)

Fig. 10. - Chiasmocleis capixaba sp. nov. (MNRIJ 17514, holotype). Dorsal (A) and ventral (B) views of

head. Hand (C). Foot (D).

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 65

Etymology. - The name of the species, a native Brazilian word here utilized as a noun in

apposition, refers to the occurrence of the species as an inhabitant of the state of Espirito

Santo.

Geographical distribution. —- Known only from the state of Espirito Santo in southeastern

Brazil (fig. 4).

Remarks. — Scattered specimens of C. capixaba were collected in pitfall traps installed in

lowland forests, at 60 m above sea level. Explosive reproduction, when numerous male and

female specimens congregate, was observed in temporary ponds inside the forest.

Chiasmocleis carvalhoi sp. nov.

Holotype. - MNRJ 17505, adult male, collected at the Horto Florestal de Santa Cruz,

municipality of Seropédica (22°44’S, 43°43/W,; ca. 40 m altitude), state of Rio de Janeiro,

Brazil, on 31 January 1995, by C. A. G. DA CRUZ, L. KRAUSE and G. VINCIPROVA.

Paratopotypes. - MNRJ 17506-17513 and 17565, collected with the holotype; MNRJ 17480-

17504, on 2 December 1968, by E. IZECKSOHN, S. T. ALBUQUERQUE and J. SAMARÂO; EI

2096-2235, on 21 February 1964, by J. JM and E. IZECKSOHN; EI 2236-2307, on November

1964, by J. JIM, S. T. ALBUQUERQUE, W. F. MENDONÇA and E. IZECKSOHN; EI 2308-2399, on

December 1964, by J. JIM, S. T. ALBUQUERQUE, W. F. MENDONÇA and E. IZECKSOHN; EI

4381-4389, on December 1965, by S. T. ALBUQUERQUE, W. F. MENDONÇA, A. LEBEDENCO, J.

JmM and E. IZECKSOHN; EI 4391-4393, on 19 October 1966, by S. T. ALBUQUERQUE, W. F

MENDONÇA and E. IZECKSOHN; EI 5332, on 17 September 1975, by E. IZECKSOHN and J. G.

Si va; EI 8937-8938, on 30 November 1979, by I. FERREIRA.

Diagnosis. — À small sized species of Chiasmocleis diagnosed by the following combination of

characters: (1) SVL 15.5-18.3 mm in males, 16.0-22.5 mm in females; (2) body trunk ovoid; (3)

snout short, tip rounded in dorsal and lateral views; (4) hand and foot not webbed in both

sexes; (5) fingers and toes lacking disks, fringed; (6) fingers and toes with lateral dermal spines

in males, absent in females; (7) dorsal and ventral surfaces of body and limbs with small,

uniformly distributed dermal spines in males, absent in females except in anal region; (8) in

preservative, color on dorsum brown with small white dots irregularly distributed; (9)

posterior sides of legs with or without a light longitudinal line; (10) venter finely marbled in

brown and pale cream.

Description. — Size small for the genus; body trunk ovoid (fig. 11); head short, slightly broader

than long; nostrils at the tip of snout, not protuberant, and directed anterolaterally; snout

short, tip rounded in dorsal and lateral views (fig. 12 A-B); internarial distance equal eye

diameter, smaller than eye to nostril distance; canthus rostralis rounded; loreal region

oblique, slightly concave; lips not flared; eyes small, slightly protruding; upper eyelid width

smaller than half of the interorbital space; interorbital area flat; cranial crests and occipital

fold absent; postorbital fold present; tympanum absent; upper jaw slightly projecting beyond

lower; mandible with truncate, trilobed anterior margin; tongue large, ovoid; choanae small,

rounded, widely separated; a small, subgular vocal sac present.

Source : MNHN, Paris

66 ALYTES 15 (2)

Fig. 11. - Dorsal and ventral views of Chiasmocleis carvalhoi sp. nov. (MNRJ 17505, holotype).

Arms slender, lacking tubercles and crests on forearm. Hand (fig. 12 C) not webbed in

both sexes; fingers lacking disks, fringed; fingers of males with lateral dermal spines, absent in

females; finger length I < II < IV < III; subarticular tubercles well developed, rounded;

supernumerary tubercles absent; palmar tubercle large, divided in two parts; thenar tubercle

small, rounded, at the base of finger I.

Legs short, robust; knee and heel lacking tubercles; tibial and tarsal ridges absent. Foot

(ig. 12 D) not webbed in both sexes; toes lacking disks, fringed; toe length I < II < V < III <

IV; toes with lateral dermal spines in males, absent in females; subarticular tubercles well

developed, rounded; supernumerary tubercles absent; an oval inner, but no outer, metatarsal

tubercle. Thigh length less than tibia length; knee and elbow widely separated with limbs

adpressed to sides of body; combined thigh and tibia length approximately 82 % of snout-

vent length in males, 79 % in females; heels slightly superposed when flexed legs held at

right angles to body; foot length approximately 66 % of snout-vent length in males, 62 % in

females.

Skin smooth above and beneath; dorsal and ventral surfaces of body and limbs with

small, uniformly distributed dermal spines in males, absent in females except in anal region.

Anal opening not modified, lacking para-anal tubercles and glands around anus.

In preservative (70 % ETOH), color on dorsum brown with small white dots irregularly

distributed; a light longitudinal mid-dorsal line on body, and a similar line on the posterior

sides of thighs, present or absent. Ventral surfaces of body and limbs finely marbled in brown

and pale cream; male throat infuscated.

Measurements of holotype. - SVL 17.3; HL 5.6; HW 5.3; IND 1.3; END 1.6; ED 1.5; UEW

1.0; IOD 2.2; THL 7.2; TL 7.4; FL 11.6.

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 67

Fig, 12. - Chiasmocleis carvalhoi sp. nov. (MNR 17505, holotype). Dorsal (A) and ventral (B) views of

head. Hand (C). Foot (D).

Source : MNHN, Paris

68 ALYTES 15 (2)

Variation. — Variation in measurements is presented in tab. 1. A light mid-dorsal longitudinal

line on body and a similar line on posterior surfaces of thighs are observed in some specimens.

These lines appear associated with a light line on ventral surfaces of arms and pectoral region,

where it assumes a V-shape.

Etymology. - The name of the species honors the late Prof. Antenor Leitäo DE CARVALHO

(1910-1985), an outstanding teacher, naturalist, and herpetologist.

Geographical distribution. - Known from the states of Rio de Janeiro and Säo Paulo in

southeastern Brazil (fig. 4).

Remarks. — Specimens of C. carvalhoi were collected in lowland forests, at 40 m above sea

level. Explosive reproduction, when numerous male and female specimens congregate, was

observed in temporary ponds inside the forest. BOGART & NELSON (1976) referred to a diploid

number of 24 chromosomes for specimens identified as C. bicegoi, collected at Itaguai, state of

Rio de Janeiro. This reference applies to C. carvalhoi.

DISCUSSION

Species of Chiasmocleis occurring in the Atlantic Rain Forest are readily separated in two

morphologically distinct groups. The first grouping involves C. leucosticta and C. capixaba,

characterized by the presence of well developed webbing on the feet. The second grouping

includes C. schubarti, C. atlantica, and C. carvalhoi, with no or only vestigial web on the feet.

Chiasmocleis leucosticta is larger than C. capixaba (see tab. 1), and males of the former

have a fully webbed hand instead of the absence of webbing observed in C. capixaba. The

latter species has a finely marbled venter with males presenting numerous, conspicuous lateral

dermal spines on digits, whereas C. leucosticta presents a roughly marbled venter and small

dermal spines on digits. The geographical distribution of these two species is largely disjunct

(see fig. 4).

The striking webbing observed in C. leucosticta and C. capixaba is found in only one

other species of the genus, C. anatipes, described by WALKER & DUELLMAN (1974) from Santa

Cecilia, Ecuador. As noted by these authors, in the event that generic distinction should be

desirable for the fully foot-webbed species, the name Nectodactylus Miranda-Ribeiro, 1924

(type-species N. spinulosus, a junior synonym of C. leucosticta) is available.

In the second grouping, C. carvalhoi is smaller than C. schubarti and C. atlantica (see

tab. 1), and presents a finely marbled venter instead of the roughly marbled venter found in the

other two species. Chiasmocleis schubarti presents the snout rounded in dorsal and lateral

views, and C. atlantica has the snout truncate in dorsal and slightly protruding in lateral views.

Moreover, males of C. atlantica have numerous, conspicuous lateral dermal spines on digits,

whereas C. schubarti presents few, small lateral spines on those digits. Chiasmocleis atlantica

and C. carvalhoi are sympatric, and both are disjunctly distributed in relation to C. schubarti

and C. leucosticta. On the other hand, C. schubarti is sympatric with C. capixaba, and largely

disjunct from C. leucosticta (see fig. 4).

Source : MNHN, Paris

Tab. 1. - Measurements of five species of the genus Chiasmocleis Méhely, 1904 from the Atlantic Rain Forest of Brazil: Chiasmocleis leucosticta

(Boulenger, 1888); Chiasmocleis schubarti Bokermann, 1952; Chiasmocleis atlantica sp. nov.; Chiasmocleis capixaba sp. nov.; Chiasmocleis

carvalhoi sp. nov. n, number of specimens; x, mean; s, standard deviation.

Chiasmocleis leucosticta Chiasmocleis schubarti Chiasmocleis atlantica Chiasmocleis capixaba Chiasmocleis carvalhoi

(Boulenger, 1888) Bokermann, 1952 Sp. nov. Sp. nov. sp. nov.

Characters| Range x s Range CT Range x s Range x s Range x 5

SVL 19.0-22.8| 20.0 | 1.26 |19.2-26.3| 23.5 | 1.87 |22.0-25.0| 23.1 | 0.80 |14.7-16.5 0.58 |15.5-18.3| 17.1 | 0.76 ©

HL 5.4-7.0 6.0 5.4-8.1 6.8 0.65 0.42 | 4.3-6.0 5.2 0.46 4

HW 5.3-6.5 5.8 6.8-8.4 7.4 0.41 0.21 | 4.4-6.1 5.3 0.40 Li

IND 1.2-1.6 LS 1.2-2.0 1.6 0.21 0.14 | 1.0-1.4 13 0.14 E

END 1.6-2.0 17 1.4-2.3 1.9 0.23 0.22 | 1.423 1.7 0.23 a

ED 1.3-1.6 LS 1.0-1.5 Le 0.22 0.16 | 1.0-1.5 13 0.16 >

UEW 0.6-1.0 0.9 1.0-1.2 1.0 0.06 0.18 | 0.8-1.0 0.9 0.10 a

I0D 2.0-2.8 2.4 2.6-3.3 2.9 0.18 0.19 | 2.0-2.3 2.1 0.11 Ë

THL 7.8-9.1 8.3 9.0-10.7 | 9.9 0.46 0.48 | 6.0-7.3 6.8 0.41 8

TL 7.8-9.6 8.8 9.8-11.4 | 10.6 | 0.42 0.58 | 6.6-7.8 73 0.32 É

FL 11.9-15.6| 13.4 11.7-17.8| 16.5 1.31 10.0-12.6 F2

Females n=15 n= È

Characters| Range x Range % s Range x s Range x | d &

SVL |21.8-25.5| 23.6 30.0-31.8| 30.6 | 1.04 |17.9-21.7| 20.2 | 1.01 |16.0-22.5| 20.2 | 1.93 5

HL 5.7-7.5 6.8 6.6-7.8 73 0.61 | 4.4-6.7 5.7 0.64 | 4.2-6.6 5.5 0.66

HW 5.7-7.4 6.4 8.1-8.2 8.1 0.06 | 5.4-6.2 5.7 0.22 | 5.0-6.4 5.6 0.40

IND 1.3-2.0 1.6 1.8-2.4 24 0.31 1.0-1.4 12 0.15 1.2-1.6 1.4 0.15

END 1.7-2.2 1.9 1.9-2.4 2.1 0.25 | 1.4-1.8 1.6 0.10 | 1.4-2.0 1.8 0.20

ED 1.4-2.0 1.6 1.6-2.0 1.8 0.21 1.0-1.9 13 0.21 1.0-2.0 1.4 0.30

UEW 0.7-1.3 1.0 1.1-1.4 13 0.15 | 0.7-1.0 0.9 0.09 | 0.8-1.0 1.0 0.07

I0D 2.5-3.0 2.7 3.6-3.7 3.7 0.06 | 2.0-2.9 2.4 0.20 | 2.1-3.0 2.4 0.28

THL 9.0-10.5 | 10.0 11.5-12.3| 11.8 | 0.44 | 6.7-8.0 3 0.43 | 6.2-9.1 7.6 0.73

TL 9.3-11.0 | 10.3 2 x 11.8-12.8| 12.3 | 0.50 | 6.4-8.3 7.5 0.61 | 6.5-9.1 7.9 0.70

15.1-17.4 14.1-20.7 20.0-21.2 10.6-12.8 9.9-14.2

Source : MNHN, Paris

70 ALYTES 15 (2)

RÉSUMÉ

L'étude taxinomique des espèces du genre Chiasmocleis de la Forêt Atlantique brési-

lienne est presentée. Chiasmocleis bicegoi Miranda-Ribeiro, 1920 est mise en synonymie de C.

albopunctata (Boettger, 1885), une espèce distribuée hors des limites géographiques considé-

rées dans le présent travail. La synonymisation de C. urbanae Bokermann, 1952 avec C.

leucosticta (Boulenger, 1888) est confirmée. Une description détaillée de C. leucosticta et de C.

schubarti Bokermann, 1952 est fournie. Trois nouvelles espèces sont décrites.

RESUMO

O estudo taxonômico das espécies do gênero Chiasmocleis que ocorrem na Floresta

Atlântica do leste do Brasil é apresentado. Chiasmocleis bicegoi Miranda-Ribeiro, 1920 é

sinonimizada com C. albopunctata (Boettger, 1885) e considerada fora da delimitaçäo geo-

gräfica abrangida pelo presente trabalho. A sinonimizaçäo de C. urbanae Bokermann, 1952

com C. leucosticta (Boulenger, 1888) é reafirmada. Chiasmocleis leucosticta e C. schubarti

Bokermann, 1952 säo redescritas e três espécies novas sâo descritas.

ACKNOWLEDGMENTS

We acknowledge Célio F. B. HappaD (CFBH), Renato N. FE10 (UFV), Ana Maria R. Cosra and

Paulo E. VANZOLINI (MZUSP), and Sérgio P. DE CARVALHO E SILVA (SPCS) for the loan of specimens

under their care; Paulo Roberto NASCIMENTO, for the drawings, and Cässia Satie Y. MURAMATSU, for

the measurements of specimens; José P. PoMBAL Jr. and Hussam ZAHER, for critically reviewing the

manuscript. Aracruz Celulose kindly permitted the searching for specimens in the preserved forests under

its care. The authors were partially supported by CNPq (Conselho Nacional de Desenvolvimento

Cientifico e Tecnolégico).

LITERATURE CITED

Boerroer, O., 1885. - Liste von Reptilien und Batrachiern aus Paraguay. Z. Naturw, 58: 213-248.

BOGART, J. P. & NELsoN, C.E., 1976. - Evolutionary implications from karyotypic analysis of frogs of the

families Microhylidae and Rhinophrynidae. Herpetologica, 32 (2): 199-208.

BOKERMANN, W. C. A., 1952. - Microhylidae da coleçäo do Departamento de Zoologia (Amphibia —

Anura). Papéis Avulsos do Departamento de Zoologia, Säo Paulo, 10 (16): 271-292.

—— 1966. — Lista anotada das localidades tipo de anfibios brasileiros. Serviço de Documentaçäo, Reitoria

da Universidade de Säo Paulo: 1-183.

BouLENGER, G. À., 1888. — À list of batrachians from the province Santa Catharina, Brazil. Ann. Mag.

nat. Hist., (6), 1 (6): 415-417.

CarvaLHo, À. L., 1954. — A preliminary synopsis of the genera of American microhylid frogs. Occ. Pap.

Mus. Zool. Univ. Michigan, 558: 1-21.

Source : MNHN, Paris

DA CRUZ, CARAMASCHI & IZECKSOHN 71

FRosr, D. R. (ed.), 1985. — Amphibian species of the world. A taxonomic and geographical reference.

Lawrence, Allen Press, Inc., and The Association of Systematics Collections: i-v +1-732.

KAsAHARA, S. & HaDDAD, C. F B., 1997. - Karyotypes of two Brazilian microhylid frogs of the genus

Chiasmocleis, including a new case of polÿploidy. J. Herp., 31 (1): 139-142.

MÉHELY, L. VON, 1904. — Investigations on Paraguayan batrachians. Ann. Mus. nat. hungarici, 2: 207-232,

1 pl.

MIRANDA-RIBEIRO, A., 1920. — Os engystomatideos do Museu Paulista (com um genero e tres especies

novos). Rev. Mus. Paulista, 12 (2): 281-288, 2 pl.

ee 1924. — De Batrachorum generibus speciebusque duobus in collectio Musei Nationalis servatis. Bol.

Mus. nac. Rio de Janeiro, 1 (4): 255-257.

PARKER, H. W., 1934. - À monograph of the frogs of the family Microhylidae. London, Trustees of the

British Museum: i-viii + 1-208.

WALKER, C. F. & DUELLMAN, W. E., 1974. - Description of a new species of microhylid frog, Chiasmo-

cleis, from Ecuador. Occ. Pap. Mus. nat. Hist. Univ. Kansas, 26: 1-6.

APPENDIX 1

LIST OF SPECIMENS EXAMINED

Chiasmockeis leucosticta. - BRAZIL: Säo Paulo: Casa Grande (MNRJ 17564); Ribeiräo Branco

(MNRIJ 17900-17904; CFBH 268, 2136, 2229-2232, 2234, 2236-2237, 2239-2243, 2245-2247, 2251-2652;

EI 8957-8958); Ilha de Säo Sebastiäo (MZUSP 9033, holotype of Chiasmocleis urbanae); Santa Catarina:

Corupä (MNRIJ 525, 5582, syntypes of Nectodactylus spinulosus); Santa Luzia e Araujos (MNRJ 17563).

Chiasmocleis schubarti. - BRAZIL: Espirito Santo: Linhares, Cérrego Juncado (MZUSP 2309,

holotype); Linhares, Sooretama (EI 2095; MNRJ 17548); Santa Teresa (EI 8939); Aracruz (MNRJ

17538, 17539, 17542-17545, 17546, 17547, 17896-17899; CFBH 2667, 2703-2704, 2710-2713; EI 8959-

8960); Minas Gerais: Marliéria, Parque Estadual do Rio Doce (MNRJ 17883-17890; UFV 2600-2601,

2603-2608, 2610, 2612-2615, 2617-2618).

Chiasmocleis atlantica. - BRAZIL: Rio de Janeiro: Nova Iguaçu, Tinguä (MNRJ 17550, holotype;

MNRIJ 17549, 17551-17554, paratypes; EI 8940-8954, paratypes); Sao Paulo: Ubatuba, Picinguaba

(SPCS 5605-5607).

Chiasmocleis capixaba.- BRAZIL: Espirito Santo: Aracruz (MNRIJ 17514, holotype; EI 8955-8956,

paratypes; MNRJ 17515-17529, 17532-17534, 17535-17537, 17891-17895, paratypes; CFBH 2668-2669,

2685, 2693-2695, 2701-2702, 2714, paratypes).

Chiasmocleis carvalhoi. - BRAZIL: Rio de Janeiro: Seropédica, Horto Florestal de Santa Cruz

(MNRJ 17505, holotype; MNRJ 17480-17504, 17506-17513, 17565, paratypes: EI 2096-2235, 2236-2307,

2308-2399, 4381-4389, 4391-4393, 5332, 8937-8938, paratypes); Arraial do Cabo, Ilha de Cabo Frio

(MNRJ 17555-17562); Duque de Caxias (MNRJ 17566-17567, 17568-17571, 17572, 17573-17575,

17576); Niterôi (MNRJ 17577, 17578); Nova Iguaçu, Tinguä (EI 4378, 4379, 4380, 4390, 4394); Sao

Paulo: Ubatuba (CFBH 1322, 1571); Ubatuba, Picinguaba (SPCS 5608-5610, 5612-5613, 5617, 5620-

5621).

Corresponding editor: W. Ronald HEYER.

Source : MNHN, Paris

Alytes, 1997, 15 (2): 72-90.

Ervthrocyte size and ploidy determination

in green toads (Bufo viridis complex)

from Middle Asia

Matthias Srôck and Wolf-Rüdiger GROSE

Institut für Zoologie, Martin-Luther-Universität Halle-Wittenberg, Domplatz 4, 06108 Halle/Saale, Germany

The sizes of erythrocytes in blood smears of green toads (Bufo viridis

complex) from diploid and tetraploid Middle Asian populations (Iran,

Turkmenistan, Uzbekistan and Kyrgyzstan) were compared by measuring

their projection areas. Significant differences in the average values of

ervthrocyte areas between diploid and tetraploid toads were found. In some

populations the values are in the overlapping range between both levels of

ploidy. Ervthrocyte areas variability, in relation to age, body size and

altitude of the habitat, are discussed in the light of literature data on green

toads and other polyploid anurans. The staining of erythrocytes with the

Feulgen reaction and the microdensitometrical determination of the DNA

content are described and considered to be an unequivocal method which

does not require to kill the animals.

INTRODUCTION

Middle Asia (for ecogeographical definition, see WALTER & BRECKLE, 1986: 233, 275) is

inhabited by diploid and tetraploid green toads (Bufo viridis complex; for taxonomic survey,

see: ROTH, 1986; KUZMIN et al., 1988; STôCK, 1995, 1997). In order to name diploid and

polyploid forms from Middle and Central Asia which are all quite closely related to Bufo

viridis, we use the term “ Bufo viridis complex”. This excludes some species (e.g., Bufo calamita

or Bufo surdus) belonging to INGER’s (1972) “ Bufo viridis group”, which was defined without

information about the existence of polyploid forms. Since the discovery of tetraploid forms

(Mazxk et al., 1976), the ploidy determination in these toads was based first on cytogenetic,

cytophotometric and electrophoretic investigations, and concerned therefore only a quite low

number of animals from each locality (BACHMANN et al., 1978; PISANETS, 1978; TOKTOSUNOV,

1984; KrJUKOV et al., 1985; BorkiN et al., 1986a-b, 1995; ORLOVA & UTESEV, 1986; ROTH &

RAB, 1986, 1987; Wnu & ZHAO, 1987; KUDRJAVCEV et al., 1988; KUZMIN et al., 1988;

MEZZERIN & PISANETS, 1990, 1995a-b; PISANETS, 1991, 19924-b). On the other hand, in

addition or exclusively, external morphological characters (which are sometimes misleading:

RoTH, 1986; Srôck, 1995, 1997) or form and size of the clutch (a questionable character too:

KUZMIN, 1995: 94; STück, unpublished) were used when the laborious methods mentioned

above had to be limited to a few specimens or to avoid killing animals (ATAEV, 1987; PISANETS,

1987, 1992a-b; KUZMIN et al., 1988; TormasTOv, 1989; MEZZERIN & PISANETS, 1990; MEYER,

1991).

Source : MNHN, Paris

Srôck & GROBE 73

The aim of the present study was to develop additional methods for ploidy determination of

many animals and suitable in field examinations in order to improve our knowledge about

diploid and tetraploid green toads. Two methods were explored in this respect: (1) the simple

microscopic measurement of projection areas of erythrocytes; (2) the microdensitometric

measurement of DNA amounts.

MATERIAL AND METHODS

SPECIMENS

Samples were taken from Middle Asian animals in four diploid (n = 36) and six tetraploid

populations (n = 98; see tab. 1, fig. 1) in spring 1993, 1994 and 1995 and autumn 1993 (for

comparison, 13 animals from Halle/Saale, Germany, were used; permission by District

Presidium Halle, 18.01.95). In these populations other investigations, such as morphological,

bioacoustical and karyological analyses, were made (STôck, 1995, 1997, 1998). The skin of

the tip of the fourth digit of each toad was scratched with a scalpel. This intervention seems

to be of a little damage for an animal: in terrarium we observed the normal feeding of a toad

as soon as a few seconds after manipulation and the wound was closed in one or two days.

A small blood dose was used to make a blood smear on a clean microscopic slide (a single

sample from each toad); in method (2) below, a “control smear”’ from an animal of known

ploidy was made on the other end of the slide. The blood smears must not be polluted with

skin mucus.

METHODS

Choice of methods

(1) In the past, the measurement of the size of cell nuclei took a leading part in the

determination of polyploid individuals and species in Amphibia (reviews in: WEI8, 1954;

HERTWIG et al., 1958). Moreover, relatively early, the empirically known ratio of genome size

(DNA amount) to cell size was used for identification of polyploid amphibians, and erythro-

cytes were favoured because of their simple shape (reviews: GÜNTHER, 1977; AUSTIN &

BoGarT, 1982). Until today, some comparative studies (OLMO & MoREsCALCHI, 1975, 1978;

KURAMOTO, 1981; HORNER & MACGREGOR, 1983) verified that, both in Urodela and in Anura,

there is a direct linear correlation between the DNA content and the nuclear volume, cell

volume and cell surface of erythrocytes. BACHMANN et al. (1978) and KUDRJAVCEV et al.

(1988) referred to greater cells of polyploid Middle Asian green toads, but with no statistical

demonstration. HERRMANN (1989) suggested a micromorphological discrimination of diploid

and tetraploid green toads using Raster Electron Microscope for distinguishing surface

structures of the skin, but this author did not exactly determine the ploidy of his specimens.

Source : MNHN, Paris

74 ALYTES 15 (2)

Tab. 1. - Populations, numbers (n) and localities of specimens of green toads (Bufo viridis

group) investigated.

Population

Ploidy Abbreviation Localities of the populations,

(diploid: 2n=22 | usedin fig. 1 altitudes, dates of investigations

tetraploid: 4 n = 44)

38°37°N 56°38'E, Turkmenistan, Kysyl-Arvatskii Rayon,

Kopet-Dag Mountains, valley approx. 25 km SE the station, S

ofthe pass, alt. 750 m, March 1994

Bacharden 38°14'N 57°3L'E, Turkmenistan, Ashchabadskaya Oblast,

2n=22 approx. 10 km W Kelyata, alt. 500 m, April 1994

NE Iran 37°38°N 55°29'E, frontier district near Turkmenistan, approx.

2n=22 50 km NE Gonbad-e-Kävüs, alt. 250 m, April 1994

Bishkek 42°53N, 74°46'E, Kyrgyzstan, Bishkek, Botanical Garden,

2n=22 September 1993

pue 39°06°N 55°06'E, Turkmenistan, Ashchabadskaya Oblast’,

: 7 stream 2-4 km SE and warm spring approx. 4 km SE, alt.

LAB 200 m, April 1994

Bol'shoi Balkhan 39%43°N 54°29'E, Turkmenistan, Nebit-Dagskii Rayon,

4n=44 Bol’shoi Balkhan Mountains, northern slope, approx. 15 km S

(and 2 n = 22?) Oglanly village, alt. 500 m, April 1994

40°35°N 66°30'E, Uzbekistan, Dzhizakhskaya Oblast”, Rayon

PR Farish, Nuratau Nature Reserve, northern slope, alt.

DT 300-1600 m, May 1995

nn 41°16'N 69°13'E, Uzbekistan, alt. 450 m, April 1995

Chatkal 41935°N 70°07'E, Uzbekistan, 80 km E Tashkent, Chatkal

4n=44 Nature Reserve, 5 km E Burchmulla, alt. 900 m, April 1995

Issyk-Kul” i 42°29°N 76°20'E, Kyrgyzstan, northern bank near village

4n=44 Sary-Kamysh, alt. 1670 m, April 1995

Bami

2n=22

Source : MNHN, Paris

> ;

4 Kashgar He

rs LS

-Gissarskiy

fr”

——— &

Kabul

Fig. 1. - Localities of the populations of green toads (Bufo viridis complex) investigated. Letters: see tab. 1. Map by Katrin ScHneper & Matthias

Srôck, modified from WALTER & BRECKLE (1991: 273). For spelling of geographical terms, see ANONYMOUS (1993).

Source : MNHN, Paris

3J0UD HIQLS

Tab. 2. - Average values of erythrocyte areas from populations investigated and of exemplary specimens and larvae which were

karyologically investigated (counting of chromosomes). Numbers of toads involved in microdensitometry. For comparison a

diploid population from central Europe (Halle/Saale) was added. *, values in the overlapping range between diploid and

tetraploid populations.

Tab. 3. - LSD test. Significance level:

Population D NElran |Bacharden| Bami | Bishkek re Danata FE. Issyk-Kul”

(altitude) aiom | C50m) | (00m) | (50m) | @50m) | Éo6m) | 20m) | :690m) (1600 m)

Ploidy 2n=2/|2n=-2|2n-2|2n-2|2n-2 dogs 4n=4|4n=44 4n=4

n of toads (blood smears) E 9 3 3 1 E 17 36

Average erythrocyte projection

areas (um):

- of populations 231.89 | 26448 | 243.79 | 283.49 | 1279.78] | 305.80+ | 34012 | 334.81

- of toads karyologically 292.49% | 330.55 | 311.14*

investigated is 2049 | à 283.64 | 27978 | 3526 | 36794 | 31728

n of toads involved in F = * a > : E 2

microdensitometry

n of larvae karyologically 2 E 2 2 E u 2 3

investigated

Halle

NE Iran *

Bacharden Q

Bishkek

Bami * +

B. Balkhan Q * * +

Tashkent + + + + +

Chatkal + + + + + +

Nuratau + + + + + +

Danata + + o + + +

Tssyk-Kul + + + o + F + +

Source : MNHN, Paris

STôck & GROBE 77

(2) “The use of a scanning and integrating microdensitometer that is capable of making

large numbers of accurate and reproducible readings from Feulgen-stained nuclei is probably

the best way of determining the DNA content of cell nuclei”? (HORNER & MACGREGOR, 1983),

but time and condition of the main influencing steps of the Feulgen reaction (fixation and

hydrolyses) “must be determined experimentally at the start of each and every programme of

microdensitometry (...) for different tissues”” (MACGREGOR & VARLEY, 1983: 233).

Description of methods

(1) Following GÜNTHER (1977) and PoLLS PELAZ & GRAF (1988), the blood smears were

only air dried. The microscopically visible projection areas of 30 or 50 randomly chosen,

normally shaped erythrocytes were measured directly. We used the image analyses system

CYDOK (Fa. Hilgers, see below) in combination with a transmission light microscope

(ZETOPAN, Reichert, Vienna; 25 X enlarging objective) in the Hautklinik at the Martin-

Luther-University. The minimum, maximum, average value and standard deviation of the

areas were calculated. Photographs were taken using Orowopan 25 (15 DIN).

(2) Some air dried blood smears from field investigations which were stored more than

one year and some few fresh blood smears (diploid: n = 5; tetraploid n = 5; tab. 2) from animals

living in a terrarium were fixed according to HORNER & MACGREGOR (1983) for 10 min in

fresh, ice cold methanol/glacial acetic acid (3/1). Slides were then processed through the

Feulgen reaction using the method of Swirr (1955). Hydrolyses for 18 min in 5 M HCI (Merck

109911) at 18°C, staining in Schiffs reagent (Merck 9034) for 90 min and washing in several

changes of sulphite rinse (10 mi 1 N HCI + 10 ml 5 % solution of Natriummetabisulphite

+ 180 ml water) followed respectively HORNER & MACGREGOR (1983) and MINZUNO &

MACGREGOR (1974). The stained preparations were dehydrated carefully through 95 % and

100 % ethanol and xylene and then mounted in a synthetic mountant and covered with

coverglasses. One or two hundred nuclei were measured, in comparison with the samples of

known ploidy used as a standard. We used the image analyses system CYDOK (Fa. Hilgers)

and a transmission light microscope with a 40 X enlarging objective at a wavelength of 539

nm. The microscopic picture is transmitted life to a screen (enlarging factor approximately

1800 X) via video-camera (effective size of the picture 420 X 420 pixels). The Feulgen dye

content (the DNA amount) of a nucleus randomly chosen by the operator (mouse click) was

determined by measuring the integrated optical density in the area covered by this nucleus.

RESULTS

ERYTHROCYTE AREAS

Erythrocyte areas in toads of both levels of ploidy

Erythrocytes are easily distinguishable from other blood cells by their ellipsoid shape.

KHaALIL & ELFEKY (1986) provided a detailed description of blood cells morphology in Bufo

viridis from Egypt.

Source : MNHN, Paris

ALYTES 15 (2)

lea

Maximum

Ideviation

Minimum

1Z=N

pioidese])

ANS]

1 S=N

{pioidese)

LT]

L 9=N

(pioidene})

AuoyuseL

L SŒ=N

{pioidesey)

Ill NEENN

J LL=N

(proideser)

ejeueq

£l=N

(& proidene})

| ueuxega

£Z=N

| (proidip) eg

£=N

(poidip)

uepieyoeg

6=N

(pioidip)

(poldip)

exusig

| en

2SBESERSIRESCSSS

RRSSRRANSRRRNRESES

EEE

S1ejew1049u esenbs ul]

Fig. 2. - Minimum, maximum, standard deviation and average value (mean) of erythrocyte size in diploid and tetraploid green toad populations.

Source : MNHN, Paris

STôck & GROBE 79

The average value of all diploid Middle Asian green toads investigated was 277.64 um?

(standarddeviation, s =19.28 um?),whileintetraploidtoadsitwas338.13 pm?(s = 27.71 um?).

A Student ! test for statistical comparison of the two means showed a significant difference

with P < 0.0001. The variation range of all diploid individuals (235 um? to 318 um?), whose

number is also smaller, is distinctly smaller than that of tetraploid toads (287 um? to 418 um?);

this was especially caused by the values measured in the population Issyk-Kul’ (see below).

Figure 2 displays the average value, the standard deviation and the variation range in

each population. Average projection areas in the populations Danata, Nuratau, Tashkent and

Issyk-Kul’ in which exemplary specimens were identified as tetraploid were mostly larger than

310 um?. On the contrary, the diploid populations Bami, NE Iran and Bacharden, as well as

the diploid specimen from Bishkek, exhibited values mostly smaller than 310 um?. The

distinction of diploid and tetraploid green toads from these populations is possible with an

error rate of only about 3.3 %: among 36 diploid toads, only 1 individual displayed a value

higher then 310 um?; among 85 tetraploids, 3 had a value lower than 310 um?. The values of

the average erythrocyte area in the population Bol’shoi Balkhan were situated in the overlap-

ping range between diploid and tetraploid toads values and are therefore difficult to use for

identification. In this population, the two investigated animals were identified as tetraploid

(erythrocyte areas 292 um? and 322 pm; see tab. 2), but the occurrence of diploid or triploid

specimens in the sample could not be fully excluded (see Discussion).

Fig. 3-7.- Sections of blood smears from two diploid and tetraploid green toads. (3) Diploid individual

from NE Iran; average value of erythrocyte projection area: 258.53 um?. (4) Diploid individual from

Bishkek; 279.78 pm. (5) Tetraploid individual from Danata; 367.94 pm°. (6) Tetraploid individual

from Issyk-Kul’; 363.67 pm2. (7) Tetraploid individual from Nuratau: 311.14 m2.

Source : MNHN, Paris

80 ALYTES 15 (2)

Taking the population Bol’shoi Balkhan into consideration, there is an overlapping

range between 287 um? (minimal average value of tetraploids; n = 98) and 318 um? (maximal

average value of diploids; n = 36). This range includes the values of eight diploids from the

population Bami and of 21 tetraploids (10 from Bol’shoi Balkhan, n = 13; 6 from Nuratau,

n = 36; 2 from Tashkent, n = 6; 2 from Chatkal, n = 5; 1 from Danata, n = 17). Therefore this

method only allowed to classify unambiguously 77.8 % of diploid and 78.6 % of tetraploid

green toads. Figures 3-4 show sections of blood smears of two diploid specimens from the

populations NE Iran and Bishkek. Examples of tetraploid specimens from the populations

Danata, Nuratau and Issyk-Kul’ are to be seen in fig. 5-7.

Erythrocyte areas of the individuals

In all specimens investigated, a high variability of erythrocyte areas was found. Minima

and maxima in a sample (one sample measured from each toad) were up to 25 % above or

below the average value. However, despite the resulting fluctuation in standard deviation

(s= 25-35 um?), we consider that the measurement of 30 or 50 erythrocyte areas gives a good

representation of the erythrocytes of an individual.

Erythrocyte areas of the populations

‘When enough animals could be tested, the variation range was found to vary between

populations: from 39 pm? between minimum and maximum value in Bol’shoi Balkhan (12%

of the average value; n = 13), through 48 um? in NE Iran (18 % of the average value; n = 9),

60 um? in Bami (21 % of the average value; n = 23), 63 um? in Danata (18,5 % of the average

value; n = 17), 72 um? in Nuratau (21,5 % of the average value; n = 36), and up to 91 um? in

Issyk-Kul’ (24,3 % of the average value; n = 21; see fig. 2). Variation covers a similar range in

the diploid and tetraploid populations. Although in some cases the number of specimens is

too low to be statistically meaningful, distinct differences of the average values between some

populations can be noticed (see tab. 2). This is clear especially in the population Issyk-Kul”

whose average value of 374 um? is considerably higher than in all other tetraploid populations

studied. To compare the average values of all populations, an analysis of variance was

performed. The average value of each diploid population was significantly different from each

tetraploid population (Multiple Range test/LSD test; P < 0.05; see tab. 3). Furthermore it was

found that the average value of erythrocyte areas in the population Issyk-Kul’ is significantly

different from every other diploid and tetraploid population. While the tetraploid populations

Danata, Nuratau, Tashkent and Chatkal seem to be quite homogeneous (but sample sizes in

Tashkent and Chatkal are small), and exhibit no significant differences between each other,

the population Bol’shoi Balkhan occupies, as expected, a special position. Its average value is

different from those of the populations Danata, Nuratau and Chatkal (but not from Taskent),

and also from each diploid population (NE Iran, Bacharden, Bami). The average value of the

population Halle from Central Europe differs from each average value of the Middle Asian

populations. With 231.89 um?, it is smaller than the minimal values of the Middle Asian

populations (NE Iran: 264.48 um?; Bacharden: 243.79 um?).

Relationship between body size, age and erythrocyte size

A ratio of erythrocyte area to snout-urostyle length within a population was considered

useful for testing a dependence of the parameter on age or body size respectively. Figure 8

Source : MNHN, Paris

STôck & GROBE 81

390

"| |

. |

370 |

Average erythrocyte area (square micrometers)

ARE ERREUR à À PES

270 À ——

@Bami, 2n = 22

ADanata, 4n = 44

XNE Iran, 2n = 22

| xIssyk-Kul', 4n = 44

| ONuratau, 4n = 44

230 |

220 + :

50 55 60 65 70 75 80 85

Snout-urostyle length (mm)

Fig. 8. - Erythrocyte size and body size in two diploid and three tetraploid green toad populations.

Source : MNHN, Paris

82 ALYTES 15 (2)

shows this relation for two diploid and three tetraploid populations. Body size, although

exhibiting high variation, showed no correlation with erythrocyte size. Therefore, body size

does not affect the identification of ploidy levels of mature animals. However, the additional

investigation of some tetraploid juveniles (snout-urostyle length 3 to 5 cm) from the popula-

tion Issyk-Kul’ showed values (327, 327 and 342 um?) which were at the lower border of the

values within this population. Four juveniles (snout-urostyle length 4.5 to 5 cm) from the

population Nuratau had similarly low values (290, 306, 316 and 317 um?); a juvenile from

Danata (3.5 cm) had the second smallest value within this population.

Erythrocyte areas, sex and altitude of the habitat

There is no noticeable sexual dimorphism in this character: within the populations, the

values of the (underrepresented) females were uniformly distributed among the values of

males. No relationship was found between erythrocyte area and altitude (see tab. 2) of toad

habitats.

MICRODENSITOMETRICAL MEASUREMENTS

Stored, air dried blood smears

The nuclei in air dried blood smears which were stored without freezing some weeks or

months were not suitable for quantitative staining with the Feulgen reaction. The older the

sample was, the less it could be stained.

Fresh fixed blood smears

On the contrary, fresh fixed blood smears exhibited very exact results. Figure 9a shows as

an example the frequency distribution of the DNA amounts measured in 100 erythrocyte

nuclei of a diploid (DNA index I) specimen from the population NE Iran. Figure 9b

demonstrates the DNA amounts of 200 nuclei of a tetraploid (DNA index II) from the

population Issyk-Kul’. Since the total DNA amount of a nucleus is measured, what could in

principle result also from an aneuploid chromosome number, in such measurements the DNA

amount of a diploid nucleus is named as DNA index 1 and relative values were analysed.

DISCUSSION

ERYTHROCYTE AREAS

Method

The measurement of erythrocyte areas yields useful results for distinguishing between

diploid and tetraploid green toads. Combined with the hitherto presented morphological

characters (PISANETS, 1978; PISANETS & SCERBAK, 1979; KUZMIN, 1995) or with multivariate

analyses of morphometrical data (Srôck, 1997), the method offers new opportunities for the

determination of Middle Asian green toads. In spite of this study including only one diploid

Source : MNHN, Paris

STôck & GROBE 83

DI1

CV 5%

0 1 2 3 4 5

DNA index

Cell number

2 3

DNA index

Fig. 9. - (a) Frequency distribution of DNA amounts of 100 erythrocyte nuclei of a diploid green toad

individual (DNA index DI 1) from population NE Iran. CV, coefficient of variation (%).

b) Frequency distribution of DNA amounts of 200 erythrocyte nuclei of a tetraploid green toad

(DNA index DI 1.96) from population Issyk-Kul'’. CV, coefficient of variation (%).

Source : MNHN, Paris

84 ALYTES 15 (2)

animal from the East of the range (Bishkek, Kyrgyzstan), with reference to BACHMANN et al.

(1978) and to the DNA measurements (based on flow cytometry) by BORKkIN et al. (1986), it

can be postulated that the method can be applied also in this region, all the more that

tetraploids in this region (Issyk-Kul”) are characterised by especially large erythrocytes. The

method can help to enlarge the knowledge about distribution of diploid and tetraploid green

toads, which is desirable to clarify taxonomic problems (BoRkIN et al., 1986).

However, this study has shown that there are populations (Bol’shoi Balkhan) whose

values of erythrocyte areas are between most of the other diploid and tetraploid populations.

In such cases, or if there are specimens with average values between about 285 and 320 um?,

it is necessary to use methods such as karyological, cytophotometrical or microdensitometri-

cal analyses (see below).

There were only few records of triploid hybrids in nature, in Danata (PISANETS, 1978;

MEZZERIN & PISANETS, 1995a-b) and near Bishkek (KUZMIN 1995: 187, without reference to

the method of ploidy determination), and only few references to “mixed populations”, in

Dushanbe (ROTH & RÀB, 1986) and in East Kazakhstan (GoLUBEv, 1990, but ploidy deter-

mination in this case seems doubtful). Consequently, we first supposed that there were only

toads of one level of ploidy at each investigated locality. In none of the populations an

inconsistency was found between ploidy of adults and ploidy of larvae using karyological or

microdensitometrical methods (see tab. 2; Srôck, 1995). Among adult animals, a few had

average values of erythrocyte areas that were situated in the overlapping range between

diploid and tetraploid toads values (marked * in tab. 2). However, all these individuals had the

same ploidy as other adult toads and tadpoles of the population. So, it seems very improbable

that there were any “mixed population” among those studied.

Variability of erythrocyte areas

In the present study, the number of red blood cells investigated per individual (30 or 50)

seems to be large enough, in particular in comparison with literature (OLMO & MORESCALCHI,

1975: 15 cells per individual; GÜNTHER, 1977: 50 cells; KURAMOTO, 1981: more than 20 cells;

AUSTIN & BOGART, 1982: more than 20 cells; PoLLs PELAZ & GRAF, 1988: 10 cells; SCHRÔER,

1996: 15 cells). The standard deviations within the samples are of the same magnitude as those

presented by KURAMOTO (1981) or PoLLs PELAZ & GRAF (1988) for some other anuran species.

High variability within the cell sample from a specimen could be caused by the occur-

rence of immature erythrocytes (erythroblasts) in the peripheral blood of Amphibia (ZAPATA

et al., 1982; WELSCH & STARCK, 1986), a presence that has been shown by KHALIL & ELFEKY

(1986) to occur in green toads of the Bufo viridis complex. Such cells have a tendency to

exhibit a circular shape. Among the cells randomly chosen for measurement, this cell type was

included too. Apparently the spleen is the principal erythropoietic organ in temperate adult

anurans (DUELLMAN & TRUEB, 1994: 180), but due to the activity of the bone marrow as an

erythropoietic site during spring (DUELLMAN & TRUEB, 1994: 180), there could be seasonal

shifts in the frequency of different erythrocyte sizes. ToKTOsUNOv (1984) noted an increased

number of erythrocytes during the cool seasons (autumn, winter, spring) in diploid green

toads from Chu valley near Bishkek (Kyrgyzstan). Because of the uniform period of investi-

gations (breeding time) in our study, seasonal effects are thought to be quite constant (see

GÜNTHER, 1977). Actually, about half of the individuals from the population Issyk-Kul’ were

Source : MNHN, Paris

Srôck & GROBE 85

investigated in autumn, but the range of the values did not diverge from the values in animals

investigated in spring. Exclusively some maxima were only detected in spring.

Although the variation range of the erythrocyte areas in all polyploid individuals in the

present study is larger than in all diploids (a result similar to GÜNTHER’s, 1977, findings in

triploid Rana kl. esculenta, but in contradiction with those of PoLLS PELAZ & GRAF, 1988, in

the same klepton), a relationship between this phenomenon and polyploidy cannot be

clarified. In triploid representatives of the Ambystoma jeffersonianum complex, AUSTIN &

BoGaRT (1982) found about the same variability of the erythrocyte areas as in diploid.

Theerythrocyte measurements by HEMMER et al. (1978) in toads of unknown ploidy from

the environs of Dushanbe (Tadzhikistan) could not be compared with the data presented here.

On the one hand, these authors measured length and breadth of the ellipsoid and then

calculated the ellipse area, and on the other hand they made a Pappenheim staining which

might result in a shrivelling of the cells. That might be the reason why their values are smaller

than those presented here. Interestingly however, HEMMER et al. (1978) registered high

variability in the whole studied sample of toads (i.e. in diploid and tetraploid individuals), a

result in agreement with ours. The differences between populations are striking too in toads of

the same level of ploidy. In Israel and Greece (areas quite small compared to the giant region

taken into consideration here), an enormously high genetic variability was found in diploid

Bufo viridis (DESSAUER et al., 1975: “the highest yet reported for any vertebrate”; KARAKOUSIS

& KYRIAKOPOULOU-SKLAVOUNOU, 1995). Therefore it is not surprising to find a great pheno-

typic variety in tetraploid green toads, particularly because during evolution, polyploidy was

presumably associated with long periods of isolation between many different populations in

high mountains (see ROTH, 1986). The phenomenon seems to be confirmed by measurements

of DNA contents in erythrocytes (BORkIN et al., 1986): these authors suggested the probable

existence of three groups of diploid and two groups of tetraploid toads in Middle Asia.

Considering the positive correlation mentioned above, a direct relationship between DNA

amount and cell volume (and erythrocyte area) is probable.

Other polyploid amphibians and their differences in erythrocyte areas

Contrary to the situation in the Ambystoma jeffersonianum complex, in which

diploid and triploid individuals can be distinguished without errors using the very large

erythrocytes of the Urodela (AUSTIN & BOGART, 1982), GÜNTHER (1990: 172) and SCHRÜER

(1996) mentioned that in some populations of water frogs it is problematical to distinguish

between diploid and triploid Rana kl. esculenta as reported by GÜNTHER (1977). Artificially

produced triploid individuals of Xenopus laevis exhibited a significant difference between the

average values but an overlapping of the absolute values of the largest cell axis compared with

diploid forms (GEORGE & LENNARTZ, 1980). MAHONY & ROBINSON (1980) found clear

differences in the average values of the erythrocyte areas in the tetraploid Australian lepto-

dactylids Neobatrachus sudelli and N. sutor compared with those of diploid N. pictus; however,

this work contains no information on variation range or intrapopular variability. BOGART &

WASSERMAN (1972), using two microphotographs, demonstrated the different sizes of erythro-

cytes in the diploid/tetraploid species pair Hyla chrysoscelis/H. versicolor, which was con-

firmed by RALIN (1977) and CasH & BOGART (1978); an extensive study of erythrocyte size in

this complex has still not been published.

Source : MNHN, Paris

86 ALYTES 15 (2)

Relation to age, body size and altitude

The slight increasing of erythrocyte size during ontogenesis till adulthood, as it seems to

develop in (diploid and) tetraploid green toads, corresponds to the findings in diploid and

triploid Rana kl. esculenta (POLLS PELAZ & GRAF, 1988) whose erythrocyte areas enlarge with

increasing body size after metamorphosis. In Bufo melanostictus, CHURCH (1961; cited by

GÜNTHER, 1977) and BANERIEE (1983, 1988) observed an enlargement of erythrocyte areas

with increased body size. In Bufo spinulosus, RuIz et al. (1989) noticed a reduction of

erythrocyte area and body size with increasing altitude, whereas erythrocyte number per

volume unit was increased (the authors compared individuals from 200 to 2700 m with

individuals living above 3200 m). The authors considered this phenomenon an adaptation to

the reduced oxygen content in the air of the high mountains. Our investigations do not show

any relationship between erythrocyte size and altitude of the habitat. The investigated green

toad population living at the highest altitude (Issyk-Kul”, 1670 m) displays the highest average

value of erythrocyte area. In tetraploid green toads from this area, TOKTOSUNOv (1984) found

a higher haemoglobin content (147 g/l) than in diploid lowland populations (112 g/l) and

considered this an adaptation of the tetraploids to their mountainous life. It is still unknown

whether tetraploid green toads which inhabit high mountains regions above 3500 m (Mazik

et al., 1976; ROTH, 1986; KUZMIN et al., 1988; TorMAsTOv, 1989), and which exhibit in higher

altitudes a reduced body size (TomAsTOv, 1989), have a reduced erythrocyte area too.

MICRODENSITOMETRICAL MEASUREMENTS

DNA amounts

The DNA amount in cells of the tetraploid green toads tested is about twice the content

in nuclei of diploid toads. This result is in agreement with those of BoRKkIN et al. (1986), who

found in tetraploids an amount slightly lower than the double of diploids.

Method

This method seems to be the only procedure usable without killing or considerably

damaging the animals to determine unequivocally their ploidy. The Feulgen process cited

above (HORNER & MACGREGOR, 1983) is suitable for erythrocytes in fresh blood smears from

Middle Asian green toads. Up to now it is unknown how long blood smears could be stored

if they were fixed as mentioned before staining them with the Feulgen reaction. A test is in

preparation. If it turns possible to store blood smears some time without loss in quantitative

Feulgen staining, this method could become a key to collect data about distribution of

different ploidy types in Middle Asian green toads without killing the specimens. Such a

method would be most useful in faunistics and other field investigations.

Source : MNHN, Paris

Srôck & GROBE 87

RÉSUMÉ

Les tailles des érythrocytes de populations de crapauds verts (complexe de Bufo viridis)

diploïdes et tétraploïdes de l’Asie centrale (Iran, Turkménistan, Ouzbékistan et Kirghizstan)

sont comparées en mesurant leur surfaces de projection à partir de frottis sanguins. Les

valeurs moyennes de ces surfaces sont significativement différentes entre les deux types de

crapauds. Dans quelques populations les valeurs moyennes sont intermédiaires entre celles

des animaux diploïdes et tetraploïdes. La variabilité des surfaces des érythrocytes par rapport

à l’âge, la taille et l'altitude, est discutée à la lumière de la littérature sur les crapauds verts et

les autres anoures polyploïdes. La coloration des noyaux des érythrocytes par la réaction de

Feulgen et la détermination microdensitométrique du contenu de l'ADN sont décrites. Cette

méthode est préconisée pour obtenir des résultats clairs sans sacrifier les animaux.

ACKNOWLEDGEMENTS

We wish to thank T. KLAPPERSTÜCK (Halle) for a lot of advise, helpful suggestions and practical

experience concerning Feulgen staining and using CYDOK analyses system. We thank E. TRAUTMANN

(Halle) for histochemical assistance, Dr. R. GÜNTHER (Berlin) for advice and literature references and

Dr. E. RorrserG (Halle) for linguistic advice. Concerning karyological investigations, Prof. Dr.

M. ScHMiD and C. STEINLEIN (Würzburg) and Prof. Dr. G. REUTER (Halle) kindly helped us. The following

persons accompanied the first author and assisted him during the field work in Middle Asia: Anja

BucHowski, Kerstin HOLLANDER, Christine QUErTscH, Martin BIEDERMANN, Armin BISCHOFF, Urs

JÂGER, Stefan MICHEL and Heiner NAGEL (Halle), Natalya Yu. BESHKO (Yangikishlak, Uzbekistan) and

Rezar ZEEYAMUSAVI (Gorgan, Iran).

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Alytes, 1997, 15 (2): 91-98. 91

What is the ecological significance

of laboratory temperature selection

in anuran Amphibia?

U. KATZ, Joy HOFFMAN & Nira GIL

Department of Biology, Technion, Israel Institute of Technology, Haifa, Israel

Temperature selection by a toad (Bufo viridis), was studied in the

laboratory. This species covers a wide geographical distribution, and is

exposed to different temperatures in its natural habitats. À temperature

gradient was constructed within a 1.2 m tunnel in the laboratory, and the

toads selected a preferred temperature around 27°C, when allowed free

choice. Acclimatization to an ambient temperature of 16°C lowered the

selected temperature by some 3-4°C. As reported in the literature, many

anuran species with different climatic backgrounds share a relatively nar-

row range of selected preferred temperature in the laboratory. We suggest

that the laboratory selected temperature represents an upper set-point that

provides anurans with the information to evade high lethal temperatures.

INTRODUCTION

Despite their high vulnerability to ambient conditions, amphibians have invaded many

regions of the globe in specific niches. Because water permeability of their moist skin is high,

it demands that they avoid permanently hot and dry regions (JG&RGENSEN, 1950). Physiological

regulatory temperature control in amphibians is poor or absent (BRATTSTROM, 1979), but they

use behavioural means to select optimal temperature (HEATH, 1970). Some amphibians that

were studied in the field regulate a relatively constant temperature behaviourally

(LiLLYWHITE, 1970; SiNsCH, 1984), while some species that inhabit dry regions burrow (KATZ,

1989; PINDER et al., 1992). In other species, protective mechanisms were developed, such as

waxing or cocooning (LOVERIDGE, 1976), that greatly reduce the water permeability of their

integument (SHOEMAKER & NAGY, 1977). In either case, in hazardous environments, most

amphibians seek shelter and only a few, adaptable species, face the ambient conditions and

remain active (SHOEMAKER & NAGY, 1977; KATZ, 1989).

Bufo viridis is one of a few adaptable anurans that can be acclimatized to a wide variety

of osmotic and thermal environmental conditions (TERCAFS & SCHOFFENIELS, 1962; KATZ &

GaBBAY, 1986; KATZ & GiL, 1997). It has a wide area of distribution, extending from

temperate south Scandinavia to hot and arid regions in Israel, Egypt and north Africa. Bufo

viridis is nocturnal and is normally active throughout the year; it burrows, but only intermit-

Source : MNHN, Paris

92 ALYTES 15 (2)

tently, in conditions of water shortage (DEGANI et al., 1984; KATz & GABBAY, 1986; HOFFMAN

& KATZ, 1994). In this study we investigated the temperature selection of Bufo viridis in

laboratory conditions, and examined the effects of temperature acclimatization and dehydra-

tion on this behaviour.

MATERIALS AND METHODS

Bufo viridis from Israel (more than 20 individuals, belonging to both sexes) were used in

this study. Average weight of the toads was 34 g (ranging between 25 and 40 g), and they were

kept in the laboratory at room temperature (20-24°C) with free access to tap water. The toads

were force-fed ox liver weekly, maintaining constant weight.

Temperature selection of the toads was determined in a temperature gradient that was

produced in an opaque plastic tunnel (130 x 14 x 14 cm) heated by two blackened 75 W bulbs,

one at each end, and cooled from the outside with dry ice at about a third of the length. Thus,

an asymmetric temperature gradient was produced, with a profile that spans over nearly 15°C

as shown in fig. 1. Relative humidity in chamber did not exceed 60 %, and the tunnel was dry.

The toads were placed singly into the centre of the tunnel, and were allowed 30 min to settle.

Changes of position were recorded (through a mirror, to avoid external interference) for

several hours, and time spent at various distances from one end were calculated in relation to

ambient temperature.

The relationship between body and ambient temperatures was studied separately, in a

chamber (10 x 6 x 6 cm) that was kept in a temperature-controlled water bath. The ambient

temperature was changed stepwise, by 2-5°C each time, only after the body (and skin)

temperature was stabilized (which took no less than 30 min). Temperature was measured with

a precision of 0.1°C by a thermistor (YSI, Ohio telethermometer) that was inserted about 2

cm into the cloaca.

RESULTS

Figure 2 shows that, in laboratory conditions, body temperature in Bufo viridis

(4 specimens) was related almost linearly (in the range 5-35°C) to the ambient temperatures at

steady state. The slope of the regression line over the whole range was y = 0.88 x - 0.07; r? =

0.95 (y is body temperature in degrees centigrade and x is ambient temperature). This

relationship deviated at the higher temperatures (range 25-35°C), having a steeper regression

line (y = 1.12 x - 7.73; r° = 0.78).

Toads that were acclimatized at room temperature (ca. 22°C), selected a preferred

ambient temperature of 27-28°C (fig. 3). The toads spent most of the time in the gentler

Source : MNHN, Paris

= 31+

u 294 |

_ Li D ——+ l

F + Ÿ = F

ZE 27 VS A

œ AUS N

Li $

où -297 D —E Z

>= 3

em ÈS £

EE 255% B: A >

A IN æ

21 8

SF Fë

19- + + + +

20 40 60 80

DISTANCE (cm)

Fig. 1.- Temperature gradients along the (120 cm) tunnel, as recorded in the individual experiments (n = 13). An asymmetric gradient was created by

placing two heat sources at each end of the tunnel, and a cooling region located in the third of the distance between them. Note that the toads

selected voluntarily a narrow temperature range, indicated by the two horizontal lines.

Source : MNHN, Paris

94 ALYTES 15 (2)

Le)

Z 301 8 À

LU)

E A)

Œ 20!

Ë 8

ü Ca

8 10!

(e]

0 + + + —+— +

0 10 20 30 40

AMBIENT TEMPERATURE (°C)

Fig. 2. - Relationship between body and ambient temperature in Bufo viridis. The regression line equation

is y = 0.88 x -0.07; r? = 0.95 (5 toads acclimatized at room temperature, ca. 22°C). Weight of the

animals was 30-35g.

gradient, with about 20 % of the total time at temperatures below 27°C. As shown in fig. 1, the

selected temperature (27-28°C) occupied no more than 15 % of the total temperature profile,

including part of the steeper gradient in the tunnel. In control experiments with no tempera-

ture gradient, 3 toads that were placed individually in the tunnel explored it over its length and

stationed themselves at the ends for most of the time. No other preferred points were

observed. This preference disappeared in the presence of the thermal gradient.

Acclimatization of the toads at 16°C for over 3 weeks resulted in a shift in the distribution

pattern of the selected temperature (fig. 3), so that now 75 % of total time was spent below

27°C. The difference in the preferred temperature between the two conditions was highly

significant (P > 0.001; n = 6). Fast dehydration (by approximately 15 % gross weight in less

than a day) also changed the distribution pattern of preferred temperature, with shorter time

spent above 27°C (fig. 4).

Source : MNHN, Paris

KATZ, HOFFMAN & GiL 95

DISCUSSION

Our experiments are in accord with previous observations on other species (HUTCHISON

& DurrE, 1992, for a review) and show that when the ectothermic terrestrial anuran Bufo

viridis is allowed a free choice of temperature in the laboratory, it selects a relatively narrow

range of ambient temperatures and spends most of the time at 26-28°C. Preliminary unpub-

lished observations that we made on the African species Bufo regularis gave similar results.

Body temperature of amphibians is determined largely by external physical conditions,

including ambient temperature, relative humidity (MELLANBY, 1942; MALvIN & Woop, 1991)

and resistance to water loss (SNYDER & HAMMERSON, 1993). Their body temperature is usually

somewhat lower than ambient, due to continuous evaporation, although in a number of

montane species body temperature is maintained nearly always above ambient (LILLY WHITE,

1970; VALDIVIESO & TAMSITT, 1974; SINSCH, 1989). In natural conditions, amphibians employ

mostly behavioural means to regulate their body temperature (LiLLY WHITE, 1970); they either

bask, as e.g. in Hyla labialis (VALDIVIESO & Tamsirr, 1974), or they. shelter, to avoid

over-heating. They must optimize between conflicting demands of temperature-dependent

biochemical reactions and water balance (BRATTSTROM, 1979).

80-

60 1

e #

le]

201

x LA Lo Un

26 8 30 32

20 22 24 2

AMBIENT TEMPERATURE (°C)

Fig. 3. - Effect of temperature acclimatization on the preferred ambient temperature in Bufo viridis.

Summed results of 6 sessions. Open bars, animals acclimatized at room temperature (ca. 22°C); dark

bars, animals acclimatized at 16°C.

Source : MNHN, Paris

96 ALYTES 15 (2)

Fos

= |

z 40!

EE. |

ù [ EUR 9 Do nu DK [Le

20 22 24 26 28 30 32

AMBIENT TEMPERATURE (°C)

Fig. 4. — Effect of dehydration on preferred ambient temperature in Bufo viridis. Summed results of 4

individuals on 4 separate occasions in each condition. Open bars, acclimatized at room temperature

(ca. 22°C); dark bars, dehydrated by 15 % for 16 h at room temperature; hatched bars, dehydrated,

water bath present in the terrarium.

Selected preferred temperature seems to be an inherent property, common to many or

most anurans, and does not seem to be related to the species ecological background. Bufo

regularis, for example, is an African species that cannot survive at temperatures below 8°C

(KATZ & Gi, 1997), and yet it displayed a similar preferred temperature as did Bufo viridis,

which survives at near freezing temperature. In many species, the laboratory preferred

temperature and the temperature activity in their habitat were found to be quite removed from

each other (CLAUSEN, 1973), although in some species these temperatures met with one

another, as was found in the aquatic Rana catesbeiana (LiLLYWHITE, 1970, 1971). The many

studies of laboratory preferred temperature, performed on a large number of amphibians,

revealed a narrow range of preferred temperature (between 26 and 30°C) which was selected

by more than 75 % of adults among over 40 species of anurans that were tested (HUTCHISON

& DuPrE, 1992). Only a few species, including juveniles, chose within the range 16-25°C, while

ca. 10 % opted for 31-34°C; this was so, despite the large differences in the experimental

methods and acclimatization protocols. This is even more pronounced in the data summar-

ized by STRUBING (1954), where only two anurans (Bombina spp.) deviated from the 26-33°C

range of 34 listed species. Therefore, temperature selection in the laboratory does not reflect

the thermal conditions of natural habitats of the animals which vary widely. Rather, it

indicates an essential requirement of some fundamental importance to anurans. Some

investigators suggested that the amphibian preferred temperature could represent ectother-

Source : MNHN, Paris

KATZ, HOFFMAN & GiL 97

mic equivalence of the endothermic set-point (HUTCHISON & DuPRE, 1992), but this thesis

does not seem to be tenable.

If the selected preferred temperature is not related to the thermal background of the

animal in nature, what then could it signify? We propose that the laboratory-selected temper-

ature represents a set-point that demarcates a limit to the tolerated temperature. This is

supported by the finding that the upper lethal temperature of most toads and frogs - with the

notable exceptions of Phyllomedusa sauvagei (-40°C) and Bufo calamita (-37°C) - is 32-33°C

(Kirk & HOGBEN, 1946), much lower than in reptiles, which are also ectothermic. Although

amphibians do not possess physiological thermoregulatory mechanisms, they seem to be

equipped with a temperature set-point and with cold- and warm-sensitive neurons in the skin

(CaBanac & Jebpi, 1971; DUPRE et al., 1986). These are necessary components in any

feed-back control system, and provide information that can be used either in physiological

temperature regulation or, if not available, for activation of behavioural escape mechanisms.

Lacking effective physiological thermoregulatory mechanisms, physical activity and metabol-

ic rate in Amphibia will continue to increase as ambient and body temperatures increase.

Therefore, an upper temperature set-point is required to supply safety command to evade

lethal temperatures.

In conclusion, we found that Bufo viridis and Bufo regularis selected a similar preferred

temperature in the laboratory, although they show entirely different sensitivity to tempera-

ture. Published data and the present study indicate that, in the laboratory, many or most

anurans select a preferred temperature from within a relatively narrow range. This tempera-

ture is often far removed from, and higher than, the one encountered in the field during their

periods of greatest activity. It is suggested that the preferred temperature represents an upper

set-point that provides the animals with the information to evade high temperatures.

REFERENCES

BRATTSTROM, B. H., 1979. - Amphibian temperature regulation studies in the field and laboratory. Am.

Zool., 19: 345-356.

CaBANAC, M. & JeDDi, E., 1971. - Thermopréférendum et thermorégulation comportementale chez trois

poïkilothermes. Physiol. Behav., 7: 375-380.

CLAUSSEN, D. L., 1973. - The thermal relations of the tailed frog, Ascaphus truei, and the pacific treefrog,

Hyla regilla. Comp. Biochem. Physiol., 44A: 137-153.

DEGANI, G., SILANIKOVE, N. & SHKOLNIK, À. 1984. - Adaptation of green toad (Bufo viridis) to terrestrial

life by urea accumulation. Comp. Biochem. Physiol., T7A: 585-587.

Durre, R. K., Jus, J. J., CRAWFORD, C. & POwEzL, T. L., 1986. - Temperature preference and responses

of cutaneous temperature-sensitive neurons during bullfrog development. Physiol. Zool., 59:

254-262.

HEATH, J. E., 1970. - Behavioral thermoregulation of body temperature in poikilotherms. Physiologist,

13: 399-410.

HorrmaN, J. & KATZ, U., 1994. — Urea production and accumulation in the green toad Bufo viridis.

J Zool., London, 233: 591-603.

Hurcuison, V. H. & DUPRE, R. K., 1992. - Thermoregulation. /n: M. E. FEDER & W. W. BURGGREN (ed.),

Environmental physiology of the Amphibia, The University of Chicago Press: 206-249.

JORGENSEN, C. B., 1950. - The amphibian water economy, with special regard to the effect of neurohy-

pophyseal extracts. Acta Physiol. Scand., 22, suppl. 78: 1-79.

Source : MNHN, Paris

98 ALYTES 15 (2)

KaTz, U., 1989. — Strategies of adaptation to osmotic stress in anuran Amphibia under salt and

burrowing conditions. Comp. Biochem. Physiol., 93A: 499-503.

KATZ, U. & GaBBAyY, S., 1986. — Water retention and volume regulation in toads (Bufo viridis) under

burrowing conditions. J. comp. Physiol., 156B: 735-740.

KaTz, U. & Gui, N., 1997. - Different temperature relations of two species of toads that coexist at the

border of their geographical distribution regions. Amphibia- Reptilia, 18: 259-268.

Kirk, R. L. & HOGBEN, L., 1946. - Studies on temperature regulation. II. Amphibia and reptiles. J exp.

Biol., 22: 213-220.

LiLLYWHITE, H. B., 1970. - Behavioral temperature regulation in the bullfrog, Rana catesbeiana. Copeia,

1970: 158-168.

--- 1971. - Temperature selection by the bullfrog, Rana catesbeiana. Comp. Biochem. Physiol., 40A:

213-227.

LOVERIDGE, J. P, 1976. — Strategies of water conservation in southern African frogs. Zool. africana, 11:

319-333.

MALVIN, G. M. & Woop,S. C., 1991. - Behavioral thermoregulation of the toad, Bufo marinus: effects of

air humidity. J exp. Zool., 258: 322-326.

MELLANBY, K., 1942. - The body temperature of the frog. J exp. Biol., 18: 55-61.

PINDER, À. W., SToREY, K. B. & ULTCH, G. R., 1992. — Estivation and hibernation. /n: M. E. FEDER &

W. W. BURGGREN (ed.), Environmental physiology of the Amphibia, The University of Chicago

Press: 250-284.

SHOEMAKER, V. H. & NAGy, K., 1977. - Osmoregulation in amphibians and reptiles. Ann. Rev. Physiol.,

39: 449-471.

SinscH, U., 1984. - Thermal influences on the habitat preference and the diurnal activity in three

European Rana species. Oecologia, 64: 125-131.

ee 1989. - Behavioural thermoregulation of the Andean toad (Bufo spinulosus) at high altitudes.

Oecologia, 80: 32-38.

SNYDER, G. K. & HAMMERSON, G. A., 1993. - Interrelationships between water economy and thermore-

gulation in the Canyon tree-frog Hyla arenicolor. J arid Env., 25: 321-329.

SrruBING, H., 1954. - Uber Vorzugstemperaturen von amphibien. Z. Morph. Okol. Tiere, 43: 357-386.

TERCAFS, R. R. & SCHOFFENIELS, E., 1962. - Adaptation of Amphibia to salt water. Life Sci., 1: 19-23.

VALDIVIESO, D. & TAMSITT, J. R., 1974. - Thermal relations of the neotropical frog Hyla labialis (Anura:

Hylidae). Life Sci. occ. Pap. r Ontario Mus., 26: 1-10.

Corresponding editor: Ulrich SINSCH.

Source : MNHN, Paris

Alytes, 1997, 15 (2): 99-103. 99

Un cas de néoténie,

dans un bâtiment désaffecté,

chez le triton alpestre,

Triturus alpestris apuanus (Salamandridae)

Mathieu DENOËL

Service d'Ethologie et de Psychologie animale, Institut de Zoologie, Université de Liège,

Quai Van Beneden 22, 4020 Liège, Belgique

A population of Triturus alpestris apuanus of the Apuane Alps (Tuscany,

Italy) was studied during July 1994. The particularity of this population is that

it breeds in an artificial site, in the dark and under cover, in a deserted

building. Moreover some neotenic newts were discovered. The recent coloni-

sation and the instability of this biotope indicate a form of labile neoteny,

appearing and disappearing spontaneously.

INTRODUCTION

La néoténie (KOLLMANN, 1884) est la conservation des caractéristiques de l’état

larvaire à un stade de développement plus avancé que la normale. La néoténie totale ou

pédogenèse concerne des animaux devenus matures dans cet état, tandis que la néoténie

partielle ne s'applique qu’à un retard de la métamorphose.

La néoténie est un problème qui soulève de nombreuses questions. On peut en effet

se demander, d’une part, quelle est la cause de l'apparition d'individus néoténiques, et

d’autre part, comment ces derniers peuvent se maintenir en syntopie avec des individus

métamorphosés (et inversement). Quelques réponses ont été données à ces questions en ce

qui concerne le genre Triturus. Suite à l'observation de populations néoténiques dans des

lacs à régime stable en altitude, certains auteurs ont attribué le maintien du pédomor-

phisme (néoténie au sens large, impliquant un retard du développement de caractères

somatiques et/ou sexuels: DuBois, 1985) à une prétendue hostilité du milieu terrestre

(Wizsur & CoLLins, 1973; DUELLMAN & TRUEB, 1985). Svos (1965) a montré qu’une

faible luminosité associée à une basse température pouvait entraver la métamorphose de

Triturus alpestris montenegrinus, tandis que l'obscurité seule provoquait la mort des tritons

(à une température de 18-20°C). Il suggère ainsi que la température basse est une des

conditions pour la résistance des tritons néoténiques à l’action inhibitrice d’une luminosité

insuffisante. Les lacs d’altitude, pour la plupart froids et obscurs, offrent ainsi des

conditions favorables à l'apparition de la néoténie. Mais le problème serait trop simple s’il

Source : MNHN, Paris

100 ALYTES 15 (2)

s’arrêtait là. En effet, il existe des lacs d’altitude entourés d’un milieu prétendu hostile où

l'on ne rencontre pas de tritons pédogénétiques (BREUIL, 1986). Selon BREUIL (1992),

celui-ci ne le serait pas obligatoirement, et ce notamment en milieu karstique où les très

nombreuses cavités offriraient des refuges adéquats aux animaux. D'un autre côté, on a

récemment découvert des populations néoténiques dans des milieux instables voire

temporaires et entourés d’un milieu terrestre apparemment favorable. Il en est ainsi en ce

qui concerne les tritons alpestres néoténiques du Nevesinjsko Polje en Bosnie (DZUKIC &

KALEZIC, 1984) et du Lago dei Due Uomini en Calabre, ce dernier lac s’asséchant même

en été (Dugois & BREUIL, 1983; Dugois, 1983; BREUIL, 1986). L’assèchement des points

d’eau contenant des populations néoténiques est un problème difficilement soluble.

Certains auteurs parlent dans ce cas de néoténie labile, celle-ci pouvant apparaître et

disparaître spontanément (FUHN, 1963; GABRION et al., 1977, 1978; BREUIL, 1992).

Dans la présente note, nous rapportons l'observation d’une petite population

néoténique dans un milieu instable et obscur.

MATÉRIELS ET MÉTHODES

La population observée occupe un complexe de bacs en béton et de canaux

partiellement remplis d’eau dans un bâtiment en construction désaffecté depuis environ 5

ans (fig. 1). Ce site devait devenir une station thermale, mais les travaux ont dû être arrêtés

à la suite d’impératifs budgétaires. Il est situé près de Castelnuovo di Garfagnana en

Toscane (Italie) et plus précisément à quelques mètres de l'étang “Il Bagno” (Lago di Prà

di Lama) (UTM: 32TPP1387). L’altitude de la station est de 357 mètres. D’après FERRACIN

et al. (1980) ainsi que BREUIL (1986), cet étang contient une population de Triturus alpestris

apuanus, mais ces auteurs n’y ont trouvé aucun individu néoténique. Toutefois, nous

n’excluons pas qu'il puisse exister des animaux néoténiques dans cette station. En effet, le

pédomorphisme n’est pas un phénomène exceptionnel chez T. a. apuanus: des cas en ont

été relevés dans les provinces de Torino, Cuneo, Savona, Genova, Piacenza, Arezzo et

Rieti (ANDREONE & DORE, 1991) et de Lucca (FERRACIN et al., 1980).

Lors de ma visite, le 17 juillet 1994, 17 bacs ainsi que le système ‘‘d’égouttage”

contournant le bâtiment contenaient de l’eau. Une vingtaine d’autres bacs et une partie du

canal d’égouttage étaient à sec. La profondeur des bacs et du canal est de 20 cm, mais celle

de l’eau n’est que de 3 à 9 cm. La superficie des bacs est de 13 à 27 m° selon ceux-ci. Il

existe des communications (tuyaux) entre certains des bacs et le canal d’égouttage, mais le

niveau d’eau ne les atteint pas toujours. Néanmoins, ce dernier a probablement déjà été

plus élevé, permettant ainsi des échanges entre les bacs. La lumière naturelle pénètre à

l'intérieur d’une partie du bâtiment, mais le canal d’égouttage est quant à lui presque

totalement obscur.

La majorité des tritons alpestres, aussi bien les adultes que les juvéniles et les larves,

ont été capturés bac après bac et relâchés directement. Tous les individus capturés ont été

mesurés vivants, sans avoir été anesthésiés, du museau à l'extrémité de la queue (L). Il

semble que la marge d’erreur n'excède pas le millimètre.

Nous avons distingué cinq groupes au sein de la population observée: (1) les larves

(individus présentant des branchies bien développées, mesurant moins de 65 mm et ne

possédant aucune tache gulaire); (2) les juvéniles (tritons métamorphosés mais encore

immatures); (3) les subjuvéniles (tritons immatures en fin de métamorphose — larves DH, Paris

DENOËL 101

Fig. 1. — Vue de l'intérieur du bâtiment montrant les bacs où ont été trouvés les spécimens

néoténiques de Triturus alpestris apuanus.

néoténiques — présentant la coloration et l’habitus des juvéniles, mais ayant encore des

branchies); (4) les néoténiques (tritons présentant un phénotype larvaire mais de grande

taille — plus de 60 mm — et possédant des taches gulaires); (5) les adultes métamorphosés

(tritons métamorphosés, de grande taille — plus de 70 mm pour les mâles, plus de 75 mm

pour les femelles — , et supposés matures selon l’examen des caractères extérieurs).

RÉSULTATS ET DISCUSSION

Sur les 131 individus capturés dans l’eau, on dénombre 99 larves, 6 subjuvéniles, 13

juvéniles, 6 néoténiques, 2 adultes mâles et 5 adultes femelles (tab. 1).

La répartition spatiale des différents membres de la population est assez hétérogène:

certains bacs ne contiennent que des larves alors que d’autres contiennent également des

juvéniles, des adultes et des néoténiques. On constate ainsi que presque la moitié des

adultes se trouvent rassemblés dans le même bac, faiblement éclairé, d’une superficie de 28

mètres carré et d’une profondeur maximale de 4 cm.

Les larves sont en moyenne de grande taille, près des deux tiers mesurant plus de

50 mm (tab. 1). Certaines de ces larves sont d’ailleurs à un stade assez proche de celui des

néoténiques. Les 6 néoténiques (tout comme les métamorphosés) ont la partie gulaire

tachetée, légèrement chez le plus petit d’entre eux (L = 60 mm) (fig. 2).

Source : MNHN, Paris

102 ALYTES 15 (2)

Tab. 1. - Valeur moyenne, écart-type et valeurs extrêmes (en millimètres) de la longueur

totale dans les 6 groupes de la population de Triturus alpestris apuanus étudiée.

Valeurs extrêmes

(minimum-maximum)

Stade (n) Moyenne

Larves (99) 50,8 30-63

Subjuvéniles (6) 51,8 à 45-85

Juvéniles (13) 70,7 49-80

Néoténiques (6) 75,8 60-88

Femelles adultes (5) 71-107

Mâles adultes (2)

Fig. 2. — Spécimen néoténique de Triturus alpestris apuanus d’une longueur totale de 80 mm.

La population observée occupe un habitat aux conditions très particulières. En effet,

il s’agit d’un milieu obscur et frais comme c’est le cas des lacs d’altitude où l’on rencontre

parfois des populations néoténiques. Ces paramètres physiques, joints à la faible densité

de proies, ont très certainement ralenti la croissance des animaux et peut-être ainsi entravé

la métamorphose. Cette supposition est appuyée par les expériences de Svog (1965) qui ont

montré qu’une telle situation pouvait effectivement entraver la métamorphose des tritons.

In natura, dans les milieux d’altitude stables, on constate également que les larves ont

tendance à passer l'hiver dans l’eau (BREUIL, 1992). Toutefois le milieu étudié diverge de

ces milieux d’altitude entre autres par son instabilité et son faible volume d’eau. De plus,

il est d’origine extrêmement récente (moins de 5 ans). Ces faits nous amènent à penser qu’il

Source : MNHN, Paris

DENOËL 103

s’agirait d’une néoténie de type labile, pouvant apparaître et disparaître spontanément.

Celle-ci aurait été induite par les conditions particulières qu'offre ce milieu artificiel, et qui

correspondent un peu à celles rencontrées dans certaines grottes. BREUIL & PARENT (1987)

ont d’ailleurs observé des Triturus alpestris veluchiensis dans une nappe d’eau souterraine.

Les cas de néoténie chez T. a. apuanus étant relativement fréquents, il est probable qu’ils

aient une certaine base génétique sous-jacente, celle-ci ne s'exprimant que lors de

conditions environnementales particulières.

RÉFÉRENCES BIBLIOGRAPHIQUES

ANDREONE, F. & Dore, B., 1991. — New data on paedomorphism in Italian populations of the Alpine

newt, Triturus alpestris (Laurenti, 1768) (Caudata: Salamandridae). Herpetozoa, 4 (3/4) :

149-156.

BREUIL, M., 1986. — Biologie et différenciation génétique des populations du triton alpestre (Triturus

alpestris) (Amphibia Caudata) dans le sud-est de la France et en Italie. Thèse de Doctorat, Univ.

Paris-Sud: 1-192.

= 1992. — La néoténie dans le genre Triturus: mythes et réalités. Bull. Soc. herp. France, 61: 11-44.

BREUIL, M. & PARENT, G. H., 1987. — Essai de caractérisation du triton alpestre hellénique Triturus

alpestris veluchiensis. 1. Historique et présentation de nouvelles données. Alytes, 6: 131-151.

Dusois, A., 1983. — Le triton alpestre de Calabre: une forme rare et menacée d'extinction. A/ytes,

2: 55-62.

— 1985. — Neoteny and associated terms. Alytes, 4: 122-130.

Dusois, A. & BREUIL, M., 1983. — Découverte de Triturus alpestris (Laurenti, 1768) en Calabre (sud

de l'Italie). Alytes, 2: 9-18.

DuecLmaN, W. E. & TRUEB, L., 1985. — Biology of amphibians. New York, McGraw-Hill, “1986”;

reprint 1994, John Hopkins University Press: i-xxi + 1-670.

Dzuxic, G. & KaALezic, M. L., 1984. — Neoteny in the Alpine newt population from the

submediterranean area of Yugoslavia. Alytes, 3: 11-19.

FERRACIN, A., LUNADEI, M. & FALCONE, N., 1980. — An ecological note on Triturus alpestris apuanus

(Bonaparte) and Triturus cristatus carnifex (Laurenti) in the Garfagnana (Lucca, Central Italy).

Boll. Zool., 47: 143-147.

FuHN, L. E., 1963. — Sur un nouveau cas de néoténie en masse du Triton vulgaire (Triturus v. vulgaris

L.). Vest. cs. spol. zool., 27 (1): 62-69.

GABRION, J., SENTEIN, P. & GABRION, C., 1977. — Les populations néoténiques de Triturus helveticus

des Causses et du Bas-Languedoc. I. Répartition et caractéristiques. La Terre et la Vie, 31:

489-506.

KOLLMANN, J., 1884. — L’hivernage des larves de grenouilles et de tritons d'Europe et la

métamorphose de l’axolotl du Mexique. Rev. suisse Zool., 1: 75-89.

Svos, M., 1965. — Neurosekretion in Triturus alpestris montenegrinus Radov. und ihre Korrelation

mit der Neotenie. Bull. Sci. Acad. R.S.F. Yougoslavie, (A), 10: 379-381.

WizBUR, H. M. & CoLLins, J. P., 1973. — Ecological aspects of amphibian metamorphosis. Science,

182: 1305-1314.

Corresponding editor: Pierre JOLY.

Source : MNHN, Paris

Alytes, 1997, 15 (2): 104.

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AIVTES

International Journal of Batrachology

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Grar, .-D. & BOLLS PeLAz, M., 1989, - Evolutionary genetics of the Rana esculenta compiex. In: R. M. DAWLEY

& J. P. BoGaRT (eds), Evolution and ecology of unisexual vertebrates, Albany, The New York State

Museum: 289-302.

InGer, R. F, Voris, H. K. & Voris, H. H., 1974. - Genetic variation and population ecology of some

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Alytes, 1997, 15 (2): 49-104.

Contents

Carlos Alberto Gonçalves DA CRUZ, Ulisses CARAMASCHI & Eugênio IZECKSOHN

The genus Chiasmocleis Méhely, 1904 (Anura, Microhylidae) in the

Atlantic Rain Forest of Brazil, with description of three new species ..… 49-71

Matthias Srôck & Wolf-Rüdiger GROBE

Erythrocyte size and ploidy determination in green toads

(Bufo viridis complex) from Middle Asia .............................. 72-90

U. KATZ, Joy HOFFMAN & Nira GIL

What is the ecological significance of laboratory temperature

Selection Ain aNUTAN AMD. 0 ee 91-08

Mathieu DENOËL

Un cas de néoténie, dans un bâtiment désaffecté, chez le

triton alpestre, Triturus alpestris apuanus (Salamandridae) ............. 99-103

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Alytes subscription: special offer to institutions ................................... 104

Alytes is printed on acid-free paper.

Alytes is indexed in Biosis, Cambridge Scientific Abstracts, Current Awareness in Biological

Sciences, Pascal, Referativny Zhurnal and The Zoological Record.

Imprimerie F. Paillart, Abbeville, France.

Dépôt légal: 4" trimestre 1997.

Source : MNHIN, Paris: