BONN ZOOLOGICAL BULLETIN — SUPPLEMENTUM (BZB-S),

formerly “Bonner zoologische Monographien”,

Bonn zoological Bulletin, Editor-in-Chief

Ralph S. Peters, Zoologisches Forschungsmuseum Alexander Koenig — Leibniz-Institut fur Biodiversitat der Tiere

(ZFMK), Hymenoptera Section, Adenauerallee 160, 53113 Bonn, Germany, tel. +49 228-9122-290,

fax: +49 228-9122-212; r.peters@leibniz-zfmk.de

Managing Editor Supplementum Series

Thomas Wesener, ZFMK, Myriapoda Section, same address as above, tel. +49 228-9122-425,

fax: +49 228-9122-212; t.wesener@leibniz-zfmk.de

The peer-reviewed series Bonn zoological Bulletin —Supplementum, formerly “Bonner zoologische Monographien’,

has existed since 1971. It is published by the Zoological Research Museum Alexander Koenig — Leibniz Institute for

Animal Biodiversity (ZFMK), Bonn. Supplements on focus topics are produced in irregular succession. The BzB-S

consists of original zoology papers too long for inclusion in our institute’s regular journal, Bonn zoological Bulletin.

Preferred manuscript topics are: systematics, taxonomy, biogeography, anatomy, and evolutionary biology. Manu-

scripts should be in American English. Authors are requested to contact the managing editor prior to manuscript

submittal.

For subscription, back issues and institutional exchange, please contact the ZFMK library (ZFMK, Bibliothek,

Mareike Kruppa, Adenauerallee 160, 53113 Bonn, Germany, tel. +49 228—9122-216, fax: +49 228—-9122-—212;

m.kruppa@leibniz-zfmk.de). The online version of BzB -S is available free of charge at the BzB homepage:

http://www.zoologicalbulletin.de.

Authors of this issue:

Sandra Franz-Guess & J. Matthias Starck

Published: Bonn zoological Bulletin — Supplementum Vol. 65, 125 pp.

Germany.

ISSN: 0302-671X

Produced by Eva-Maria Levermann, Bonn, Germany; E.Levermann@leibniz-zfmk.de; emlevermann@netcologne.de.

Printed and bound by Verlag Natur & Wissenschaft, Postfach 170209, 42624 Solingen, Germany.

Cover illustration:

Eukoenenia spelaea (Peyerimhoff, 1902). Light microscopic micrograph of a histological cross-section through the

prosoma of a female at the level of the rostrosoma, including cheliceral and pedipalpal articulation. Please see caption

of Figure 37 in this publication for details.

Microscopic anatomy of

Eukoenenia spelaea (Peyerimhoff, 1902)

(Arachnida: Palpigradi: Eukoeneniidae)

Sandra Franz-Guess & J. Matthias Starck

Bonn zoological Bulletin Supplementum 65: 1—125 ©ZFMK

Contents Volume 65

Abstract 3

Key words 3

Introduction 4

Material & Methods 6

Results 9

Discussion 68

Acknowledgements 94

References 94

Appendix | 103

Appendix | Ta

f\\t KOENIG Bonn zoological Bulletin — Supplementum 65: 1-125 August 2020

2020 - Franz-Guess S. & Starck J.M. ISSN 0302-671

https://doi.org/10.20363/BZB-S-2020.65 http://www.zoologicalbulletin.de

Research article

urn:|sid:zoobank.org: pub:09780A FE-1686-46DC-A629-5784EFOES9E7

Microscopic anatomy of Eukoenentia spelaea (Peyerimhoff, 1902)

(Arachnida: Palpigradi: Eukoenentidae)

Sandra Franz-Guess! & J. Matthias Starck”*

"2 Ludwig-Maximilians-University (LMU) Munich, Department of Biology II, Biocenter Martinsried, Grobhadernerstr. 2,

D-&2152 Planegg-Martinsried, Germany

* Corresponding author: Email: starck(@/lmu.de

'urn:lsid:zoobank. org:author:98 DFBB07-6DD5-41C0-B21D-05298086A710

2urn:Isid:zoobank.org:author:529CA71E-01B6-407A-AA B0-4B0E4014057E

'https://orcid.org/0000-0003-2826-1248

*http://orcid.org/0000-0001-7882-9482

Abstract. Eukoenenia spelaea (Peyerimhoff, 1902) is a troglobiont palpigrade found in caves of the European Alps. In-

dividuals are small, reaching a maximum body length of 1.5 mm (if measured without the terminal flagellum). They lack

eyes and breathing organs, but have unique sensory organs. Morphological studies of Palpigradi date back the late 19th

and early 20th century, but data on microscopic anatomy and comparative morphology is incomplete. Their small body

size causes reduction, simplification and loss of organ systems, and their paedomorphic evolution results in some hyper-

plesiomorphic features (i.e., more plesiomorphic than the euchelicerate body plan) making their phylogenetic placement

among arachnids difficult.

In this study, we use serial sectioning for light microscopy, transmission and scanning electron microscopy, and

3D-reconstructions to provide a complete account on the microscopic anatomy of Eukoenenia spelaea. We describe seve-

ral new morphological features. For some already known structures we provide detailed morphological evidence allowing

for new interpretations in the light of evolutionary morphology: (1) two prosomal shields, the pro- and metapeltidium,

divide the prosoma dorsally. The traditionally described “mesopeltidium” is a sclerotization of the pleural membrane, but

not a tergite. (2) The ventral plate is probably an osmoregulatory organ. (3) The prosternum consists of the fused sternites

of segments 2-4. (4) The frontal organ and trichobothria have a unique morphology among euchelicerates. (5) We provide

evidence for a tripartite syncerebrum. (6) The adult heart lacks innervation, lacks ostia, a pericardium, and shows an early

developmental state of muscle differentiation. (7) The rostrosoma is not associated with chelicerae or pedipalps. (8) The

midgut is simple and sac-like; a hindgut is missing. (9) The coxal organ (tubule and glandular section) has no lumen. (10)

Females have an unpaired ovary with only few large eggs. (11) Aflagellate sperm have a prominent vacuole.

The small body size of Eukoenenia spelaea (and probably of all palpigrades) resulted in modification, reduction or even

loss of structures like: (1) a distinct differentiation into endo- and exocuticle is lacking over large regions of the body; (2)

absence of respiratory organs; (3) several muscles groups are reduced; (4) the prosomal ganglia are proportionally large

and display a high level of fusion; (5) neuron size ranges at the minimum size for arthropods; (6) the coxal organ has a

reduced number of cells; (7) absence of Malpighian tubules; (8) ovaries are unpaired; (9) small number of eggs. Some

organs showed a typical paedomorphic morphology: (1) the heart lacks ostia, a pericardium, and the ultrastructure of the

musculature resembles that of the hearts of early developmental stages of other arthropods; (2) the midgut is a simple sac

with no epithelial differentiation; (3) the reappearance of ancient plesiomorphies like (1) the anterior oblique muscle, (11)

an almost complete set of posterior oblique muscles in the prosoma, (111) the deutocerebral connectives embracing the eso-

phagus, and (iv) the segmental chord of ganglia in the opisthosoma are interpreted hyperplesiomorphic, i.e., features that

were rather present in the body plan of euarthropods and already reduced in euchelicerates. Hyperplesiomorphic features

may be interpreted as resulting from “reverse recapitulation” indicating paedomorphosis by developmental truncation.

— The morphological characters revealed in the present analyses were used to perform a phylogenetic analysis. Several

autapomorphic characters were recognized to characterize Palpigradi (e.g., the prosoma is dorsally divided in only two

peltidia; the anterior ventral sclerite of the prosoma is assigned to segments 2-4; the frontal organ; the lateral organ; the

preoral cavity, mouth, and pharynx are inside a simple cuticular cone shaped rostrosoma; lack of a hindgut; the ventral

plate; the ultrastructure of the trichobothria; anterior oblique suspensor muscle). A phylogenetic sister group relationship

was found with Acaromorpha with which they share: the opening of the excretory pore on or near the coxa of append-

age III (= leg 1); the lack of a postcerebral suction pump; aflagellate sperm that lack an acrosomal filament; the muscle

topography of leg 4; the tubule of the coxal organ is differentiated in a proximal and a distal segment; the presence of a

myogenic heart; the lack of the arcuate body in the syncerebrum. — Based on that sister group relationship, it becomes

probable that evolutionary miniaturization did not occur in the stem lineage of Palpigradi, but in the lineage leading to the

common ancestor of a clade Palpigradi + Acaromorpha. We suggest that Palpigradi with their simple and hyperplesiomor-

phic morphology represent an offshoot of this early lineage with little morphological variation, while the lineage leading

to the Acaromorpha resulted in a species rich diversification based on a paedomorphic morphology.

Key words. Chelicerata, evolution, miniaturization, morphology, paedomorphosis.

4 Sandra Franz-Guess & J. Matthias Starck

INTRODUCTION

Palpigradi

Palpigradi is an enigmatic group of arachnids contain-

ing an estimated number of 107 extant species and

two fossil species assigned to two taxa, Eukoenentidae

Petrunkevitch, 1955, and Prokoeneniidae Condé, 1996.

Eukoeneniidae includes 4 extant genera, 1.e., A/lokoene-

nia Silvestri, 1913, with one species; Eukoenenia Borner,

1901, with 86 species; Koeneniodes Silvestri, 1913, with

eight species, and Leptokoenenia Condé, 1965, with

five species (Harvey 2003; Giribet et al. 2014; Bu et al.

2019; Souza & Ferreira 2013; Barranco & Mayoral 2007,

2014). Prokoeneniidae includes two extant genera, L.e.,

Prokoenenia Borner, 1901, with six species and Tria-

dokoenenia Condé, 1991, with one species. Fossil genera

are Electrokoenenia Engel & Huang, 2016 (Eukoeneni-

idae) and Paleokoenenia Rowland & Sissom, 1980 (Eu-

koeneniidae or incertae sedis) with one species each (Har-

vey 2002). Palpigrades are distributed in the pantropical

region between 48° N and 40° S with most species in

Africa and Europe (Condé 1996; Harvey 2003), and sev-

eral new species recently described for South America

(Souza & Ferreira 2010, 2011a, b; 2012; 2013). Species

live in the upper soil layer or are troglobiont, especially

those in Central Europe.

Palpigradi are small, the largest species reaching a

body length of 3 mm (K4stner 1931a; Millot 1949a; Gi-

ribet et al. 2014; Dunlop 2019), including a prominent

long terminal flagellum (it is relatively short in species of

the genera Allokoenenia and Leptokoenenia) which they

hold parallel to the ground while walking and hold erect

during short stops (Kova¢é et al. 2002) sometimes moving

it laterally (Souza and Ferreira 2010; Ferreira and Sou-

za, 2012). They have no eyes, book lungs, or tracheae.

Palpigrades are characterized by a unique set of sensory

organs (frontal organ, lateral organs). They also carry nu-

merous sensory setae and trichobothria as known from

other arachnids. The prosomal dorsal shield is divided

into three sclerites, a feature that is only known from So-

lifugae and Schizomida.

Morphological studies of Palpigradi date back to the

late 19" and early 20" century [Eukoenenia angusta

(Hansen, 1901), Eukoenenia florenciae (Rucker, 1903),

Eukoenenia grassii (Hansen, 1901), Eukoenenia mirabi-

lis (Grassi and Calandruccio, 1885) (Hansen & Sgrensen

1897; Wheeler 1900, Hansen 1901; Borner 1904, Buxton

1917; Kastner 1931a; Millot 1942; 1943), Eukoenenia si-

amensis (Hansen, 1901), Prokoenenia chilensis (Hansen,

1901), and Prokoenenia wheeleri (Rucker, 1901) (Han-

sen 1901, Kastner 1931a)]. The main focus of these stud-

ies was on the external morphology. Some aspects of the

internal morphology were described by Rucker (1901),

Borner (1904), Buxton (1917), Kastner (1931a), Mil-

lot (1942, 1943, 1949a), and van der Hammen (1977a,

Bonn zoological Bulletin Suppl. 65: 1-125

b, 1982) using either dissections, or cleared specimens,

or paraffin histology. Alberti (1979a) analyzed the ultra-

structure and development of spermatozoa in P. wheeleri,

Ludwig & Alberti (1992) documented the ultrastructure

of the midgut in P. wheeleri, and Smrz et al. (2013) stud-

ied the gut content of Eukoenenia spelaea using light mi-

croscopy.

It has been frequently noted that Palpigradi are rich in

plesiomorphic features [reviewed in Dunlop & Alberti

(2008)] thus obscuring a phylogenetic analysis. Morpho-

logical character analyses and gene sequencing render di-

verging and often incompatible phylogenetic hypotheses

of euchelicerate relationships (Fig. 1). Among the pub-

lished phylogenetic hypotheses, Arachnida and Tetrapul-

monata, as well as Pedipalpi (~Amblypygi + Uropygi)

and Uropygi (=Thelyphonida+Schizomida) at the inter-

ordinal level, are those clades that have repeatedly been

confirmed. All other higher order group relationships are

debated including the phylogenetic position of Palpigra-

di. Van der Hammen (1977a) classified Palpigradi to-

gether with Actinotrichida (Acariformes) as “Epimerata”

(also suggesting a diphyletic origin of the Acari; van der

Hammen 1977a, b, 1982). Weygoldt & Paulus (1979a;

Fig. 1A) considered Palpigradi to be the sister group

to Holotracheata. Shultz (1990) considered Palpigradi

as sister group to Tetrapulmonata (Fig. 1B). However,

Shultz (2007a) presented a more complete analysis of

morphological characters (including fossils) resulting in

either a largely unresolved consensus tree (Fig. 1D; fig. 1

in Shultz 2007a) or, alternatively, a monophyletic taxon

Arachnida with the topology ((Palpigradi (Acaromorpha

(Tetrapulmonata (Haplocnemata, Stomothecata)); fig. 5

in Shultz (2007a). Giribet et al. (2002) considered mor-

phological data, fossil record and gene sequences, and

placed palpigrades on an unresolved polytomy together

with Acari, Ricinulei, and Tetrapulmonata (Fig. 1C). Re-

sults from gene sequence analyses by Regier et al. (2010)

showed Palpigradi and Acariformes together as sister

group to all other arachnids (Fig. 1E). Pepato et al. (2019)

combined molecular data and morphological data and re-

covered Palpigradi as sister group to a clade containing

Solifugae and Acariformes (Cephalosomate = (Palpi-

gradi (Solifugae+Acariformes)), however, as they state,

with relatively low support. Garwood & Dunlop (2014)

included fossil material in their morphological data set

and placed palpigrades as sister group to an unresolved

taxon containing Solifugae, Acariformes, Parasitiformes,

Ricinulei, and Tetrapulmonata (Fig. 1F). Giribet et al.

(2014) conducted a comprehensive phylogenetic analysis

of Palpigradi (29 species) based on gene sequences and

suggested a sister group relationship to Scorpiones and

Thelyphonida. Finally, Sharma et al. (2014) and Sharma

(2018) placed Palpigradi as sister to Parasitiformes, but

pointed out that there was little support. — In summary,

Palpigradi appear to take a rather basal position among

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 5

Xiphosurida Xiphosurida Xiphosunda

Scorpiones Opiliones : Solifugae

Uropygi Scorpiones Pseudoscorpiones

Amblypyai Solifugae Scorpiones

Araneae : Pseudoscorpiones Opiliones

Palpigradi a Ricinulel Palpigradi

Solifugae - Acari Ricinulei

~ Pseudoscorpiones Palpigradi Acari

7 Opiliones Araneae Araneae

Ricinulei Amblypygi Amblypyqi

Acari Schizomida Schizomida

Thelyphonida Thelyphonida

Xiphosurida Xiphosurida Xiphosurida

Opiliones Palpigradi Opiliones

Scorpiones Acariformes Scorpiones

Solifugae Opiliones Pseudoscorpiones

Pseudoscorpiones Parasitiformes Palpigrad

Palpigradi Pseudoscorpiones Solifugae

Ricinulei Ricinulei Acariformes

Acari Solifugae Parasitiformes

Araneae Scorpiones Ricinulei

Amblypygi Araneae Araneae

Schizomida Amblypygi Amblypygi

Thelyphonida Schizomida Schizomida

Thelyphonida Thelyphonida

M, F

Fig. 1. Published phylogenetic hypotheses about euchelicerate relationships. A. Phylogeny by Weygoldt & Paulus (1979a) based

on morphological characters. Scorpiones are the sister group to all other arachnids, with two major clades, 1.e., Tetrapulmonata

and Apulmonata. B. Phylogeny by Shultz (1990) based on morphological characters. Arachnida are divided into two clades, 1.e.,

Dromopoda and Micrura. C. Phylogeny by Giribet et al. (2002) based on morphological characters, fossil records, and gene

sequencing. The phylogeny of Micrura is largely unresolved. D. Phylogeny by Shultz (2007a) based on morphological characters

of extant and fossil records. Except for Tetrapulmonata, the relationships of euchelicerate clades are not resolved. E. Phylogeny by

Regier et al. (2010) based on gene sequencing. Palpigradi are positioned at the base and sister group to Acariformes. Scorpiones

is sister group of Tetrapulmonata. F. Phylogeny by Garwood & Dunlop (2014) based on morphological characters and fossil

records. While the base of the tree is resolved, the relationships between Solifugae, Acariformes, Parasitiformes, and Ricinulei is

unresolved. Abbreviations: F = fossil record analysis; M = morphological character analysis; S = gene sequence analysis.

arachnids, but an explicit hypothesis about the phyloge-

netic position of Palpigradi remains concealed.

Miniaturization

Palpigradi are small (1-3 mm) when compared to most

other arachnids. Only Acari [Parasitiformes (0.2—30 mm)

and Acariformes (0.08—-14 mm)] have smaller median

body length (Dunlop 2019). Species of Micryphantinae,

Bonn zoological Bulletin Suppl. 65: 1-125

i.e., a taxon of Linyphiidae (Araneae) reach a similar

body length of 1-4 mm. All other arachnids reach larger

body sizes, e.g., Scorpiones (up to 210 mm body length),

Solifugae (up to 70 mm body length), Amblypygi (up to

45 mm body length), or Thelyphonida (up to 75 mm body

length). Extending the comparison to Euchelicerates and

including fossil forms one encounters even larger body

sizes. The observed trend in some euchelicerate groups

towards a smaller body size might be considered evolu-

©ZFMK

6 Sandra Franz-Guess & J. Matthias Starck

tionary miniaturization. Evolutionary miniaturization has

been described for a number of invertebrate and verte-

brate taxa (e.g., Hanken & Wake 1993; Polilov 2015a,

2016; Polilovy & Makarova 2017; Dunlop 2019). As an

evolutionary process, miniaturization results from direc-

tional selection on a large ancestor towards small body

size; but the biological significance of scaling depends on

the context in which size variation 1s studied. Reduction

of body size may come along with a characteristic mor-

phological pattern, e.g., (allometric) reduction of organ

size, structural simplification (e.g., midgut differentiation

simplified in insects; Polilov 2015a, 2016), complete loss

of a structure or organ (e.g., lack of respiratory organs,

lack of a heart in insects; Polilov 2015a, 2016), and pro-

portionally large brains with small neurons.

A possible evolutionary mechanisms leading to re-

duced body size is heterochrony, specifically paedomor-

phosis (neoteny, progenesis/developmental truncation)

resulting in the retention of larval structures within the

adult (Gould 1977; Alberch et al. 1979). Progenesis /

truncation is the accelerated maturation of an organism

with respect to the overall somatic development. This is

in contrast to neoteny that also produces paedomorphic

adults, but results from retardation of organ development

with respect to the overall development. Postdisplace-

ment is the delayed start of development of an organ

system at unchanged developmental rate and may re-

sult in paedomorphic appearance of adults (Gould 1977;

Alberch 1980; McNamara 1986). According to Gould

(1977) and McNamara (1986) all three processes pro-

duce paedomorphic adults, but only progenesis may lead

to reduced body size. Alberch et al. (1979) introduced a

more dynamic model of heterochrony also considering

growth rate functions. In this model, all three process-

es potentially resulted in the same paedomorphic mor-

phologies and reduced body size, and it was impossible

deducing the ontogenetic mechanism from the study of

adult organisms. The model by Alberch et al. (1979) also

suggested that progenesis would result in reappearance

of plesiomorphic features, what they called “reverse re-

capitulation”. While this model is based on the assump-

tions of a strict anagenetic concept of evolution, it allows

explicit predictions about the cumulative appearance of

plesiomorphic features in a derived phylogenetic context.

Aims of the study

This study aims at providing a treatise of the complete

microscopic anatomy of Eukoenenia spelaea (Pey-

erimhoff, 1902). By providing a detailed microscop-

ic anatomical documentation of the species, we aim at

re-evaluating the morphology of organ systems and body

tagmatization of palpigrades based on explicit morpho-

logical evidence. We then proceed by providing a mor-

phology based hypothesis about the phylogenetic posi-

tion of Palpigradi among Euchelicerata and contribute to

Bonn zoological Bulletin Suppl. 65: 1-125

the ground pattern of euchelicerates/arachnids. This may

be complicated by the fact that Palpigradi are rather char-

acterized by plesiomorphic features than shared derived

traits that would allow for a straightforward phylogenetic

analysis. Therefore, we will test if paedomorphosis and

evolutionary miniaturization may be associated with

morphological simplification and/or enrichment of ple-

siomorphic features, thus creating the difficulty of ana-

lyzing their phylogenetic position among the arachnids.

Supposing we find proof of evolutionary miniaturization

of Palpigradi and paedomorphosis, the re-appearance of

an array of plesiomorphic characters can become helpful

in a phylogenetic analysis.

MATERIALS AND METHODS

Specimens

Eukoenenia spelaea is a troglobiont species found

in caves of the Karst region in the European Alps and

Carpathians (Christian et al. 2014), where they inhabit

the sediments on the cave floor. Twenty-two specimens

of Eukoenenia spelaea (Peyerimhoff, 1902) were col-

lected in the Ardovska Cave (Slovak Aggtelek Karst,

48°31'18.613" N, 20°25'13.798" E) in April, July, and

October 2016 and in August 2018. Eukoenenia spelaea

occurs in four subspecies in Europe (Blick & Christian

2004). For Slovakia, FE. spelaea vagvoelgyii (Szalay,

1956) has been determined as subspecies.

Access to the cave is restricted except for occasional

visits by scientists, thus, it is largely undisturbed and has

no artificial lighting. The temperature within Ardovska

Cave ranges from +7.9 to +10.8 °C, and the humidity is

approx. 97% (Kovaé et al. 2002, 2014). The cave con-

sists of numerous narrow passages as well as few larger

caverns. It is characterized by the presence of stalactites

and stalagmites of various sizes (Fig. 2A). The habitat of

Eukoenenia spelaea are small patches of sediment con-

sisting of mineral sediment, pieces of charcoal (brought

in by scientists for an experimental setup on cave fauna),

marten excrements, and bat guano (Fig. 2B).

The body length of the collected specimens without the

flagellum was 1.23 mm + 0.13 mm for adult females and

1.03 mm + 0.13 mm for adult males. Nine animals (six fe-

males and three males) were fixed in buffered paraform-

aldehyde (PFA 5% in 0.1 mol I' phosphate buffered sa-

line, pH 7.4). These animals were used for light micro-

scopic (LM) histology and scanning electron microsco-

py (SEM). Ten animals (all females) were fixed using

buffered glutardialdehyde (GDA 2.5% in 0.2 mol I!

phosphate buffered saline, pH 7.4). These samples were

used for transmission electron microscopy (TEM). Three

specimens were fixed using a modified Karnovsky’s fix-

ative (GDA 2.5% and PFA 1.5% in 0.1 mol I"! phosphate

buffered saline, pH 7.4). All animal material (includ-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) -

Fig. 2. Ardovska Cave, Slovakia. A. The cave is characterized by narrow passageways and large halls, where stalactites and

stalagmites form the typical internal surface sculpturing ofa karst cave. B. Close-up photograph of the habitat of Eukoenenia spelaea.

The sediment consists of mineral sediments, pieces of charcoal, marten excrements, and bat guano.

ing unsectioned specimens) is stored at the Zoological

State Collection in Munich, Germany (project numbers

ZSMS20190030—-ZSMS20190051).

Scanning electron microscopy

Scanning electron microscopy (SEM) was used to doc-

ument the external anatomy of the animals. Fixed spec-

imens were dehydrated through a graded series of ace-

tone. The samples were then dried using the CO,-critical

point drying method. Samples were mounted on alumi-

num stubs and sputter coated for 200 s with gold. Images

were captured using a LEO 1430VP SEM (LEO Elek-

tronenmikroskopie GmbH, Oberkochen, Germany) and

the software SmartSEM (version 5.07, Carl Zeiss AG,

Oberkochen, Germany).

Histology

Specimens were washed four times in phosphate buff-

ered saline (0.1 mol I"') over a period of 20 minutes, post

fixed in 1% osmium tetroxide for two hours and washed

again (four times, 20 minutes each) in phosphate buff-

ered saline to remove excess osmium tetroxide. Sam-

Bonn zoological Bulletin Suppl. 65: 1-125

ples were dehydrated through graded series of acetone

(30—100%) and then embedded in Glycidether 100 (Carl

Roth GmbH + Co. KG, Karlsruhe, Germany). Histolog-

ical semithin sections were cut at 1.5 um, 1 um and 500

nm thickness using an RMC MTXL ultra-microtome

(Boeckeler Instruments, Inc., Tucson, Arizona, USA)

equipped with a histo Jumbo diamond knife (DIATOME

Ltd, Biel, Switzerland). In order to obtain serial sections,

the ventral side of the trimmed specimen block was cov-

ered with a thin layer of ethyl acetate/methyl cyclohex-

ane glue (Pattex Kraftkleber Classic, Henkel AG & Co.

KGaA, Dusseldorf, Germany) mixed with xylene in a

1:1 mixing ratio. The section bands were then collect-

ed in water, attached to a glass slide and dried. Sections

were stained using Rudeberg staining solution (Rtdeberg

1967). Light microscopic images were taken with an au-

tomated Olympus BX61VS microscope and DotSlide

software (Olympus, Hamburg, Germany) or an Olympus

BX5I1TF microscope (Olympus, Hamburg, Germany)

equipped with a microscope camera (UCMOS camera,

ToupTek Photonics, Hangzhou, P.R. China). Image cap-

turing was processed via ToupView (ToupTek Photonics,

Hangzhou, P.R. China). Image analysis was done using

©ZFMK

8 Sandra Franz-Guess & J. Matthias Starck

OlyVIA (version 2.9, Build 13735, Olympus Soft Imag-

ing Solutions GmbH, Munster, Germany).

Transmission electron microscopy

For transmission electron microscopy (TEM), ultrathin

sections were cut at 50 nm thickness using an RMC

MTXL ultra-microtome (Boeckeler Instruments, Inc.,

Tucson, Arizona, USA). Sections were collected on cop-

per triple slot grids and contrasted using uranyl acetate

and lead citrate following standard protocols (Reynolds

1963). For transmission electron microscopy, a Morgag-

ni 268 electron microscope (FEI Company, Hillsboro,

OR, USA) and MegaView III CCD — iTEM-SIS software

(Olympus, Soft Imaging System GmbH, Munster, Ger-

many) for image capture was used.

Image processing

All light microscopic (LM) and transmission electron

microscopic (TEM) images were processed using Im-

ageJ (version 1.50d, NIH, USA) and Adobe Photoshop

CC 2014 (Adobe Systems Incorporated, San Jose, CA,

USA). Image processing included background subtrac-

tion (LM), removal of non-relevant embedding materi-

al (TEM), and contrast enhancement. Assembly of im-

ages was done using Image Composite Editor (version

2.0.3.0, Microsoft Corporation, Redmond, WA, USA).

Addition of labels and scale bars were done using Adobe

Illustrator CC 2014 (Adobe Systems Incorporated, San

Jose, CA, USA). To aid recognition of organs in some

LM and TEM images, a color overlay was applied. The

color code of the LM images corresponds with the color

coding used in the schematic drawings.

Schematic drawings were created with Adobe IIlus-

trator CC 2014 (Adobe Systems Incorporated, San Jose,

CA, USA). They are strictly based on histological seri-

al sections and 3D-reconstructions of the organ systems

using Amira 6.0.0 (Mercury Computer Systems Inc.,

Chelmsford, MA, USA). Thus, the topography, the size

and the shape of structures and organs are documented

as precisely as possible based on the combination of two

methods. The degree of interpretative illustration 1s min-

imal.

Phylogenetic analysis

The phylogenetic analysis is based on Shultz (2007a).

He analyzed 59 euchelicerate taxa (41 extant, 18 fos-

sil) coded for 202 binary and unordered multistate mor-

phological characters. The matrix published by Shultz

(2007a) was supplemented with additional data collect-

ed in this study, and several character states were added

and updated according to new interpretations presented

Bonn zoological Bulletin Suppl. 65: 1-125

here (Appendix I: Tab. 6, Appendix IT). Schultz (2007a)

used a certain logic of construction of characters, 1.e.,

some characters we consider as one character would be

described as two, e.g.: carapace with distinct peltidial

sclerites: 0 = absence; | = present, number of peltidial

sclerites: 0 = two peltidia; 1 = three peltidia; inapplicable

due to absence of sclerites. We consider such character as

dependent of each other with restricted degrees of free-

dom and therefore consider them different states of one

character (applies to characters 6, 32, 116, 131, 194, 198

in our table 6 (Appendix I)).

The newly described fossil Chimerarachne yingi was

also included in the analysis based on the description of

Wang et al. (2018). Eukoenenia spelaea was regarded

as a separate taxon to allow independent analysis of its

characters. Otherwise, the analysis was set up identical

to Shultz (2007a), i.e., using Tree Analysis using New

Technologies (TNT), but, the newer version 1.5 was

used (Goloboff et al. 2008; Goloboff & Catalano 2016).

The non-arachnid euchelicerate taxa, 1.e., Xiphosurida,

were chosen as outgroup. “Traditional” tree searches

with 1000 replicates using TBR branch swapping were

performed. Bootstrap percentages (Felsenstein 1985)

and Bremer support (Bremer 1994) were calculated in

TNT. Absolute bootstrap percentages were determined

by 1000 pseudo-replicates, which were analyzed by ten

random-addition replicates using TBR branch swapping,

each. Absolut Bremer support was determined by mea-

surement of the difference between the unconstrained

and constrained minimal-length trees. Implied weights

analysis (Goloboff 1993) was performed to examine ho-

moplasy effects. Implied weights analyses with constant

of concavity (k) values of 1-6 were conducted using

“traditional” search based on 1000 replicates using TBR

branch swapping. Lower k-values are weighted more

strongly against homoplastic characters while higher

k-values are weighted less.

Terminology

We wish to keep terminology neutral and simple with-

out implications of unproven “homologies”. Therefore,

numbering of the body segments starts with | for the first

segment (the “ocular” segment) and not with zero as used

by several authors (Millot 1949b; van der Hammen 1989;

Shultz 2007b; Dunlop & Lamsdell 2017).

The terminology of the articles of the pedipalps and

legs is confusing and loaded with homology implica-

tions and inductive evolutionary reasoning (e.g., van der

Hammen 1977b; Boxshall 2004; Waloszek et al. 2005;

Dunlop & Alberti 2008; see also Tab. 8). Therefore, arti-

cles of appendages are numbered starting with 1 for the

most proximal article (Tab. 8). Possible homologies are

discussed.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 9

RESULTS

Individuals of Eukoenenia spelaea are small and delicate

with an elongate body shape, a long terminal flagellum,

and a light, almost translucent body (Figs 3-4). The

body length of the analyzed specimens varied between

0.90 mm and 1.35 mm (without the flagellum). The en-

tire body is covered by a dense pubescence (Pub; Fig. 9),

i.e., filiform cuticular microstructures ranging in length

between 2 um and 3 um. Numerous large, spiked senso-

ry setae (SS; Figs 3-4, 9; Tabs 1—4) are arranged on the

body surface. The number and position of the setae was

the same in all individuals. However, the number of se-

tae associated with the genitalia differed between males

and females (Tab. 3). We also observed a slight sexual

dimorphism in the shape of the genitalia, the body length

(females 1.23 + 0.13 mm, males 1.03 + 0.13 mm), the ex-

tent of imbrication of the opisthosomal tergites, and the

musculature of prosoma and opisthosoma (see below).

External morphology

The tagmatization of Eukoenenia spelaea (Figs 3—5) fol-

lows the typical euchelicerate body plan with a prosoma

and an opisthosoma. The prosoma (seven segments) car-

ries the extremities, 1.e., the chelicerae, the pedipalps and

four pairs of legs (Ch, PP, LI-4; Figs 3-5). The opistho-

soma (11 segments) is subdivided into an anterior meso-

soma (seven segments) and a posterior metasoma (four

segments). The metasoma carries a terminal flagellum

with 15 articles (Figs 3B, 4B). The flagellum has approx-

imately the length of the entire body.

Prosoma dorsum

The seven segments of the prosoma are partially merged

forming topographically distinct sclerites; however, the

borders between sclerites differ on the dorsal and the

ventral sides of the prosoma (Figs 3-5). Dorsally, the

prosoma is covered by three sclerites that have tradition-

ally been considered tergites and termed pro-, meso- and

metapeltidium. As will be shown below, the propeltidi-

um is the common dorsal sclerite (tergite) of segments

1-6 (not 1—5 as considered traditionally) with a posterior

imbrication. The posterior end of the propeltidium tapers

off and partially covers the mesopeltidium as well as the

most anterior part of the metapeltidium (PrPlt, PaPIt,

MtPlt; Figs 3A, C, SA—B). The mesopeltidium 1s repre-

sented by a pair of small, poorly sclerotized, dorsolateral

sclerites in the region of leg 3, and partially covers the an-

terior part of the metapeltidium. We will show below that

the mesopeltidium is not a tergite, but a sclerotization of

the dorsolateral pleural membrane. We therefore suggest

the term “/ateral dorsal plate” which will be used from

here on. The metapeltidium is the tergite of segment 7,

roughly triangular shaped with the pointed end towards

anterior, and a broad margin towards posterior. It extends

from the region between leg 2 and 3, 1.e., at the begin-

ning of the overhang of the propeltidium, to the end of

the prosoma (Figs 3A, C, 5A—B). Each prosomal sclerite

carries a fixed number of setae in the same topographic

positions (Tab. 1).

Prosoma ventrum

The ventral side of the prosoma has four sclerites

(Fig. 3D). A large anterior sclerite covers segments 2-4

(Figs 3D, 5C). This element has been termed deutotri-

tosternum and was considered to contain ‘epimera’ of

segments 2 and 3 (van der Hammen 1982). However,

we provide evidence that it contains 3 prosomal sclerites

related to segments 2-4. At its anterior margin, it has a

medial concavity, which embraces the ventral plate (VP;

Fig. 3D). Segments 5—7 carry individual segmental scleri-

tes (Figs 3D, 5C). They cover the entire region between

the first articles of the legs. All ventral prosomal sclerites

are free of setae, with the exception of the larger anterior

sclerite, which carries six setae (Tab. 1).

Table 1. Eukoenenia spelaea, number of setae located on the prosoma. The mesopeltidium is termed here “lateral dorsal plate”

(see discussion). The fused sternite of segments 2-4 is termed here “prosternum” (see discussion).

Number

Propeltidium 14(29)"

Lateral dorsal plate 7

Metapeltidium 6

Prosternum 6(5)’

“It is widely reported in the literature that individuals of £. spelaea have 20 setae on propeltidium (e.g.,

Christian et al. 2014, Condé 1956, Condé 1972). We give those literature values in brackets.

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

10 Sandra Franz-Guess & J. Matthias Starck

Ventral plate

In ventral view, the large lower lip covers the mouth

opening and the bases of the chelicerae (Fig. 3D). Direct-

ly behind the posterior end of the lower lip, located in a

notch of the anterior margin of the prosternum is a large

ventral plate with a unique morphology (VP; Figs 3D,

6). The plate is externally covered with uniform cuticu-

lar teeth pointing to the posterior. They are approx. 2 um

long, 0.4 um thick at the base and pointed (cT; Fig. 6B).

Rostrosoma

We use the term “rostrosoma” as a topographic descrip-

tion of the body region carrying the moth opening and

associated structures; the term as used here does not im-

ply a-priori homologies to the rostrosoma of other cheli-

cerates (e.g., van der Hammen 1989). The rostrosoma is a

prominent tubular structure hosting the preoral cavity, the

mouth opening, and anterior parts of the pharynx. It orig-

inates ventromedial between the bases of the chelicerae

and protrudes towards anterior, where it is nestled be-

tween the basal articles of the chelicerae (ROS; Fig. 3D).

Anterior, the rostrosoma extends into an upper and a

lower lip (Fig. 7). The upper lip forms lateral walls over-

arching the lower lip. These lateral walls emerge right

from the point where upper and lower lip separate from

the cuticular tube and extend to the anterior end of the

lower lip (LL, UL; Fig. 7F). There, the left and the right

lateral walls merge forming an anterior overhang in front

of the lower lip. This anterior overhang of the upper lip

is covered with a coarse pubescence on the outside and

fine-toothed transversal ridges on the inside (tR; Fig. 7B,

D-F). Five small setae insert on each side at the ventral

margin of the upper lip (Fig. 7E). The lower lip extends

into the anterior overhang of the upper lip where its cu-

ticle also forms fine-toothed, transversal ridges. These

transversal ridges probably function as counterparts to

the transversal ridges on the inner side of the upper lip

(tR; Fig. 7D). Fine-toothed, transversal ridges cover the

ventral side of the lower lip. Each of these ridges interca-

lates laterally with two sections of elongated, hook-like

teeth (fT, hT; Fig. 7E).

The mouth opening is small and located where upper

and lower lip merge into a cuticular tube. Here, two scle-

rotized knobs reach from the inner surface of the upper

lip to the lower lip (*, Fig. 7C—D, F). The cavity anterior

to these knobs 1s the preoral cavity. The pharynx with its

associated musculature is housed in the tubular part of

the rostrosoma.

Sensory organs on the prosoma

The prosoma carries several sensory organs, 1.e., the

frontal organ, the paired lateral organs, numerous senso-

ry setae, and trichobothria. The frontal organ is located

Bonn zoological Bulletin Suppl. 65: 1-125

dorsally on the apex of the prosoma and Is partially cov-

ered by the propeltidium (FO; Fig. 3A, C). The frontal

organ is unpaired and has a base that carries two modi-

fied setae. Each of these setae is approx. 25 um long and

has a diameter of approx. 5 um. The cuticular surface of

the frontal organ carries a honeycomb pattern (Fig. 8A)

formed by cuticular ridges and pits.

The lateral organs are left and right dorsolateral under

the propeltidium, above the base of the pedipalps (LO;

Figs 3A, C). However, an explicit association with a

prosomal segment is not possible because prosomal seg-

ments 1-6 are fused dorsally. Each lateral organ consists

of four modified setae. These setae are similar in shape

and size to the setae of the frontal organ (approx. 25 um

long and 4.5 um in diameter; Fig. 8B). Their cuticular

surface also carries a honeycomb pattern like the frontal

organ. The surface of the pits, which are surrounded by

the cuticular ridges, has cuticular grooves.

The entire animal carries numerous sensory setae. The

topographic arrangement of sensory setae on the sur-

face of the body was the same in all individuals stud-

ied (Fig. 3; Tabs 1-4). The spiked setae can reach up to

120 um in length. Each sensory seta inserts on a circu-

lar socket (diameter approx. 2 um) in the cuticle and is

flexible (Fig. 9). The diameter of the sensory setae de-

creases gradually from base (approx. 1.2 um) to tip (ap-

prox. 0.8 um).

In Eukoenenia spelaea, trichobothria are exclusively

located on the first pair of legs. The topographic distribu-

tion of trichobothria on the leg is as follows: article 4 car-

ries one, article 6 carries four, and articles 8 and 10 carry

one trichobothrium each (Fig. 3A). The trichobothria on

articles 4, 8, and 10 are oriented toward dorsolateral and

distal, 1.e., away from the body median line. The four

trichobothria on article 6 are arranged along a cuticular

groove on the surface of the article. Of those four tricho-

bothria, two insert distal to the cuticular groove, and the

other two proximal (cGR, Fig. 3A). All trichobothria root

in a cuticular, cup-shaped socket (bothrium, diameter ap-

prox. 6 um) with a toothed rim (tBR, Tr; Fig. 9). The

cuticular teeth surrounding the socket are approx. | um

long and 0.2 um thick at the base (Fig. 9B). The max-

imum deflection of the seta before touching the cuticu-

lar teeth of the socket is 25—30°. The seta is longer (ap-

prox. 250 um) and thinner (diameter consistently approx.

0.8 um) than other sensory setae.

Chelicerae

The chelicerae are proportionally large, and are orient-

ed straight towards anterior. They have three articles,

i.e., the basal article, the fixed digit, and the movable

digit, which together, form a chela (FD, MD; Figs 3A,

4C, 10A). The basal article has approximately the same

length as the chela. The propeltidium overlaps parts of

the basal article. The basal article is covered in pubes-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 1]

Fig. 3. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing based on light microscopic and scanning electron microscopic

images. A. Dorsally, the prosoma is divided into a pro-, para- (see discussion), and metapeltidium. The opisthosoma tapers off in

segment 15. The chelicerae consist of three articles. Arabic numbers indicate leg articles, starting with 1 for the most proximal article.

The frontal organ (green) and the lateral organ (orange) are largely covered by the propeltidium. Trichobothria are highlighted in

red. B. The flagellum. Long setae are less numerous than spikes and located more proximal on the articles. C. Lateral view of a

female. The frontal organ is nested underneath the anterior end of the propeltidium. D. Ventral view of a female. The prosternum

(see discussion) is a fusion of ventral sclerites associated with the chelicerae, pedipalps and the 1‘ leg. The previously undescribed

ventral plate (blue), is located between the rostrosoma and prosternum. Only large spiked setae are depicted. Abbreviations: Ch

= Chelicerae; ChBA = cheliceral basal article; cGr = cuticular groove; chGr = cheliceral groove; FD = fixed digit; FO = frontal

organ; GL = genital lobe; GO = genital operculum; L1—4 = leg 1-4; LL = lower lip; LO = lateral organ; MD = movable digit; MtPIt

= metapeltidium; PaPIt = lateral dorsal plate; PP = pedipalp; PrPlt = propeltidium; PSt = prosternum; ROS = Rostrosoma; St =

sternum; Ter = tergite; UL = upper lip; VP = ventral plate.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Sandra Franz-Guess & J. Matthias Starck

Table 2. Eukoenenia spelaea, number of setae located on each article of the extremities, excluding trichobothria on leg 1.

bs a aiake nes a Sp Art.4 Art.5 Art.6 Art.7 Art.8 Art.9 Art.10 Art. 11

Chelicera 6(10)! 6(7)! — na n.a. na n.a. na na. n.a. n.a.

Pedipalp 19 9 7(8) 10 3 6 1 6 20(24)P sna. n.a.

Leg 1 io) 13 9 9 9 9(8)" 4(3)'" 5 4(5y 6 22(25)

Leg 2 13(14)!? 3 5 5 5 4 11 na na n.a. n.a.

Lég3 13 3 5 2) 5 4 11 na na n.a. n.a.

Leg 4 10(9)!? 3 3 5 4(5) 4 4(5)° 7 n.a. n.a. n.a.

'Christian et al. (2010), ? reviewer comment, unpublished material

Table 3. Eukoenenia spelaea, number of setae located on opisthosomal segments (S) 8-19. The number of setae on the genital

plates are listed separately.

S8 S9 S10 S11

Tergite — 8 2 12!

Sternite — 4 — 4

Genitalia 9 n.a. 22 n.a. n.a.

Genitalia 3 na. 32 n.a. n.a.

S12 S13 S14 S15 S16 S17 S18

2! 12! 14 12 10 8 8

4 4 2, 4 4 n.a. n.a.

n.a. n.a. n.a. n.a. n.a. n.a. n.a.

' The taxonomic literature represents distinctly different numbers, probably because tergite and sternite are differently deliminated. The border

between both sclerites is extremely difficult to see. Based on our histological samples, we recognize that the tergites reach far to the ventral site

(Fig 3); taxonomic standard is tergite with 8 setae, sternite with 10 setae (with variability possible; Christian pers. comment)

Table 4. Eukoenenia spelaea, number of setae and spikes located on each article of the flagellum.

Art. Art. Art. Art. Art. Art. Art. Art Art. Art. Art. Art. Art. Art. Art.

1 Z 3 4 5 6 ii 8 9 10 11 12 13 14 [5

Setae 4 11 10 10 10 8 8 8 8 8 7 % it . 6

Spikes 14 14 14 — 13 - 13 - 12 - - — — -

cence. Two cuticular grooves span along the entire length

of the ventral side of the basal article. The lateral walls of

these grooves are knobbed (arrows; Fig. 10B), whereas

their inner ridge is smooth like the rest of the ventral side

of the basal article. The medial face of the basal articles

is concave providing space for the rostrosoma (Figs 3D,

7A).

The main body of the fixed digit is covered in pubes-

cence, however, the medial faces of the chelicerae are

smooth (*; Fig. 10A). We counted twelve setae on each

chelicera: six on the basal article and six on the fixed dig-

it (the taxonomic literature reports 10 and 7 setae on basis

and hand, respectively; Christian et al. 2010). The mov-

able digit has no setae (Figs 3, 10; Tab. 2). The teethed

sections of the movable and fixed digits carry eight serrat-

Bonn zoological Bulletin Suppl. 65: 1-125

ed teeth each (sT; Fig. 1OA, C—D). The distance between

the teeth is approx. 1—1.5 um, and approx. 0.05—0.1 um

between the serrations on the teeth. The tips of the digits

are elongated and almost as long as the toothed section

of the digits. The tips cross each other (Figs 3, 10A, C).

Pedipalps and legs

All extremities carry numerous setae and a terminal claw

(Tab. 2). The pedipalps and the first leg are both distinct-

ly longer than the other legs, have more articles, and are

directed to the anterior. The pedipalps and leg | are palp

shaped. In this species, only leg 1 carries trichobothria

(Fig. 3A). — The pedipalps are divided into nine articles.

Leg 1 is the longest pair of extremities, it has 11 articles.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 13

100um

Fig. 4. Eukoenenia spelaea Peyerimhoff, 1902), overview images. A. Dorsal view of a female, light microscopy. B. Lateral view

of a flagellum, light microscopy. C. Lateral view of a female, light microscopy. Sternites cannot be clearly seen due to the drying

process. D. Ventral view of a male, scanning electron micrograph. Abbreviations: A = anus; cGr = cuticular groove; Ch = Chelicerae;

ChBA = cheliceral basal article; chGr = cheliceral groove; FD = fixed digit; GL = genital lobe; GO = genital operculum; LI1+4 = leg

1-4; LL = lower lip; MD = movable digit; MtPlt = metapeltidium; PaPlt = lateral dorsal plate; PP = pedipalp; PrPlt = propeltidium;

ROS = Rostrosoma; SS = sensory seta; Sp = spike; St = sternite; Ter = tergite; UL = upper lip.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

14 Sandra Franz-Guess & J. Matthias Starck

A PrPit PaPit MiIPIt

PP L1 Mesosoma Metasoma

Prosoma Opisthosoma

Fig. 5. Eukoenenia spelaea (Peyerimhoff, 1902), segmentation. Prosomal structures are marked in shades of red, opisthosomal

structures are marked in shades of blue. Every hue marks a segment. A. Dorsal view. The propeltidium (horizontal hatching lines)

extends to the level between the 3 and 4" leg. The lateral dorsal plate (vertical hatching lines) is located at the level of the 3"

leg. The metapeltidium (diagonal hatching lines) covers the dorsum of the 7" prosomal segment, but extends anteriorly to the

level between leg 2 and 3. The mesosomal segments 8—14 carry segmental tergites and sternites; the metasomal segments 15-18

have sclerotized rings instead of tergites and sternites. B. Lateral view. In lateral view, the prosomal segments are difficult to

discern as there are no visible cuticular structures indicating segment borders. Opisthosomal segments are clearly delimited by their

sclerites. C. Ventral view. Fused sclerites of the cheliceral segment, the pedipalpal segment and the segment of the 1“ leg form a

prosternum. Segments 5—7 carry individual ventral sclerites. Numbers indicate segments. Abbreviations: L1—4 = leg 1—4; MtPlt =

metapeltidium; PaPlt = lateral dorsal plate; PP = pedipalp; PrPlt = propeltidium; PSt = prosternum.

Fig. 6. Eukoenenia spelaea (Peyerimhoff, 1902), scanning electron micrographs of the ventral plate. A. Ventral view of the ventral

plate. The cuticular teeth are arranged in even rows pointing posteriorly. B. Close-up image of the cuticular teeth. The teeth are

elongated and taper off at the tip. Abbreviations: cT = cuticular teeth; ROS = rostrosoma; VP = ventral plate.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 15

ante

am... oe e-erist

Neen sane ae = sath eat

we mivatten sas rpms

— Seow, re _

Fig. 7. Eukoenenia spelaea (Peyerimhoff, 1902), rostrosoma. A. Light micrograph of a cross-section through the rostrosoma anterior

to the mouth opening (inset). The rostrosoma is nestled between the cheliceral basal articles. B. Transmission electron micrograph

of cross-section of the anterior overhang of the upper lip (inset). The toothed ridges are spaced regularly. C. Light micrograph of

a slightly oblique longitudinal section of the rostrosoma. The mouth opening lies posteriorly of the lateral protrusions (asterisk)

of the upper lip. D. Lateral view of the rostrosoma, schematic drawing based on longitudinal and cross-section light micrographs.

Parts of the upper lip (red) have been removed to display the mouth opening. The grey area indicates that upper and lower lip

are fused. E. Ventral view of the rostrosoma, scanning electron micrograph. The lower lip is covered in finely toothed ridges and

hook-like teeth (inset). F. Ventral view of the rostrosoma, schematic drawing based on cross-section light micrographs as well as

scanning electron micrographs. Parts of the upper and lower lip have been removed to display the close association of the lateral

protrusions with the mouth opening. Abbreviations: ChBA = cheliceral basal article; ff = fine tooth; hT = hook-like tooth; LL =

lower lip; MO = mouth opening; P = pharynx; PP = pedipalp; PrPlt = propeltidium; tR = toothed ridge; asterisk = lateral protrusions

of functional upper lip; UL =upper lip; ULO = lip overhang; VP = ventral plate.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

16 Sandra Franz-Guess & J. Matthias Starck

Fig. 8. Eukoenenia spelaea (Peyerimhoff, 1902), scanning electron micrographs of the frontal organ and lateral organ. A. Ventral

view of the frontal organ. It consists of a base and two modified setae. The cuticular structure of the base is irregular. The setae

carry a cuticular honeycomb pattern. B. Ventral view of the lateral organ. It consists of four modified setae. The setae carry the

same honeycomb surface pattern like the frontal organ. The cuticular ridges form pits with grooved openings in the cuticle (inset).

Abbreviations: Ch = chelicera; cGr = cuticular groove; cP = cuticular pit; cR = cuticular ridge; FOB = frontal organ base; FOS =

frontal organ seta; LOS = lateral organ seta; PrPlt = propeltidium.

The most proximal articles of the pedipalps and leg 1 are

elongate as compared to those of the following legs. Ar-

ticle 6 of leg 1 shows a cuticular groove which spans the

entire article diagonally (Figs 3A, 4C). Legs 2 and 3 are

located on segments 5 and 6, respectively. They are both

oriented towards lateral and consist of seven articles. In

both legs, the proximal article is short and appears wid-

er than those of the other legs. Leg 4, 1s located in seg-

ment 7. It is oriented posteriorly and consists of eight

articles. Its proximal article 1s elongated and borders the

posterior ventral sclerite.

The articulations between articles of the legs are locat-

ed dorsally in all appendages and all articles (Fig. 11A—

C). In figure 11, we used the formalized schematic of van

der Hammen (1977a) to document the position of joints

and muscle/tendon attachments for a later comparative

discussion. Despite this, the articulations between arti-

cles are rather like a broad ridge in the proximal articles

(1/2-4/5, Fig. 11D—E) and rather like a point articulation

in the distal articles (5/6—x/y). The membrane connecting

the articles displays a knobbed surface (Fig. 11F). The

articulations between the first article and the body could

not be clearly determined.

Fig. 9. Eukoenenia spelaea (Peyerimhoff, 1902), scanning electron micrographs of the different types of sensory hairs.

A. Trichobothria and sensory hairs on articles 6—8 of leg 1. There are noticeable differences in length and thickness between sensory

setae and trichobothria. B. Close-up of the bases of a sensory seta and a trichobothrium. The sensory seta is thicker and its spikes

are longer than in the trichobothrium. However, the cuticular socket of the sensory seta is an approx. 3-fold smaller than that of the

trichobothrium. The protuberances of the pubescence have no sockets. Abbreviations: Pub = pubescence; SS = sensory seta; tBR =

toothed bothrial rim; Tr = trichobothrium.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 17

Fig. 10. Eukoenenia spelaea (Peyerimhoff, 1902), scanning electron micrographs of the chelicerae. A. Dorsal view of the fixed

and the movable digits. The inward oriented part of the main body of the fixed digit is free of pubescence (asterisk). The basal

article displays cuticular grooves (see B). B. The ridges of the lateral basal article grooves are knobbed (arrows), the central ridge is

smooth. C) The tips of the cheliceral digits are crossed. D. Close-up image of the teethed section of the cheliceral digits. The teeth

are serrated. Abbreviations: FD = fixed digit; MD = movable digit; Pub = pubescence; sT = serrated tooth.

Opisthosoma

The opisthosoma has 11 segments and is subdivided

into a mesosoma (segments 8—14) and a metasoma (seg-

ments 15—18; Figs 3-5). Segment borders are recognized

by the anterior and the posterior margins of the tergites

and sternites, respectively (Figs 3—4). Mesosoma and

metasoma are distinguished by their sclerites, 1.e., seg-

ments of the mesosoma carry dorsal and ventral sclerites,

and segments of the metasoma carry cuticular rings sur-

rounding the entire segments. In the mesosoma, segment

8 is short and has a smaller diameter than the following

six segments. In segments 9-17, the dorsal sclerites are

imbricated forming posterior overhangs that reach over

the anterior part of the following segment. In females,

these overhangs are thicker than in males (SD; Fig. 12A—

B), 1.e., the base of the overhang is up to 20 um wide

while the distal part tapers off gradually (Fig. 12A). In

Bonn zoological Bulletin Suppl. 65: 1-125

males, the base of the overhang is 5 um wide and the

distal part is evenly thin (diameter max. | um; Fig. 12B).

The segments of the metasoma, 15—18, decrease in di-

ameter (Fig. 5). Attached to the last segment is a sclero-

tized ring, which is the base of the flagellum (Fig. 13).

The anus is located between segment 18 and the annular

basal article of the flagellum, 1.e., ventral to the base of

the flagellum. The number of setae on each segment were

consistent in all individuals, except for the sex-specific

number of setae on the genital plate (Tab. 3).

Opisthosomal sclerites

The tergites of the mesosomal segments expand far later-

ally (Figs 3C—D). The tergites of segments 8 and 9 reach

to the lateral midline where they meet their respective

sternites. In segments 10-14, the tergites reach further

down to the ventral side (Fig. 12C), and the sternites of

these segments are reduced to small medial sclerites.

©ZFMK

18 Sandra Franz-Guess & J. Matthias Starck

= =)

== 5

Fig. 11. Eukoenenia spelaea (Peyerimhoff, 1902), frontal views of pedipalp, leg 1 and leg 2 from the right side of the prosoma.

Contrast enhanced light micrographs. The icons follow the formalized scheme of van der Hammen 1977a) documenting the

position of the joints and muscle attachment sites for each article. Articles of the appendage are numbered, the black dot in the icon

shows the position of the hinge, red arrow heads indicate the muscle/tendon attachment. A. Pedipalp. Muscle attachment in the

joint between articles 2 and 3 are lateral. The muscle attachment at the following joint (3/4), has shifted to latero-dorsal. All distal

muscle attachments are ventral. B. Leg 1. Two or three muscle attachment sites can only be found in the joints connecting articles

3/4 and 5/6, respectively. Single muscle attachment in all other joints is predominately located ventral and ventrolateral. C. Leg 2.

Only two joints with muscle attachment sites are found in this leg. In the joint between articles 3 and 4, the attachment is located

laterally, in the joint between articles 5 and 6, the attachment is ventral. All other joints lack any muscle attachments. — continued.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 19

20um

— a Se

| Boyle Oey

| Sneed

sige TEENY sr

: ra

er oo

P14,"

MK ETI) radd

LL ST hh

i ay,

LA) eee

;

\ 4

. '

)

3 “S

- —

4 *

4 —

Mis

- S

= ~

& = = SR

= we

a ae

Fig. 11 continued. Eukoenenia spelaea (Peyerimhoff, 1902), joints on leg 2. Scanning electron micrographs. Numbers indicate the

leg’s articles. D. Overview of leg 2. Arrows indicate the location of the joints. Between the proximal articles (1/2, 2/3, 3/4, 4/5)

the joint is broader than between the distal articles (5/6, 6/7). The same configuration can be found in the legs and the pedipalp.

E. Articulation between articles 4 and 5. The articulation is a broad ridge. F. Close-up oblique view of the joint between articles 3

and 4. The membrane connecting both articles has a knobbed surface (arrows).

Each segment of the metasoma, 15-18, carries a cuticular

ring surrounding the entire segment (Figs 3, 5).

On the ventral side of the mesosoma, the sternites dif-

fer between segments. The sternites are more distinct

in males than in females. In both sexes, the cuticle of

the sternites of segments 8—14 is weakly sclerotized and

forms folds, which are particularly well developed on the

lateral side of the sternite (Fig. 12C—E). The sternites of

segments 8 and 9 cover the entire ventral side of the ani-

mal’s body (Fig. 3C—D). The ventrum of segment 9 also

carries the genital plate, which gives it a unique morpho-

logy (see below; Fig. 14). In segment 10, the sternite is

Bonn zoological Bulletin Suppl. 65: 1-125

restricted to the middle part of the ventral side (Fig. 3D).

The anterior part of this sternite is also involved in build-

ing the genital plate, the rest of the sternite is shaped

evenly. The sternites of segments 10—14 overlap posteri-

orly. A groove is vaguely perceptible between the tergite

and the sternite (Fig. 3D).

Genital segment

In females, the external genital apparatus is formed by

the genital operculum and the paired genital lobes (GL,

GO; Figs 3C—D, 14A—C) which, however, are almost

©ZFMK

20 Sandra Franz-Guess & J. Matthias Starck

Fig. 12. Eukoenenia spelaea (Peyerimhoff, 1902), structure of the cuticle. A. Female, light micrograph of a sagittal section of the

opisthosoma. Two segments are shown with their dorsal sclerites. The sclerites overlap broadly. The base of the sclerite duplicature

is between 10 um and 20 um wide. The distal part of the duplicature is thicker in females than in males (see B). B. Light micrograph

of longitudinal section of the opisthosoma cuticle of a male. The base of the duplicature is approx. 5 um wide. The distal part of

the duplicature is max. 1 um in thickness. C. Light micrograph of a cross-section through segment 11 (inset). The ventral sclerite

carries cuticular folds laterally. Orange lines indicate the border of tergite and sternite. D. High power light micrograph of the

lateral region of the sternite from C. The cuticular folds are clearly visible. E. Transmission electron micrograph of cross-section of

the same region as D. The cuticle in this area is thin compared to the neighboring areas. Abbreviations: C = cuticle; cF = cuticular

folds; EC = epidermal cell; MG = midgut; N = nucleus; OV = ovary; Pub = pubescence; SD = sclerite duplicature; St11 = sternite

of segment 11; Terl1 = tergite of segment 11.

Fig. 13. Eukoenenia spelaea (Peyerimhoff, 1902), structure of the terminal ring. A. Light micrograph of longitudinal section

through the terminal structures. The sclerotized ring is articulated with segment 18 and the following flagellar article. B. Scanning

electron micrograph of the sclerotized ring, viewed from posterior. The membranous fold between segment 18 and flagellar base

lacks pubescence but shows a knobbed structure. Four short setae insert on the sclerotized ring. Abbreviations: A = anus; fA =

flagellar article; mF = membranous fold; S18 = segment 18; sR = sclerotized ring; sS = short seta.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 21

Fig. 14. Eukoenenia spelaea (Peyerimhoff, 1902), genital structures. A. Scanning electron micrograph of female genitalia in

slightly oblique dorsal view. The genital operculum covers the genital lobes. B. Schematic drawing of female genitalia in dorsal

view. The extension of the genital operculum is elongated. C. Schematic drawing of female genitalia in lateral view. The finger-like

genital lobes originate at the junction of segments 9 and 10. D. Scanning electron micrograph of male genitalia in dorsal view. The

number of setae is larger in comparison to the female genitalia. E. Schematic drawing of male genitalia in dorsal view. The base of

the first pair of genital lobes is broadest compared to genital lobes 2 and 3. At the base, all genital lobes are closely together, at their

tips, the lobes are spread farther apart. F. Schematic drawing of male genitalia in lateral view. The first pair of genital lobes has a

rounded tip, whereas lobes 2 and 3 are pointed. The paired fusules originate on genital lobe 1. These fusules extend posterior past

genital lobe 3. Abbreviations: FS = fusules; GL = genital lobe; GO = genital operculum.

entirely covered by the genital operculum. The genital

operculum is formed by the sternite of segment 9; the

genital lobes originate at the junction of segments 9 and

10. At its posterior end, the genital operculum shows

a short extension, whose sides arch upward to create a

tube-like structure. The genital operculum carries 16 se-

tae, the genital lobes carry three setae each (Fig. 14A—C;

Tab. 3).

In males, the external genital apparatus consists of three

pairs of genital lobes. Two pairs of genital lobes emerge

from the central and posterior part of segment 9, prob-

ably representing the genital operculum, followed by a

third pair of genital lobes originating at the border to seg-

ment 10. The first pair of genital lobes has a broad base

and two finger-like processes. At the tip of each process

are two adjoining fusules (FS, GL1; Fig. 14D-—F). The

second pair of genital lobes, originating from the poste-

rior part of segment 9, is located just posterior to the first

pair and has triangular-shaped lobes (GL2; Fig. 14D-F).

Bonn zoological Bulletin Suppl. 65: 1-125

The third pair of genital lobes is similar in shape to the

second pair, but ends in two needle-like processes (GL3;

Fig. 14D-—-F). The tips of the lobes are in contact with the

tips of the preceding lobes. A total of 32 setae are located

on the genital lobes: 18 on the first pair, six on the second

pair, and eight on the third pair (Fig. 14D-F; Tab. 3).

Flagellum

The flagellum is a long terminal attachment to the metaso-

ma and carries numerous flexible setae and inflexible

spikes. It is extremely delicate and fragile. Only one in-

tact flagellum was obtained from all analyzed specimens.

It consists of 15 articles and has a total length of 1.40 mm

(Fig. 3B). The flagellar base article is a sclerotized ring

and is connected with segment 18 with a membranous

fold, which displays a knobbed surface (mF; Fig. 13).

This base article carries four short setae (approx. 20 um

each) in a square arrangement. The other articles are sim-

©ZFMK

22 Sandra Franz-Guess & J. Matthias Starck

ilar to each other in shape, but vary in size, number of _ the spikes 1s, in contrast to that of the setae, smooth. The

setae, and number of spikes. The general number of setae length of a spike (approx. 30 um) is about one-sixth the

and spikes decreases from proximal to distal (Tab. 4). length of a seta (approx. 200 um; Figs 3B, 4B).

The setae are located posteriorly on each article. The

spikes are located towards the apical end. The surface of

Re ee a

a i

nee

moe

Eas,

Fig. 15. Eukoenenia spelaea (Peyerimhoff, 1902), epidermis. A. Transmission electron micrograph of the cuticle and epidermis on

the ventral side of the prosoma. The epidermal cells are arranged as squamous epithelium with flattened nuclei. B. Light micrograph

of the ventral part of a cross-section through the prosoma at the level of the pedipalp (inset). The epidermal cells under the ventral

plate are modified. C. Transmission electron micrograph of the epidermis cells under the ventral plate showing microvilli which

extend into the cuticle. D. Transmission electron micrograph of the epidermis under the ventral plate. The nuclei and mitochondria

are located basal in the cells, whereas the apical part carries microvilli. The basal part of the cells contains glycogen granules

(arrows). The light layer within the cuticle (asterisk) is a fixation artefact. Abbreviations: C = cuticle; EC = epidermal cell; EnC =

endocuticle; EpC = epicuticle; ES = esophagus; ExC = exocuticle; M = mitochondria; MF = muscle fiber; MV = microvilli; N =

nucleus; NP = neuropil; Pub = pubescence; PrC-B = type B pericarya; PrC-D = type D pericarya; sEC = specialized epidermal cell;

VP = ventral plate.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 23

Internal morphology

Epidermis

The epidermis of Eukoenenia spelaea is a typical sin-

gle-layered, squamous epithelium with elongated, heter-

ochromatin-rich nuclei. The thickness of the epidermis

varies over the body. In the tightly packed prosoma, the

squamous cells can be thin with a height of less than

1 um. In these regions, the heterochromatin-rich nuclei

of the epidermal cells are also flattened (Fig. 15A).

The epidermis cells of the ventral plate differ from

those of the rest of the body. In light microscopy, they ap-

pear as a homogenously stained cytoplasmic region be-

tween the subesophageal ganglion and the cuticle (SEC;

Fig. 15B). These modified epidermal cells occupy the en-

tire region of the ventral plate (Figs 3D, 15B, D). Trans-

mission electron microscopy reveals cellular polarity

with the nuclei and mitochondria located basally (M, N;

Fig. 15D). The basal part of the cells contains glycogen

granules and has membranous invaginations, 1.e., a basal

labyrinth. The apical borders of the cells form numerous

microvilli (MV; Fig. 15C) that extend into the cuticle,

tightly connecting the epidermal cells with the overlay-

ing cuticle. Cuticular pores associated with the microvilli

were not found. The apical part of the specialized epi-

dermal cells is almost completely void of glycogen gran-

ules. The cuticle covering the specialized epidermal cells

carries smooth cuticular teeth. These cuticular teeth are

part of the exocuticle and are covered by electron-dense

epicuticle (EpC, ExC, VP; Fig. 15C).

Cuticle

The cuticle of Eukoenenia spelaea, like that of other ar-

thropods, consists of two layers: the procuticle and the

epicuticle (EpC, ProC; Fig. 16). The thickness of the pro-

cuticle is about 0.2 um in the region of the epidermal

folds of the opisthosoma (Fig. 12E) and 1.5 um in the

region of the prosomal sclerites (Fig. 16E). In contrast to

the rest of the body, the procuticle of the prosomal scleri-

tes and the ventral plate is stratified into an endo- and an

exocuticle (EnC, ExC; Figs 15C—D, 16E). The exocuticle

is approx. 0.3 um thick. Pore canals of different shape

and size are found within the procuticle (PC; Fig. 16).

The epicuticle is uniform in thickness (0.01 um), but dif-

fers in electron density. While overall the epicuticle is

electron-dense, it 1s electron-translucent in the esopha-

gus (Fig. 37D), on the frontal (Fig. 27B), and the late-

ral organ, as well as some regions of the opisthosoma

(Fig. 16F). The pubescence, which occurs over almost

the entire body, represents cuticular surface sculpturing

and is formed by procuticle, but it appears to lack an epi-

cuticle (Figs 12E, 16C).

Bonn zoological Bulletin Suppl. 65: 1-125

Endosternite

The endosternite lies in the center of the prosoma. It ex-

tends from the region of the 1* leg to the region of the 4"

leg. The two lateral arms of the endosternite form a hor-

izontal V in an anterior-posterior axis, with the opening

towards anterior (EBr; Fig. 21A). The anterior transverse

bridge connects both lateral arms (EaB; Fig. 21). The

lateral arms continue towards posterior to the posterior

transverse bridge at the level of the 3 leg (EpB; Fig. 21).

A medial central bridge (EcB; Fig. 21A) connects the an-

terior and the posterior bridges. Posterior to the level of

the 3™ leg, the endosternite narrows and makes a concave

bend towards ventral (EU; Fig. 21B). It then broadens

into a stylized W in an anterior-posterior axis, with the

opening towards posterior, before terminating in the last

prosoma segment (EpS; Fig. 21A).

The endosternite is cellular (ESt; Fig. 17A). The endo-

sternal cells are surrounded by an approx. 1.5 um thick

extra-cellular matrix (Fig. 17B, D). This matrix is the

point of attachment for a large number of muscles. These

muscles attach to the ECM with hemidesmosomes (ar-

rows; Fig. 17B, D). The endosternite cells have a central

heterochromatin-rich nucleus, which is surrounded by

the cytoplasmic compartment containing all other cellu-

lar organelles. The cytoplasm stains light in light micros-

copy and is electron-translucent in transmission electron

microscopy (Fig. 17A—B). The rough endoplasmic retic-

ulum does not form flattened cisternae, but large com-

partments filled with fine granules (Fig. 17B—C). Few

glycogen granules and free ribosomes are also found in

the cytoplasm.

Muscle ultrastructure

The somatic musculature of Eukoenenia spelaea is trans-

versely striated, and each fiber consists of several myo-

fibrils (MF, MyF; Fig. 18A—B). The visceral muscula-

ture is also striated, but muscle fibers are small, usually

containing only one, single myofibril (Fig. 18D). In the

somatic muscles, the Z-line, A-band and I-band are clear-

ly visible in longitudinal section (A, J, Z; Fig. 18B). On

their surface, the muscle fibers have membrane invagina-

tions at positions corresponding with the Z-lines of the

sarcomeres (arrowheads; Fig. 18B). These invaginations

probably lead into the T-tubular system. The sarcoplas-

mic reticulum is well developed (SR; Fig. 18C). Both

components, the T-tubular system and the sarcoplasmic

reticulum, form diads (arrows, Fig. 18C inset) throughout

the muscle fiber. The nuclei are positioned centrally and

are surrounded by a perinuclear cytoplasmic compart-

ment that contains all cellular organelles. Mitochondria

are scattered throughout the muscle fibers (M; Fig. 18C).

— The prosoma contains prominent musculature associ-

ated with the endosternite and the body wall as well as

musculature in the legs (Figs 19-21; Appendix I: Tab. 5).

©ZFMK

24 Sandra Franz-Guess & J. Matthias Starck

Fig. 16. Eukoenenia spelaea (Peyerimhoff, 1902), transmission electron micrographs of the cuticle. A. Cross-section of the basal

article of the chelicera. The procuticle is contains numerous thin pore canals. B. Cross-section of the rostrosoma. The electron-

translucent procuticle appears single layered and has only few pore canals. The epicuticle appears electron-dense. C. Cross-section

of the propeltidium. The pore canals in the procuticle widen towards the surface of the cuticle and show electron-dense content. The

area below a process of the pubescence is electron-translucent (asterisk). The pubescence consists of electron-dense procuticle. D.

Cuticle of a leg. The procuticle shows few pore canals. The electron-dense epicuticle appears frayed. E. Cuticle of prosomal sternum

associated with leg 2. The layering of the procuticle in endo- and exocuticle is clearly visible. The endocuticle is multi-layered as

well. The exocuticle is electron-denser than the endocuticle F. Cuticle of the opisthosoma. The procuticle is approx. 0.4 um thin.

The epicuticle is electron-translucent. Abbreviations: EnC = endocuticle; EpC = epicuticle; ExC = exocuticle; PC = pore canal;

ProC = procuticle; Pub = pubescence.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 25

Yon WEE ere.

Fig. 17. Eukoenenia spelaea (Peyerimhoff, 1902), endosternite. A. Light micrograph of a cross-section of the middle position

at the level of the 2™ leg. The cells of the endosternite have a central nucleus, peripheral cytoplasm and substantial extracellular

matrix around the cells. B. Transmission electron micrograph of the endosternite. Cytoplasmic extensions of the endosternite

cells extend into the ECM. Musculature attaches to the ECM by hemidesmosomes (arrows). C. High power transmission electron

micrograph of the rough endoplasmic reticulum surrounding the nucleus from B. The RER forms large compartments instead of the

typical flattened cisternae. D. Transmission electron micrograph of the extracellular matrix of the endosternite. Muscles attach with

hemidesmosomes (arrows). The ECM has a granular character. Abbreviations: ECM = extra cellular matrix; ESt = endosternite; M

= mitochondrion; MF = muscle fiber; MG = midgut; N = nucleus; RER = rough endoplasmic reticulum; SubEG = subesophageal

ganglion; SupEG = supraesophageal ganglion.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

26 Sandra Franz-Guess & J. Matthias Starck

The musculature of the opisthosoma is less prominently

developed than in the prosoma (Figs 19-20).

Musculature of the prosoma

The description of the musculature of the prosoma fol-

lows major topographic relationships, 1.e., muscles of the

extremities, muscles of the body wall, and muscles as-

sociated with the endosternite. This formal distinction is

for descriptive purposes and does not intend to be a func-

tional interpretation. Detailed descriptions with points of

origin and insertion for each muscle are given in table 5.

Possible antagonists are described according to the to-

pography of their origin and insertion points.

Musculature of the extremities

The musculatures of the chelicerae, the pedipalps and legs

1-4 are described for the right side. Thirteen muscles in-

sert on the chelicera. Three extrinsic muscle strands (C1,

C3, and C4; Tabs 5, 10) originate dorsal from the pro-

peltidium and insert at the basal article of the chelicerae.

These muscles might move the chelicerae up and down,

with muscles Cl and C3 probably being antagonists to

C4 (Figs 19B, 20A). The basal article of the chelicera

contains three strands of prominent intrinsic muscula-

ture (C5—7) and two minor intrinsic muscle strands (C2

and C8). Muscles C5 and C7 are possible antagonists to

C6 (Figs 19A, 20A). The movable digit of the chelicera

as well as the tip of the fixed digit are free of muscula-

ture. Cheliceral muscles C9-13 insert with tendons on

the movable digit immediately distal to the joint between

movable and fixed digit. They probably close the chela.

No musculature with antagonistic topography was found,

thus hemolymph pressure and/or elasticity presumably

provide the necessary antagonism for opening the chela

(Figs 19A, 20A).

The pedipalp has a total of five muscles (PP1—PP5) in

its proximal articles and five tendons (PP6t—PP10t) in its

distal articles (Figs 11A, 19A—B, 20A, C; Tabs 5, 12).

One extrinsic muscle (PP1) originates dorsal from the

propeltidium. Like the pedipalp, leg 1 has muscles (LI1—

LI8) in the proximal articles but tendons (LI9t—LI14t)

in the distal articles. In both extremities, the tendons are

attached to the cuticle by pulleys in each article. The

two extrinsic muscles of legs 1-3 (LI1, LI2, LH1, LH2,

LIW1, and LUI2), originate from the propeltidium and the

endosternite, respectively (Figs 19A—B, 20A, C; Tabs 5,

12). In addition, legs 2 and 3 have seven (LI3—LH9)

and eight (LI3—LIII10) intrinsic muscles, respectively.

Leg 4 has 11 intrinsic (LIV4—LIV14) and three extrinsic

(LIV1—LIV3) muscles. Most extrinsic muscles originate

from the endosternite (except PP1, LI, LII1, and LI;

Figs 19A—B, 20A, C; Appendix I: Tab. 5). The determina-

tion of origin and insertion of intrinsic leg muscles from

the analysis of serial sections of pedipalps and legs sug-

Bonn zoological Bulletin Suppl. 65: 1-125

gests that some pairs of intrinsic muscles might function

as antagonists, while other muscles have no antagonist

and hemolymph pressure might provide the antagonistic

function. In the pedipalp, articles 7-9 have no intrinsic

antagonistic pair of muscles. The same 1s true for articles

6-11 of leg 1. The muscle and tendon of article 6 span the

entire article. Articles 6 and 7 1n legs 2 and 3, and articles

6-8 in leg 4 also have no intrinsic antagonistic pair of

muscles (Figs 19A, 20C). For comparative purpose, it is

important to note that no leg musculature originates from

the ventral sclerites of the prosoma.

Musculature of the body wall

Thirteen muscles (P1—13) originate from the body wall,

but are not directly associated with the extremities or the

endosternite. Prosomal muscles 1 and 2 are associated

with the upper lip of the rostrosoma. The paired P2 mus-

cles might act as antagonists to each other to enable lat-

eral movement of the upper lip. An antagonistic muscle

for Pl was not recognized. The muscles associated with

the pharynx (unpaired P3 and paired P4) function as dil-

atators of the pharynx. The circular muscle of the phar-

ynx wall might serve as their antagonist (Figs 19B, 20A,

37A).

The majority of the other prosomal muscles (P5—12)

originates dorsal, and inserts posterolateral on the body

wall (Figs 19B, 20A; Appendix I: Tab. 5). Sexdimorphism

in the musculature of the body wall can be found in P11

and P12. Muscles P11 and P12 are actually extensions of

the dorsal longitudinal muscle system of the opisthoso-

ma into the prosoma. In females, the longitudinal proso-

mal muscle P11 is paired and elongated; it connects the

propeltidium with the metapeltidium. It has a thin lateral

branch that insert on the pleural membrane posterior to

leg 3. In males, muscle P11 is paired, but shorter as com-

pared to females. Thickness and branching of the mus-

cle, however, show no differences between females and

males (Figs 19B—C, 20A-B). In females, muscle P12 is

largely reduced, unpaired, short, and does not extend into

the following segment. In males, this muscle 1s paired

and connects the metapeltidium with the first opisthoso-

mal segment (Figs 19B, 20B; Appendix I: Tab. 5).

Musculature of the endosternite

Dorsal suspensor muscles: Dorsal suspensor muscles are

diagnosed as the segmental muscles originating from the

endosternite and inserting on the dorsal shield of their

prosomal segments. Because segmental borders are not

recognizable on the endosternite or the dorsal shields of

Palpigradi the assignment of muscles to segments re-

mains to some degree arbitrary. However, the relation-

ship to the prosomal extremities is always clear, thus our

description uses the topographic relationship to the pro-

soma appendages as landmark for segment assignment.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 27

Fig. 18. Eukoenenia spelaea (Peyerimhoff, 1902), musculature. A. Cross-section through the prosoma, light microscopy. Striated

muscles fibers are arranged in a bundle, the nuclei are located centrally. The Z-lines are clearly visible. B. Prosomal muscle fiber,

transmission electron micrograph. The Z-lines as well as the A-band and I-band are clearly visible. The lateral invaginations of

the sarcolemma (arrowheads) represent the T-tubular system. C. Prosomal muscle fiber, transmission electron micrograph. The

sarcoplasmic reticulum (SR) displays narrow as well as widened areas. The nucleus is located in the center. The T-tubular system

together with the SR form dyads throughout the muscle (inset, arrows). D. Visceral muscle of the midgut, transmission electron

micrograph. The muscles are delicate in comparison with the somatic muscles. Abbreviations: A = A-band, J = I-band; M =

mitochondrion; MF = muscle fiber, MyF = myofibril; N = nucleus; S = sarcolemma; SR = sarcoplasmic reticulum; Z = Z-line.

Five muscles (E3, E5, Ell, E14, E17) originate from

the dorsal side of the endosternite and insert dorsally on

the body wall (Fig. 21; Tabs 5, 7). Muscle E3 originates

from the anterior end of the endosternite (pedipalpal seg-

ment) and extends straight dorsally where it inserts on

the propeltidium; it is recognized as the dorsal suspensor

muscle of the third segment (pedipalpal segment). Mus-

cle E5 originates posterior to E3 in the region of leg 1

and extends dorsally but with a slight posterior orienta-

tion; it also inserts on the propeltidium. Muscles E11 and

E14 originate posterior of leg 2 and posterior of leg 3,

respectively. Both muscles converge to the same poste-

rior insertion point on the posterior part of the propeltid-

ium. Muscle E17 originates from the endosternite in the

region of leg 4, extends straight dorsal and inserts on the

metapeltidium.

Bonn zoological Bulletin Suppl. 65: 1-125

Anterior oblique suspensor muscle: Muscle E6 origi-

nates dorsal from the endosternite close to the origin of

muscle E5 (dorsal suspensor of the 4" segment) and ex-

tends to the pleural membrane where the chelicera artic-

ulates with the prosoma. Based on the topography of its

origin and insertion, it 1s diagnosed as the only anterior

oblique suspensor muscle found in the prosoma (Fig. 21;

Tabs 5, 7).

Posterior oblique suspensors muscles: The posteri-

or oblique suspensor muscles originate dorsal from the

endosternite and insert posterolateral on the body wall.

We found four segmental muscles (E4, E12, E15, E20) in

such a topographic position (Fig. 21; Tabs 5, 7). Muscle

E4 originates from the endosternite in the topographic

neighborhood to leg 1, and inserts posterior on the pro-

peltidium in the region of leg 2. Muscles E12 and E15

originate in topographic relationship to legs two and

©ZFMK

28 Sandra Franz-Guess & J. Matthias Starck

i ra

rr]

PLATT ERAN EUAN LAN OLA BAN

EAL

bALL

Fig. 19. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of a dorsal view of the musculature. The drawing is based

on serial cross-section light micrographs and 3D-reconstructions of the musculature. Female/Male indicates that the musculature

is identical in both sexes. Female or male indicates sex dimorphism of musculature. A. In the prosoma, only extrinsic muscles of

the extremities originating from the endosternite, and the intrinsic muscles of the walking legs, the pedipalps and the chelicerae are

shown; all other prosomal musculature is shown in B (males) and C (females). Tendons in the chelicerae, pedipalps, and leg! are

highlighted in red. Tendons are labeled by a terminal ‘t’ in their name. In the opisthosoma, only the ventral longitudinal muscles, the

dorsoventral muscles, and transverse muscles are show. The dorsal longitudinal, the intersegmental and the flagellar musculature

are not shown (see Fig. 19C, 20 A, B for those muscles). Inset: cross-section of the mid-section of segment 11 of a female. Female-

specific opisthosomal muscles are marked orange. Abbreviations: C2—13 = cheliceral muscle; DV 1—5 = dorsoventral muscle; Gf =

genital muscle female; LI2—14t = leg 1 muscle/tendon; LII2—9 = leg 2 muscle; LII1—10t = leg 3 muscle; LIV1—14 = leg 4 muscle;

PP2—10t = pedipalps muscle/tendon; TI1—2 = transversal muscle 1; TI[1—2 = transversal muscle 2; TII]1—2 = transversal muscle

3; TIV1—2 = transversal muscle 4; TV1—2 = transversal muscle 5; TVI1 = transversal muscle 6; V = ventral muscle. — continued.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 29

Bia

Piz

Py Pe PO POD Pit

PS PeT UN) Pe LH

Fig. 19 continued. B. Male. Serial segmental muscles, longitudinal muscles and intrinsic prosoma muscles are shown, for other

musculature see figure 19A. Sex-dimorph male prosomal and opisthosomal musculature is marked blue. In the opisthosoma,

only the ventral longitudinal muscles, the dorsoventral muscles and transverse muscles are show. The dorsal longitudinal, the

intersegmental and the flagellar musculature are not shown (see Figs. 19C, 20A,B for those muscles). Inset: cross-section of the

mid-section of segment 11. C. The female specific pattern of muscle anatomy is shown for the prosoma. In the opisthosoma, only

the dorsal longitudinal, the intersegmental and the flagellar musculature are shown. Ventral longitudinal muscles, the dorsoventral

muscles and transverse muscles are not show (see Figs. 19A, 20 A, B for those muscles). Sex-dimorph musculature is marked

orange (female). Abbreviations: C1—4 = cheliceral muscle; D1—2 = dorsal muscle; DV1—5 = dorsoventral muscle; F1—2 = flagellar

muscle; Gm1—4 = genital muscle male; JI1—4 = intersegmental muscle 1; JI[11—-6 = intersegmental muscle 2; JII1—6 = intersegmental

muscle 3; JIV1—6 = intersegmental muscle 4; JV1—6 = intersegmental muscle 5; JVI1—5 = intersegmental muscle 6; JVII1-4 =

intersegmental muscle 7; JVIII1—3 = intersegmental muscle 8; JIX1 = intersegmental muscle 9; LI] = leg 1 muscle; LI] = leg

2 muscle; P1-13 = prosomal muscle; PP1 = pedipalpal muscle; TI1—2 = transversal muscle 1;TII1—2 = transversal muscle 2;

TIII1—2 = transversal muscle 3; TIV1—2 = transversal muscle 4; TV1—2 = transversal muscle 5; TVI1 = transversal muscle 6; V =

ventral muscle.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

30 Sandra Franz-Guess & J. Matthias Starck

HH 0 8 Oe fd

fd Fd Bd be

Eid thd Old Gh Bo dd Od

aTwwa4

Mh rep ep mu ee OT aa

a fv for

Gur er ur

AL 2 A 8 Ob i A LAO

ALL PALL Wd, ETL

TAL EAL

Fig. 20 Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of a lateral view of the musculature based on serial cross-

section light micrographs and 3D-reconstruction. A. Female; the right body side is shown. Tendons within the chelicerae are

highlighted in red. Sexually dimorphic opisthosomal muscles for females are marked orange. B. Male; only the right body side is

shown. Sexually dimorphic prosomal and opisthosomal musculature is marked blue. Abbreviations: C1—13 = cheliceral muscle;

D1-2 = dorsal muscle; DV1—5 = dorsoventral muscle; F1—3 = flagellar muscle; Gf = genital muscle female; Gm1—4 = genital

muscle male; JII—4 = intersegmental muscle 1; JI[1—6 = intersegmental muscle 2; JUI1—6 = intersegmental muscle 3; JIV1-6 =

intersegmental muscle 4; JV1—6 = intersegmental muscle 5; JVI1—5 = intersegmental muscle 6; JVII1—4 = intersegmental muscle

7; JVII1—3 = intersegmental muscle 8; JIX1—2 = intersegmental muscle 9; LH2 = leg 2 muscle; LIII2 = leg 3 muscle; LIV1-3 =

leg 4 muscle; P = pharynx; P1—13 = prosomal muscle; TI1—2 = transversal muscle 1; TI[1—2 = transversal muscle 2; THI1—2 =

transversal muscle 3; TIV1—2 = transversal muscle 4; TV1—2 = transversal muscle 5; TVI1 = transversal muscle 6; V = ventral

muscle. — continued.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 31

Fig. 20 continued. C. Schematic drawings of the intrinsic musculature of pedipalp and legs based on serial cross-section light

micrographs and 3D-reconstruction. Frontal view of the pedipalps and legs. Tendons are labeled by a terminal ‘t’ in their name.

Abbreviations: LI3—14t = leg 1 muscle/tendon; LII3—9 = leg 2 muscle; LII3—10t = leg 3 muscle; LIV4—14 = leg 4 muscle; PP2—10t

= pedipalps muscle/tendon.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

32 Sandra Franz-Guess & J. Matthias Starck

E10 6EW 6 6EW@ «6E

Fig. 21. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawings of the endosternite musculature based on serial cross-

section light micrographs and 3D-reconstruction. No sex-dimorphism was found. Only axial segmental muscles originating from

the endosternite are shown. Color coding is as follows: orange = dorsal suspensor muscles, purple = anterior and posterior oblique

suspensor muscles, dark red = lateral suspensor muscles, blue = ventral suspensor muscles, yellow = muscles extending anterior or

into the opisthosoma. Endosternite musculature associated with the legs (LI2, LII2, LIV1-—3) is only shown with its point of origin

(yellow shaded). A. Dorsal view. B. Lateral view. Abbreviations: El1—20 = endosternite muscle; EaB = endosternite anterior bridge;

EBr, endosternite branch; EcB = endosternite central bridge; EpB = endosternite posterior bridge; EpS = endosternite posterior

section; EU = endosternite upturned U section; P = pharynx.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 33

three, respectively. Both muscles insert on the metapelti-

dium (next to the insertion of dorsal suspensor E17).

Muscle E20, which is possibly also part of the posterior

oblique suspensors, originates at the posterior end of the

endosternite and inserts laterally at the pleural membrane

between the prosoma and segment 8 (Fig. 21; Tabs 5, 7).

Lateral suspensor muscles: The lateral suspensors

originate lateral from the endosternite and insert lateral

at the pleural membrane. We diagnosed four muscles in

two clusters (E10, E16, E18, E19) as lateral suspensor

muscles (Fig. 21; Tabs 5, 7). Muscles E16, E18, and E19

are clustered and originate in the same region of leg 4.

Muscle E10 is located in the region of leg 2.

Ventral suspensor muscles: The ventral suspensors

(El, E2, E7, E9) originate ventral from the endoster-

nite and insert ventral at the pleural membrane (Fig. 21;

Tabs 5, 7). Muscles El and E2 originate both at the an-

terior end of the endosternite (Fig. 21). Muscle E2 is as-

sociated with the palpal segment; muscle E1 that has an

anterior and a posterior branch and is the only muscle

which inserts straight ventral, is associated with leg 1.

The anterior branch of this muscle inserts at the interseg-

mental membrane between the ventral plate and the pro-

sternum; the posterior branch inserts on the prosternum

(Fig. 21; Tabs 5, 7). Muscles E7 and E9 both originate

from the endosternite in the region of leg 2 (anterior to

lateral suspensor muscle E10), and insert anterolateral on

the pleural membrane between the basis of leg 1 and leg

Two muscles were observed that do not fit into the pat-

tern of BTAMS. Muscle E8 originates medial from the

anterior endosternal bridge and extends to the postero-

ventral end of the pharynx (Fig. 21; Appendix I: Tab. 5).

Endosternal muscle E13 originates from the lateral

branch of the endosternite and extends into the opistho-

soma where it inserts at the border of segment 8 and 9

(Fig. 21).

Musculature of the opisthosoma

In Eukoenenia spelaea, the opisthosomal musculature

consists of the dorsal longitudinal, dorso-ventral, in-

tersegmental, transversal, genital, ventral longitudinal

and flagellar musculature. The dorsal longitudinal, dor-

so-ventral, and ventral longitudinal musculature are

elements of the box truss muscle system (Shultz 2001,

2007b). Sexdimorphism can be found in the dorso-ven-

tral, transversal, genital, and ventral longitudinal mus-

culature. The following description is an account on the

general topography of the musculature. Detailed descrip-

tions of origin and insertion for each muscle are given in

table 5. Possible antagonistic functions are hypothesized

according to the topography of muscle origin and inser-

tion.

Dorsal longitudinal musculature: The dorsal longitu-

dinal musculature is divided into a paired anterior strand

Bonn zoological Bulletin Suppl. 65: 1-125

(D1) and a paired posterior strand (D2; Figs 19C, 20A—

B; Appendix I: Tab. 5). The anterior strand, D1, origi-

nates dorsolateral from the metapeltidium. From there, it

passes into the opisthosoma and inserts dorsal in segment

10, lateral to the body median line. Muscle D2 originates

dorsal in segment 9, medial to D1, and inserts on the an-

terior margin of the tergite of segment 17. Consequently,

both muscles, D1 and D2, overlap in segments 9 and 10

(Figs 19C, 20A—B). The topography of these two mus-

cles suggest that their contraction moves the opisthoso-

ma upward against the prosoma in segments 8-10. — The

dorsal longitudinal muscle system extends into the proso-

ma (muscles P11, P12).

Ventral longitudinal musculature: The ventral longitu-

dinal musculature consists of a pair of strands (V) extend-

ing from the posterior margin of the sternite of segment

8 to the border between segments 17 and 18. It branches

in segments 9 and 11 with both branches converging to a

ventro-lateral insertion in the posterior region of segment

9 (Figs 19A—B, 20A—B; Appendix I: Tab. 5). In females,

the ventral longitudinal muscle has a more ventrolater-

al position except in the genital region (Figs 18A, 19A,

20A). In males, the muscle is more medial than in fe-

males but more ventrolateral in the metasoma, 1.e., seg-

ments 15—18 (Figs 19A, 20A, 19B, 20B).

Segmental dorso-ventral musculature: The first opistho-

somal segment (segment 8) is void of dorso-ventral mus-

cles. Segmental dorso-ventral musculature (DV1—DV5)

is found in segments 9-13. These muscles are paired and

located in the anterior part of each segment; they span the

opisthosoma from dorsal to ventral. Dorsal, they origi-

nate just lateral to the strands of the dorsal longitudinal

musculature. Ventral, they insert just medial of the ven-

tral longitudinal musculature. The dorso-ventral muscles

of segments 9 and 10 (DV1 and DV2) are sexually di-

morphic. In females, DV1 and DV2 are simple strands

with no branching (Figs 19A, 20A; Appendix I: Tab. 5).

Muscle DV2, however, bends slightly toward posterior

before inserting just posterior to the base of the genital

lobes. In males, both muscles branch ventrally. The ante-

rior branch of DV1 inserts on the dorsal side of the ven-

tral overlap of segment 9. Muscle DV2 splits just dorsal

of the genital atrium. Its anterior branch then inserts lat-

eral on the center of the genital atrium’s anterior-posteri-

or axis (Figs 19B, 20B; Appendix I: Tab. 5). The posteri-

or branch inserts just posterior to the base of the third pair

of genital lobes. In both sexes, DV5 branches, with the

posterior branch inserting close to the transversal muscle

TV (Figs 19A—B, 20A-B).

Intersegmental musculature. The intersegmental

musculature forms several parallel strands that span

the pleural membranes in anterior-posterior direction

(Figs 19C, 20A—B; Appendix I: Tab. 5). These parallel

muscle strands are evenly spaced from dorsal to ventro-

lateral along the body wall. There is a total of nine sets

of intersegmental muscle strands (JI-JEX). Six strands,

©ZFMK

34 Sandra Franz-Guess & J. Matthias Starck

SupEG NL?

NLS SubEG = AIL

Fig. 22. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of the nervous system based on serial cross-section light

micrographs and 3D-reconstruction. A. Dorsal view. The supraesophageal ganglion is the prominent feature of the dorsal prosoma

in the area of the propeltidium. It gives rise to the cheliceral nerves as well as several lobes (asterisk) into the anterior part of the

propeltidium and into the rostrosoma. The subesophageal ganglion is larger than the supraesophageal ganglion and is the prominent

feature of the ventral prosoma. It extends into the opisthosoma into segment 9. The perikarya layer (orange) is patchy all over the

prosomal ganglial mass, leaving areas of neuropil (yellow) exposed. The distal articles of pedipalps and leg 1 are filled with sensory

cells of the sensory setae and trichobothria (orange). Paired opisthosomal ganglia are found in segments 11-14. The opisthosomal

commissures (arrows) and the nerve leaving the last opisthosomal ganglion are drawn as dashed lines because their topography

could not be resolved by histology. B. Lateral view. The prosomal ganglia extend through most of the prosoma, especially in

the area of the propeltidium. The thickness of the leg nerves decreases in the extremities from proximal to distal. Abbreviations:

asterisk = ganglial lobes; NC = nerve cord; NCh = cheliceral nerve; NL1-4 = leg nerve 1-4; NPP = pedipalpal nerve; OG =

opisthosomal ganglion; SubEG = subesophageal ganglion; SupEG = supraesophageal ganglion.

on each side of the opisthosoma, are present in segments

9/10-12/13 (JM1—6, JINI1-6, JIV1-6, and JV1-6). Set JVI

consists of five strands, JVII of four strands, and JVIII of

three strands. JLX, between segments 16 and 17 consists

of only one strand of muscles on each side. Segment 8

does not have any intrinsic musculature. Interestingly, we

found four small muscle strands in the anterior half of

segment 9, JI1—4, in a position similar to other interseg-

mental muscles, but not bridging over to segment 8. All

other sets, JII-JIX, originate from the posterior part of

One segment, span the segmental border, and insert in

the anterior half of the following segment (Figs 19C,

20A-B; Appendix I: Tab. 5). There is no intersegmen-

tal musculature spanning between segments 17 and 18

(Figs 19C, 20A-B).

Bonn zoological Bulletin Suppl. 65: 1-125

Genital musculature: The most distinct sexual differ-

ence of the opisthosomal musculature can be found in

the genital musculature. Whereas females have only one,

paired genital muscle (Gf), males possess four pairs of

genital muscles (Gm1l—Gm4). In females, the genital

muscle is located inside the genital operculum, lateral to

the body median line. It inserts on the dorsal opercular

wall, which is oriented toward the genital opening. Mus-

cle Gf then splits into two branches and inserts anterior

and posterior on the ventral opercular wall (Figs 19A,

20A; Appendix I: Tab. 5).

The male genital musculature consists of four thin,

paired muscle strands associated with the genital lobes

and the genital atrium. Muscles Gm1—Gm3 are grouped

together and originate from the ventral wall of the atri-

um. The most anterior muscle (Gm1) extends ventrally

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 35

10pm

Fig. 23. Eukoenenia spelaea (Peyerimhoff, 1902), light microscopic cross-sections of the nervous system. A. Area of the border of

segments 4 and 5. The prosomal ganglia fill a large portion of the prosoma. The perikarya layer (orange line) forms an incomplete

cortex to the more central neuropil (yellow line). Large type-D perikarya are present. B. Cross-section of the area of leg 3. In

the posterior section of the subesophageal ganglion the central neuropil is reduced. The surrounding perikarya layer consists of

predominantly small type-B neurons, but is incomplete. In some areas, the neuropil is in direct contact with musculature and

epidermis. C. Light micrograph of a cross-section through segment 9 with the genital atrium. At their origin, the paired nerves

supplying the posterior section of the body are enlarged. They are located dorsal to the genital atrium and accessory gland. D. Area

of the border of segments 11 and 12, just anterior to the opisthosomal ganglia of segment 12. The nerve cords of the rope ladder

system of the opisthosomal nervous system are located ventral, and medial of the dorsoventral musculature. Abbreviations: AG =

accessory gland; CxG = coxal organ glandular section; ES = esophagus; ESt = endosternite; GA = genital atrium; GL1 = genital

lobe 1; L3 = leg 3; MG = midgut; MtPIt = metapeltidium; NC = nerve cord; PaPIt = lateral dorsal plate; PrPlt = propeltidium;

SubEG = subesophageal ganglion; SupEG = supraesophageal ganglion; T = testes.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

36 Sandra Franz-Guess & J. Matthias Starck

and inserts on the anterior wall of the first genital lobe.

The muscle pairs Gm2 and Gm3 insert on the second pair

of genital lobes and possibly move them up and medial.

Muscle Gm4 originates from the lower third of the gen-

ital atrium. It inserts on the ventral body wall (Figs 19B,

20B).

Transversal musculature: The transversal musculature

(TI-TVI) consists of paired strands of two parallel mus-

dNF

cles in segments 9-14. The transversal muscles are locat-

ed ventral, below the nerve cord; they are missing in the

metasoma. The transversal musculature spans the short

pleural membrane between the lateral extensions of the

tergite and inserts on the sternite. There are paired trans-

versal muscle strands per body half with the exception of

TI in males and TVI in both females and males, which

consist of only one strand per body half (Figs 19A-—B,

NPP

Fig. 24. Eukoenenia spelaea (Peyerimhoff, 1902), commissures in the syncerebrum. A. Light micrograph of a slightly oblique

cross-section in the region of the pedipalp and leg 1.The perikarya partially surrounds cheliceral muscle C4 and endosternal muscle

E8. B. Simplified and schematic drawing of the section in (A) to highlight the protocerebral commissure (perikarya layer, dark

blue; neuropil, light blue). The protocerebral commissure is dorsal to the esophagus. Abbreviations: C4 = cheliceral muscle; ChC =

cheliceral commissure; NF = descending fiber; E8—9 = endosternal muscle; ES = esophagus; L1—2 = leg 1-2; NL1 = nerve of leg

1; NPP =nerve of pedipalp; PCC = protocerebral commissure; PP = pedipalp; PPC = pedipalpal commissure. — continued.

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 37

20A, B; Appendix I: Tab. 5). Muscle TI] of females in-

serts ventrolateral and is possibly involved in widening

of the genital opening. The ventral strands THI2, TIV2,

and TV2 are longer in females than in males. Muscle TVI

is located more posterolateral in segment 14. It is as short

as the ventral transversal muscle strands of the male,

and it is shorter than the dorsal strands of the transversal

fg ‘

; . aie

: dNF ES

dNF

muscles in the other segments of both females and males

(THI1, TIV1, and TV1; Figs 19A—B, 20A—B).

The last opisthosomal segment carries the flagellar

musculature. It consists of three paired thick parallel mus-

cle strands (F1—F3). The muscles are arranged parallel to

the body axis and encircle almost the entire segment. The

most ventral part of segment 18 is free of musculature.

Fig. 24 continued. C. Light micrograph of a slightly oblique cross-section through the region between pedipalp and leg 1. The

perikarya layer of the synganglion partially envelops the cheliceral muscle C4 and muscle E8. D. Schematic drawing of the

perikarya layer and neuropil in the region between pedipalp and leg 1 based on C. The cheliceral commissure is more pronounced

than all other commissures within the synganglion. The lateral nerve fibers are potentially part of the circumesophageal commissure.

Abbreviations: C4 = cheliceral muscle; ChC = cheliceral commissure; dNF = descending fiber; E8—9 = endosternal muscle; ES =

esophagus; L1 = leg 1; NL1 = nerve of leg 1; PP = pedipalp. — continued.

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

38 Sandra Franz-Guess & J. Matthias Starck

dNF

NL1

Fig. 24 continued. E. Light micrograph of a slightly oblique cross-section in the region between leg | and 2. The pedipalpal

commissure is located between esophagus and muscle E8. The broad base of the nerve of leg 1 is clearly visible. F. Schematic

drawing of the perikarya layer and neuropil based on E. The delicate pedipalpal commissure is nestled between the esophagus and

endosternal muscle E8. Abbreviations: dNF = descending fiber; E8 = endosternal muscle 8; ES = esophagus; L1—2 = leg 1-2; NL1

= nerve of leg 1; PPC = pedipalpal commissure.

Muscles F1—F3 insert anterior on the basal article of the

flagellum. Muscle Fl might be associated with the up

movement of the flagellum; F2 and F3 are possibly asso-

ciated with down and lateral movement (Figs 19A, 20B). Nervous system

The flagellum has no intrinsic musculature.

The nervous system of Eukoenenia spelaea consists of a

prominent prosomal synganglion, which occupies large

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 39

Fig. 25. Eukoenenia spelaea (Peyerimhoff, 1902), schematic

drawing of the anterior region of the synganglion. The small

protocerebrum with its commissure has a position between

the roots of the cheliceral nerves leaving the supposed

deutocerebrum. The cheliceral commissure is located dorsally

of the esophagus. The pedipalpal commissure is located ventral

of the esophagus. Abbreviations: ChC = cheliceral commissure;

ES = esophagus; NLI = nerve of leg 1; NPP = nerve of

pedipalp; PCC = protocerebral commissure; PPC = pedipalpal

commissure.

parts of the prosoma. It is the origin for the prosomal

nerves and for a pair of parallel opisthosomal nerve cords

(Figs 22-23). The synganglion consists of the suprae-

sophageal ganglion (syncerebrum) and the subesopha-

geal ganglion. The supraesophageal ganglion reaches

from the base of the chelicerae to the posterior end of

the propeltidium (SupEG; Fig. 22). Using standard light

microscopic histology, we identified three commissures:

(1) the most anterior commissure is located dorsal to the

esophagus in the anterior region of the syncerebrum. This

commissure 1s possibly the protocerebral commissure

(PCC; Figs 24A—-B, 25). (2) The second commissure is

more prominent and also located dorsal to the esophagus.

It is possibly the cheliceral commissure (ChC; Figs 24C—

D, 25). (3) The posterior commissure is located just ven-

tral to the esophagus between esophagus and endosternal

muscle E8. It probably represents the pedipalpal com-

missure, because nerve fibers from the commissure lead

to the base of the pedipalpal coxae (PPC; Figs 24E-F,

25). — The presumed protocerebral part of the syncere-

brum is small. An arcuate body, a constitutive element

of the ground pattern of the euchelicerate brain, was not

found.

The subesophageal ganglion is larger than the supra-

esophageal ganglion. It extends from the base of the pedi-

palps into the opisthosoma to the genital plate (SubEG;

Fig. 22). The ganglial lobes of the respective legs can be

distinguished. Neuropil around the esophagus (circume-

sophageal connective) connects the supra- and subesopha-

geal ganglia (Fig. 37C) just anterior to the anterior endo-

sternal bridge. The supraesophageal ganglion is never

Bonn zoological Bulletin Suppl. 65: 1-125

in direct contact with the endosternal bridge because the

midgut separates both structures (Fig. 17A).

We did not find a hemolymph space surrounding the

ganglia, which is typical for (larger) euchelicerates

(Figs 23A, 26A—B). The ganglia are surrounded by a

glial cell sheath, the perineurium, and a thin extracellular

matrix, 1.e., the neurilemma. The neurilemma has a uni-

form thickness of approx. 50-100 nm around all struc-

tures adjacent to the ganglia, e.g., esophagus and muscles

(Figs 26A-B, 37C).

A patchy and incomplete cortex of the perikarya

(Figs 22, 23A) surrounds the neuropil of the syngangli-

on. In the supraesophageal ganglion, the perikarya layer

extends far anterior, sending out several lobes into the

rostrosoma (*; Fig. 22). The supra- and subesophageal

ganglion are penetrated by muscle strands which origi-

nate from the endosternite and attach dorsal, ventral, and

lateral to the body (Figs 17A, 23B, 24A, C, E).

Following the neuron categorization by Babu (1985),

two types of neurons can be recognized: (1) type B neu-

rons. The nucleus of type B neurons is approx. 1-2 um

in diameter. They are predominantly found in the cortex

of the posterior part of the subesophageal ganglion, and

in the opisthosomal ganglia (Fig. 26C—D). In light mi-

croscopy, their nucleus stains darker and their cytoplasm

lighter. The nucleus is the prominent structure of the neu-

ron in transmission electron microscopy, the cytoplasm

is largely reduced. (2) Type D neurons. These neurons

are larger with a nucleus of approx. 3—4 um in diameter

(Figs 15A—B, D, 26C). A thin layer of cytoplasm, like in

type B neurons, surrounds the nucleus of type D neurons.

They can be found in the cortex of the supraesophageal

ganglion and the anterior part of the subesophageal gan-

glion. Their nucleus stains lighter in and the cytoplasm

darker in light microscopy (Figs 15B, 26C). Cell organ-

elles, glycogen granules and free ribosomes are present

in the cytoplasm of both types of neurons. Neurons of

type A (typically associated with the mushroom bodies of

the visual center) and type C (neurosecretory) were not

identified. The neuropil consists of axons and dendrites

from the cortical neurons, and nerve fibers with microtu-

bules, free ribosomes and glycogen granules (Fig. 26B).

Several nerves originate from the syncerebrum. The

cheliceral nerve (NCh; Figs 22, 25), originates lateral on

each side of the supraesophageal ganglion just above the

pharynx (Fig. 22B). The nerves of the pedipalps and legs

14 originate laterally from the circumesophageal con-

nectives and decrease in diameter in article 2 (NLI1-4,

NPP, PrC-B, PrC-D; Figs 22, 26C). In the pedipalp and

leg 1, the number of type B and D neurons increases

towards the more distal articles. Therefore, their distal

articles are almost entirely filled with nerve cells. In con-

trast, legs 2-4 have only a few neurons distributed along

the leg, with musculature being the prominent feature

within the leg (Fig. 22). Several lobes originating from

the supraesophageal ganglion reaching anterior in the

©ZFMK

40 Sandra Franz-Guess & J. Matthias Starck

Fig. 26. Eukoenenia spelaea (Peyerimhoff, 1902), nervous system. A. Transmission electron micrograph of the supraesophageal

ganglion just below the body wall. A thin neurilemma (arrow) separates the perikarya from the epidermis. B. Transmission electron

micrograph of the subesophageal ganglion in close neighborhood to a muscle. The thin neurilemma separates the muscle and the

ganglion (arrow). C. Light micrograph of a cross-section through a distal article of leg 1. Neurons are dispersed throughout the

leg. The enlarged nuclei are surrounded by darker staining cytoplasm. D. Transmission electron micrograph of an opisthosomal

ganglion. The connective nerve (yellow line) lies dorsal to the ganglion (orange line) and is thicker in diameter than the ganglion

itself. One of the adjacent nerve fibers is possibly the commissure. Abbreviations: C = cuticle; cNF = connective nerve fibers; EC

= epidermal cell; MF = muscle fiber; NF = nerve fiber; PN = perineurium; PrC-B = type B perikarya; PrC-D = type D perikarya;

T = tendon.

prosoma (*; Fig. 22). These lobes cannot be associated

with sensory structures like the frontal organ or lateral

organs.

From the posterior end of the subesophageal ganglion

in segment 9, two parallel nerve cords extend into the

opisthosoma (Fig. 22). These nerve cords contain four

pairs of opisthosomal ganglia in segments 11-14 and

their respective connectives (OG; Fig. 22). More ante-

rior in the opisthosoma, the nerve cords have a larger

diameter than further distal (Figs 23C—D). Each opist-

Bonn zoological Bulletin Suppl. 65: 1-125

hosomal ganglion consists of approx. 25 type B neurons

(Fig. 26D). The connectives are thicker in diameter than

the ganglia and connect to the ganglia on their dorsal side

(cNF; Fig. 26D). Additional nerve fibers are located me-

diolateral of the connective (NF; Fig. 26D). These nerves

might represent commissures connecting the left and

right opisthosomal ganglia. However, distinct commis-

sures were not identified using light microscopy. The ori-

gin of the nerves supplying the sensory setae of segments

15-18 and the flagellum could also not be identified.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 4]

Fig. 27. Eukoenenia spelaea (Peyerimhoff, 1902), frontal organ. A. Light microscopic cross-section through the modified setae of

the frontal organ. The frontal organ is located in a medial position at the anterior end of the prosoma, but partially covered by the

propeltidium. B. Transmission electron micrograph of a cross-section of the cuticular wall of a modified seta of the frontal organ.

The procuticle is generally electron-dense, but electron-translucent in the root of the cuticular ridges (green line). The procuticle of

the cuticular ridges and of the wall pores/grooves is less electron-dense and covered by epicuticle. The epicuticle extends between

the pores/grooves. Abbreviations: cGr = cuticular groove; Ch = chelicera; cR = cuticular ridge; EpC = epicuticle; FO = frontal

organ; PrPlt = propeltidium; ProC = procuticle.

Sensory organs

Sensory organs of Eukoenenia spelaea are the frontal or-

gan, the lateral organs, the sensory setae, and the tricho-

bothria. They all have the same basic structure: (1) an

outer cuticular structure, 1.e., hair or modified hair, (2)

two or more sensory cells, and (3) several enveloping

cells. The nervous elements are the same in all sensory

organs: (1) the inner dendritic segment containing the

mitochondria, (2) the ciliary segment with the typical

9 x 2 +0 arrangement of microtubules, and (3) the outer

dendritic segment with more or less loosely arranged mi-

crotubules in varying numbers.

Frontal organ

The frontal organ is located medial at the most anteri-

or part of the prosoma, just below the propeltidium, and

dorsal to the chelicerae (FO; Figs 3A—B, 8A, 27A). It

has a broad base with two finger-like sensory setae

that extend to the anterior (Figs 3A, 8A, 27A). The se-

tae are approx. 25 um long and have a diameter of ap-

prox. 5—10 um. Cuticular ridges on the surface of the

frontal organ form a hexagonal honeycomb pattern.

Transmission electron micrographs show that these

ridges reach deep into the procuticle, with their base be-

ing electron-translucent (cR; Fig. 27B). The cuticle of the

area surrounded by the ridges ts rich in cuticular grooves

and covered with an electron-translucent epicuticle (cGr,

Bonn zoological Bulletin Suppl. 65: 1-125

EpC; Fig. 26B). The procuticle is electron-dense at the

base, but is less electron-dense in the more apical parts

that form the grooves (ProC; Fig. 27B). The cuticle of

the ridges is 0.35—0.5 um thick, in the area between the

ridges it is only 0.2 um thick. The cuticular grooves are

0.1 um deep. The diffusion distance through the cuticle

is therefore approx. 0.1 um. The base of the frontal organ

(Fig. 28A, J) is filled with electron-dense material and

displays fewer pores than the setae.

In the base, there are wide spaces between the cells

of both dendrite pairs, which might be part of the recep-

tor lymph cavity. The dendrites reaching into the left and

right setae differ from one another. In the right seta, we

find two mitochondria-rich dendrites (diameter 0.2 um)

enclosed by nine enveloping cells (IDS 1/2, EnvC;

Fig. 28A, J). The electron density of the cytoplasm of

both dendrites and the enveloping cells is equally high

and similar to the electron density of the surrounding

dense material. Distally, the dendrites branch cylindrical-

ly multiple times (dBr; Fig. 28B, K). The diameter of the

branches varies between 0.1 and 2.5 um. The dendritic

branches show an irregular arrangement of microtubules

and are enveloped in dense material (DM; Fig. 28B, K).

Some dendritic branches have small, electron-dense re-

gions, or droplets as well as deteriorated vacuoles and

lamellar bodies (*; Fig. 283B—E). The dense material is

mostly found just underneath the setal wall.

In the left seta, we find also two mitochondria-rich den-

drites. However, the mitochondria are larger than in the

©ZFMK

42 Sandra Franz-Guess & J. Matthias Starck

Fig. 28. Eukoenenia spelaea (Peyerimhoff, 1902), transmission electron micrographs of cross-sections through the frontal organ. A.

Section through the basis of the frontal organ containing two distinct units of two dendrites, each surrounded by enveloping cells.

The dendritic units are surrounded by a thick layer of dense material. B. Right seta. The dendrites have branched cylindrically. In

cross-section, cuticular ridges and pits are distinguishable. A lamellar body is closely associated with a dendritic branch (asterisk).

C. Proximal part of the left seta. In addition to the cylindrically branched dendrite, a dendrite with flattened branches is present.

In the left seta, lamellar bodies are found in close association with dendrites as well (asterisk). D. Medial part of the left seta. The

dendritic branches have become more flattened. E. Proximal part of the left seta. The flattened dendritic branches have become

lamellate. F. Close-up of the flattened dendritic branches in C. The microtubule doublets are clearly visible. G. Close-up of the

flattened dendritic branches in D. The microtubules are now all single microtubuli. H. Close-up of the flattened dendritic branches

in E. No microtubules are present. Abbreviations: cP = cuticular pit, cR = cuticular ridge; dBr = dendritic branch; DM = dense

material; EnvC = enveloping cell; IDS1-4 = inner dendritic segment 1-4; MT = microtubule; ODS = outer dendritic segment. -

continued.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 43

Fig. 28 continued. Same images as figures A—H but with colored overlay. J. Basis of frontal organ. The inner dendritic segments 1

and 2 of the right seta (green line) are enveloped in nine enveloping cells (yellow line). The inner dendritic segments 3 and 4 that

reach into the left seta (blue line) have 12 enveloping cells (yellow line). The dendrites and their enveloping cells are packed in a

thick layer of dense material (orange overlay). K. The right seta is filled with a large number of dendritic branches (green line). The

dense material (orange line) is a thin layer in the periphery. L. The cylindrically branched dendrite 3 (green line) of the left seta has

differently sized branches. Dendrite 4 has a lamellar type of branching. M. As the dendrites increase their branching, the amount

of dense material is reduced. N. Distally, dendrite 3 is reduced to two small branches, whereas the lamellate branches of dendrite 4

(blue line) take up most of the space within the seta. Dense material (orange overlay) is reduced to a thin layer at the setal wall. O.

The flattened dendritic branches of dendrite 4 at the basal part of the left seta are still similar in thickness to cylindrically branched

dendrites. P. The arrangement of the flattened branches of dendrite 4 becomes more regular, the branches themselves become

more flattened. Q. Distally, the dendritic branches of dendrite 4 are now so flat, that they appear as lamella. Abbreviations: dBr =

dendritic branch; dBrs = dendritic branches; DM = dense material; EnvC = enveloping cell; IDS1—4 = inner dendritic segment 1-4.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

44 Sandra Franz-Guess & J. Matthias Starck

Fig. 29. Eukoenenia spelaea (Peyerimhoff, 1902), lateral organ. A. Light micrograph of a cross-section at the level of the chelicerae

and pedipalps of a male. The lateral organs are located laterally beneath the propeltidium. Each modified seta is attached separately

to the body. B. Transmission electron micrograph of the eight inner dendritic segments (green line) associated with the four lateral

organs of one body half. The microtubules are arranged irregularly. C. Transmission electron micrograph of a cross-section of a

modified seta. The dendrites are branched cylindrically. Like in the frontal organ, the cuticular wall consists of cuticular ridges,

which build the honeycomb pattern, and cuticular pits, which carry the wall pores. D. Same as C with colored overlay. The dendritic

branches vary in size. The dense material (orange overlay) is largely located towards the inside, while the dendritic branches are

oriented towards the setal wall. Abbreviations: Ch = chelicera; cP = cuticular pit; cR = cuticular ridge; dBr = dendritic branch; DM

= dense material; 1ChS = intercheliceral septum; IDS = inner dendritic segment; LO = lateral organ; M = mitochondrion; MT =

microtubule; P = pharynx; P3—4 = prosomal muscle; PP = pedipalp = PrPlt = propeltidium; ROS = rostrosoma.

right seta (diameter 0.5 um). The dendrites are surround-

ed by twelve enveloping cells (IDS 3/4, EnvC; Fig. 28A,

J). Dendrites and enveloping cells have the same cyto-

plasm electron density. The widenings between the en-

veloping cells are larger than in the right receptor and

could be part of the receptor lymph cavity. In the seta,

one dendrite branches and folds, and displays an irregular

arrangement of microtubule doublets IDS 4, ODS, MT;

Fig. 28C, F, L, O). Along the seta, the ranching, flatten-

ing and folding of this dendrite increases from proximal

to apical. Within the branches, the microtubuli doublets

Bonn zoological Bulletin Suppl. 65: 1-125

become single microtubuli and the arrangement becomes

regular (Fig. 28D, G, M, P). Towards the distal end of the

seta, the dendrite forms concentrically wrapped laminae.

Within this lamellate section, microtubules are no lon-

ger recognizable (Fig. 28E, H, N, Q). Many vacuoles in

the center of the dendrite show deterioration and indicate

incomplete fixation. The second dendrite branches cylin-

drically and is pushed towards the periphery of the seta

by the first dendrite (Fig. 283C—E, L—N). The second den-

drite contains numerous deteriorated vacuoles and lamel-

lar bodies. However, there are more of these in the left

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 45

seta than in the right seta (*; Fig. 28C—E). Regions where

vacuoles are present show also small electron-dense

droplets. Similar to the right seta, the dense material is

mostly found just below the setal wall. However, in the

Lateral organ

Lateral organs are located at the left and right side on the

prosoma, just below the propeltidium and anterior to the

left seta the amount of dense material decreases from

basal to apical (Fig. 28C—E, L—N).

base of the pedipalps (LO; Figs 3A, C, 29A). Each lateral

organ consists of four single, finger-like modified setae

extending anteroventral (Figs 8B, 29A). The setae are

arranged slightly diagonally from anterior to posterior

Fig. 30. Eukoenenia spelaea (Peyerimhoff, 1902), sensory seta. A. Schematic drawing of the microscopic anatomy of the sensory

seta based on transmission electron micrographs. Seven dendrites are associated with the seta, two dendrites extend into the hair

shaft. The distal part of the hair shaft has double walls and wall pores. B. Transmission electron micrograph of a longitudinal

section of the basal part of a sensory seta. The outer dendritic segments extend into the hair shaft. Abbreviations: C = cuticle; dHS

= distal hair shaft; DM = dense material; DS = dendritic sheath; EC = epidermal cell; ExC = exocuticle; iC = inner cuticle; IDS =

inner dendritic segment; 1EnvC = enveloping cell of the inner receptor lymph cavity; iP = inner pore; iRLC = inner receptor lymph

cavity; oC = outer cuticle, ODS = outer dendritic segment; oEnvC = enveloping cell of the outer receptor lymph cavity; oP = outer

pore; oRLC = outer receptor lymph cavity; pHS = proximal hair shaft; ShC = sheath cell; TB = tubular body.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

46 Sandra Franz-Guess & J. Matthias Starck

Fig. 31. Eukoenenia spelaea (Peyerimhoff, 1902), transmission electron micrographs of sections through a sensory seta. A. Cross-

section through the base of the seta, through the inner receptor lymph cavity. Seven dendrites are arranged around the microvilli

of the central enveloping cell. B. Cross-section through the base of a sensory seta at the level of the outer dendritic segments. Six

outer dendritic segments are arranged in a circle with the seventh dendrite located centrally. The central dendrite has a reduced

number of microtubule doublets. C. Longitudinal section through the base of a sensory seta. The dendritic sheath extends into

the hair shaft. Two of the original seven dendrites continue into the hair. Arrows indicate an artefact. D. Cross-section through

the proximal hair shaft containing two dendrites (circled by green line). E. Cross-section through the apical hair shaft. The two

dendrites are located centrally within the hair. The hair itself has an inner and outer cuticle with wall pores. The outer pore is open,

the inner pore is clogged with electron-translucent material. The inner wall is covered with epicuticle. Abbreviations: DM = dense

material; DS = dendritic sheath; EnC = endocuticle; EpC = epicuticle; ExC = exocuticle; iC = inner cuticle; IDS = inner dendritic

segment; 1EnvC = enveloping cell of the inner receptor lymph cavity; iP = inner pore; M = mitochondrion; MV = microvilli; oC =

outer cuticle; ODS = outer dendritic segment; oEnvC = enveloping cell of the outer receptor lymph cavity; oP = outer pore; TB =

tubular body.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 47

Fig. 32. Eukoenenia spelaea (Peyerimhoff, 1902), trichobothrium. A. Light micrograph of a cross-section through leg 1, with a

longitudinal section of a trichobothrium. The hair shaft is located within a cuticular socket. The outer receptor lymph cavity is

separated from the socket by a cuticular membrane. B. Scanning electron micrograph of the basal part of the trichobothrium. The

hair shaft is covered with small cuticular spikes which are arranged regularly along the shaft. A row of cuticular teeth forms the

margin of the bothrium. Abbreviations: cH = cuticular helmet; cMB = cuticular membrane; cS = cuticular socket; oRLC = outer

receptor lymph cavity; pHS = proximal hair shaft; tBR = toothed bothrial margin.

along the ventral side of the propeltidium. The size of the

setae is similar to the setae of the frontal organ with an

approximate length of 25 um and an approximate thick-

ness of 4.5 um. Like in the frontal organ, the cuticle is

rich in cuticular grooves and reinforced with honeycomb

patterned cuticular ridges (cP, cR; Fig. 29C). The ultra-

structure of the cuticle is the same as in the frontal organ.

The nerve supplying the lateral organ consists of eight

dendritic units (IDS; Fig. 29B). This suggests that two

dendrites extend into each seta. The enveloping cells

could not be identified, because the transmission elec-

tron microscopic section of the nerve is too far proximal.

The dendrites branch cylindrically within the setae (dBr;

Fig. 29C—D). The branches display an uneven thickness,

which ranges between 0.1 and 4.2 um and show only

few microtubules. Like in the frontal organ, the dendritic

branches have accumulations of dense material between

them (DM; Fig. 29C—D). The dense material is largely

located towards the inside of the setae. In contrast to the

frontal organ, all dendritic branches of the lateral organ

contain numerous small electron-dense droplets as well

as few electron-translucent droplets. Deteriorated vacu-

oles were not found.

Sensory setae

Sensory setae are found all over the body and flagellum

(Tabs 1-4). At their insertion, the procuticle is differenti-

Bonn zoological Bulletin Suppl. 65: 1-125

ated into an endocuticle and an electron-dense exocuticle

(EnC, ExC, ProC; Figs 30B, 31C) probably providing

some flexibility. The epicuticle is electron-translucent.

At the base of each seta, we found seven sensory cells in

a circular arrangement around a central enveloping cell.

The enveloping cell has apical microvilli, which extend

into the inner receptor lymph cavity ((EnvC, iRLC, MV;

Figs 30A, 31A). The inner dendritic segment of the sen-

sory cells is characterized by the presence of mitochon-

dria (IDS, M; Fig. 31A).

The outer dendritic segments of the sensory cells are

characterized by a specific arrangement of their micro-

tubules (ODS; Figs 30A, 31B). In cross section, six den-

drites are in circular arrangement around a smaller central

dendrite. The six peripheral dendrites have a 9 x 2 + 0

arrangement of microtubule, but the small central den-

drite displays a 6 x 2 + O arrangement (Fig. 31B). The

outer dendritic segments extend through the outer re-

ceptor lymph cavity. They are wrapped by the envelop-

ing cell(s) and embedded in an electron-dense dendritic

sheath. This sheath is present only in the region of the

cuticular socket and the basal part of the seta (DS, oRLC;:

Figs 30, 31C).

Two of the seven dendrites of a sensory seta extend

to the tip of the seta; the other dendrites form a num-

ber of tubular bodies at the seta’s point of insertion (TB;

Figs 30A, 31C). The two dendrites extending to the tip

of the seta are unbranched and surrounded by dense ma-

©ZFMK

48 Sandra Franz-Guess & J. Matthias Starck

Fig. 33. Eukoenenia spelaea (Peyerimhoff, 1902), trichobothrium. A. Schematic drawing of the microscopic anatomy of a

trichobothrium, reconstruction based on TEM-sections. The cuticular socket of the bothrial wall extends down to the sheath cells.

The cuticular helmet is open allowing the outer dendritic segments to extend into the hair shaft. B. Transmission electron micrograph

of a cross-section of the distal hair shaft. Of the four dendrites entering the hair shaft proximally, only one dendrite extends to the

distal part of the hair. The outer dendritic segment is enveloped by dense material. C. Transmission electron micrograph of a

longitudinal section of the socket area. The sheath cells extend into the basal part of the cuticular socket. Four of the five inner

dendritic segments (green line) extend into the hair shaft. The dendritic sheath terminates at the cuticular helmet. Abbreviations: cH

= cuticular helmet; cMB = cuticular membrane; cS = cuticular socket; dHS = distal hair shaft; DM = dense material; DS = dendritic

sheath; EC = epidermal cell; EnvC = enveloping cell; [DS = inner dendritic segment; iRLC = inner receptor lymph cavity; ODS =

outer dendritic segment; M = mitochondrion; MV = microvilli; oRLC = outer receptor lymph cavity; pHS = proximal hair shaft;

ShC = sheath cell; tBR = toothed bothrial rim.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 49

terial (Figs 30, 31C—E). The number of microtubules in

the dendrites decreases towards the tip of the seta. At

the distal part of the seta, the cuticle wall is doubled and

has wall pores (iC, oC, iP, oP; Figs 30A, 31E). The ex-

act number of wall pores could not be established. The

outer cuticular wall is approx. 0.05 um thick, while the

inner cuticular wall is only approx. 0.1 um. The pores of

the outer wall are completely open, whereas the pores of

the inner wall are plugged and covered with epicuticle

(Figs 30A, 31E).

Trichobothria

In Eukoenenia spelaea, trichobothria are found exclu-

sively on leg 1. The hair of each trichobothrium is nest-

ed in a cup-shaped cuticular socket, 1.e., the bothrium,

which is divided by a thin cuticular membrane into an in-

ner and an outer compartment (cMB, cS; Figs 32A, 33A,

C). The apical opening of the bothrium is ornamented by

a ring of cuticular teeth (tBR; Figs 32, 33A, C). The di-

ameter of the outer compartment is approx. 6 um, and is

surrounded by epidermal cells. The diameter of the inner

compartment below the membrane is approx. 4.5 um.

The receptor lymph cavity is divided into two parts, the

A {—_— ED

<<) ra

eS ff

4 sy,

1g a

MO P CxTd CxS ES MGD CxTp

} =] fh -

in, :

Ke \ si} 2 a ‘

inner and the outer receptor lymph cavity GRLC, oRLC;

Fig. 33A, C). The outer receptor lymph cavity has a basal

and an apical part. The apical part of the outer receptor

lymph cavity is formed by a cup-shaped cuticula extend-

ing the bothrium basal to the cuticular membrane that

spans across the bothrium. The basal part is located at

the base of the inner compartment (Fig. 33A, C) and is

surrounded by enveloping cells with microvilli (oEnvC;

Fig. 33A, C).

The inner receptor lymph cavity is located centrally

between the inner dendritic segments. The cuticular wall

of the inner compartment tapers off at the base, leaving

a narrow opening where the dendrites enter the inner

compartment (Fig. 33). Like in the outer receptor lymph

cavity, the enveloping cells of the inner receptor lymph

cavity have microvilli GEnvC, MV; Fig. 33C). A total of

five dendrites is associated with a trichobothrium (IDS,

ODS; Fig. 33A, C). The dendrites enter the outer receptor

lymph cavity and are enveloped by a dendritic sheath,

which is built by sheath cells (DS, ShC; Fig. 33A, C).

The dendrites connect to the helmet (cH). The helmet

is a cuticular structure connected to the base of the hair

shaft, which is located centrally in the outer compartment

of the socket (pHS; Fig. 33, C). In contrast to described

H MG Rs

Fig. 34. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of the heart, the alimentary system, and the coxal organ based

on serial cross-section light micrographs and 3D-reconstruction. A. Dorsal view. The heart (red) extends from the posterior end

of the prosoma to segment 14. The prosomal midgut (green) has two lateral diverticula in the area of leg 2 and 3, just posterior to

where the esophagus terminates. The opisthosomal midgut is a sac with lateral indentations where the dorsoventral musculature

extends between tergites and sternites. The saccule (orange) of the coxal organ is located in the region of leg 1; the proximal

(brown) and distal tubule (yellow) of the coxal organ extend into segments anterior and posterior; the proximal tubule extends as far

posterior as into segment 9, the distal tubule extends into the coxa of leg 1. The excretory duct opens posterior to the basal article

of leg 1. B. Lateral view. The heart has a flattened appearance within a segment and has a roundish diameter at the junction of two

segments. The anal opening lies in the membranous fold between segment 18 and the flagellum’s basal article. Abbreviations: A =

anus; CxS = coxal organ saccule; CxTd = coxal organ distal tubule; CxTp = coxal organ proximal tubule; ED = excretory duct; ES

= esophagus; H = heart, MG = midgut; MGD = midgut diverticula, MO = mouth opening; P = pharynx; Rs = rectal sac.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

50 Sandra Franz-Guess & J. Matthias Starck

trichobothria of other arachnids, the helmet is open and one outer dendritic segment is present inside the shaft. It

ciliary sections of four dendrites continue through the is surrounded by dense material (DM; Fig. 33B). Cuticu-

opening into the proximal part of the shaft (Fig. 33). The — lar pores could not be observed on the hair shaft.

arrangement of microtubules is 9 x 2 + 0. Distally, only

- . ees

= 24

ee mS —_—.

Fig. 35. Eukoenenia spelaea (Peyerimhoff, 1902), cross-sections through the heart. A. Light micrograph of the heart in segment 11

of the opisthosoma. The overall shape of the heart is flattened. The heart is surrounded by hemolymph and connective tissue. The

arrow indicates an artefactual separation of the cuticle from the body. B. Light microscopic image of the heart between segment

9 and 10. The lumen of the heart is open and extended as compared to the flattened appearance in a more central position of a

segment (see A). Abbreviations: CT = connective tissue; HC = heart cells; HL = heart lumen; HLy = hemolymph; MG = midgut;

N = nucleus.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 51

Heart

The heart is a vestigial muscular tube located in the

dorsal midline of segments 7-14 (H; Fig. 34). Its cross-

sectional diameter changes along the anterior-posterior

axis within each segment. In the middle of a segment,

the cross-section of the heart 1s dorso-ventrally flattened,

but it is more roundish with a wide lumen in the region

between neighboring segments (Fig. 35). Ostia, which

are part of the ground pattern of the heart in arthropods,

were not found. Using transmission electron microscopy,

we did not detect any hemolymph cells in the heart lu-

men. The heart tube consists of one thin layer of circular

musculature. Over its entire length, the heart consists of

approximately 80 cells (Fig. 35). The tissue surrounding

the heart 1s difficult to diagnose using light microscopy,

and probably represents hemolymph and connective tis-

sue. We did neither find a pericardial space nor a dilator

musculature.

In transmission electron microscopic cross-sections, the

heart tube appears as a syncytium with loosely scattered,

irregular bundles of contractile filaments (Fig. 36A).

However, a sarcoplasmic reticulum could not be clearly

identified. The myofibrils seem to be interrupted and the

filaments are irregularly scattered throughout the fibril

(arrowheads; Fig. 36B—C). A sarcomere structure is bare-

ly recognizable, with the membrane of the muscle cells

forming lateral infoldings wherever a Z-line is located

(arrows; Fig. 36B—C). These membrane infoldings might

represent structures equivalent to the T-tubular system.

The muscle cell is enlarged in the region of the hetero-

chromatin rich oblong nucleus. Few mitochondria of

various sizes are dispersed throughout the muscle cell. A

nerve for stimulation of the heart muscle was not found.

Digestive tract

The digestive tract consists of the foregut (mouth open-

ing and pharynx within the rostrosoma, and esophagus),

the midgut, the prosomal midgut diverticula, and the

rectal sac. A postcerebral stomach, as reported for other

arachnid groups, is not present. The pharynx and esoph-

agus are lined with a cuticle intima. The midgut, the pro-

somal midgut diverticula and the rectal sac have no cuti-

cle lining. A cuticle-lined hindgut, typical for arthropods,

is not present. The midgut was free of identifiable food

particles. The anal opening is located ventral in the mem-

brane between segment 18 and the flagellar base ring (A;

Fig. 34).

Pharynx

The pharynx begins at the mouth opening. It is

X-shaped with the two upper arms of the X wide open,

while the lower arms have no open lumen (P; Fig. 37A).

Four strands of musculature attach to the pharynx form-

ing a precerebral suction pump: two strands of lateral

musculature (P4), one strand of dorsal musculature (P3),

Fig. 36. Eukoenenia spelaea (Peyerimhoff, 1902), cross-sections through the heart. A. Transmission electron micrograph of a

cross section through the heart. The lumen is reduced. No distinct pericardium was found. The heart is located adjacent to the

midgut. B. Transmission electron micrographic close-up of a myofibril. Dark areas, similar to Z-lines (arrows), are associated with

the infoldings of the cell membrane. Between these areas are muscle filaments visible (arrowheads). C. Schematic drawing of A.

The cell is enlarged in the area of the nucleus. The thick filaments (arrowheads) are distributed irregularly within the myofibril.

Abbreviations: CT = connective tissue; HL = heart lumen; M = mitochondrion; MG = midgut; MyF = myofibril; N = nucleus.

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

52. Sandra Franz-Guess & J. Matthias Starck

and one strand of endosternal musculature (E8; Fig. 21). and P4 (Figs 19B, 20A). The lumen of the pharynx is

A layer of circular musculature is located around the covered by a thick cuticle (EnC, EpC, ExC; Fig. 37A,

pharynx. The circular muscle fibers are located alternat- B). The cuticle consists of an electron-translucent endo-

ing between the muscle fibers of pharyngeal muscles P3 cuticle and an electron-dense exocuticle. The exocuticle

Fig. 37. Eukoenenia spelaea (Peyerimhoff, 1902), pharynx and esophagus. A. Light micrograph of a cross-section through a female

at the level of the pedipalpal articulation. The pharynx is X-shaped; however, the lower shanks of the pharynx have no open lumen,

thus, in cross-section, the actual open lumen of the pharynx resembles a “V”. B. Transmission electron micrograph of the cuticular

intima of the pharynx. The exocuticle is heterogeneous electron-dense and thicker than the electron-translucent endocuticle. C.

Transmission electron micrograph of a cross-section through the esophagus. The lumen in the lateral branches of the esophagus is

largely reduced/collapsed (white arrowheads). Two nuclei but no sarcolemma can be found indicating the syncytial character of the

circular muscle cell. Two nerve fibers are located ventrolateral. The esophagus is surrounded by the neurilemma (black arrows),

which is produced by the perineurium. D. Close-up transmission electron micrograph of the cuticular intima of the esophagus in

C. The endocuticle is the thickest of the three cuticle layers. The epicuticle is electron-translucent. Abbreviations: Ch = chelicera;

cMC = circular muscle cell; EnC = endocuticle; EpC = epicuticle; ESL = esophagus lumen; ExC = exocuticle; L1 = leg 1; IMC

= longitudinal muscle cell; M = mitochondrion; MCN = muscle cell nucleus; N = nucleus; NF = nerve fiber; NP = neuropil; P

= pharynx; P3/P4 = prosomal muscle 3/4; PL = pharynx lumen; PN = perineurium; PP = pedipalp; PrPlt = propeltidium; ROS =

rostrosoma.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 59

Fig. 38. Eukoenenia spelaea (Peyerimhoff, 1902), midgut in the opisthosoma. A. Light micrograph of a cross-section through

segment 10 of a male. The midgut (green line) is a simple sac. Secretory cells can be easily distinguished from digestive cells. B.

Transmission electron micrograph of the midgut epithelium. Secretory cells are recognized by the electron-dense secretion granules

(asterisk). The microvilli of the secretory and digestive cells extend into the midgut lumen. Apical microtubuli are more numerous

in digestive cells than in secretory cells. The digestive cells have large electron-translucent excretory vesicles. C. Transmission

electron micrograph of the semicircular midgut musculature. The muscle cells are located within indentations between digestive

cells. D. Transmission electron micrograph of the longitudinal midgut musculature. The thin muscle cells can be found spread

sparsely along the midgut. Abbreviations: AT = apical microtubule; cMC = circular muscle cell; DC = digestive cell; ExV =

excretory vesicle; IMC = longitudinal muscle cell; LV = lipid vesicles; MG = midgut; MGD = midgut diverticula; MV = microvilli;

N = nucleus; SC = secretory cell; StC = storage cell.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

54 Sandra Franz-Guess & J. Matthias Starck

Fig. 39. Eukoenenia spelaea (Peyerimhoff, 1902), rectal sac. A. Light micrograph of a sagittal section through the metasoma of a

female. The rectal sac (encircled by a green line) fills almost the entire metasoma. The nuclei of the high prismatic cells are located

mostly basally. The lumen of the rectal sac is narrow. A distinct ectodermal, cuticula cover is missing. B. Light microscopic, slightly

oblique cross-section of the rectal sac (green line) of segments 16-18. Small droplets are located mostly apically in the cells of the

rectal sac. C. Transmission electron micrograph of the rectal sac epithelium. The dense microvilli of the rectal sac epithelial cells

extend into the lumen. Abbreviations: A = anus; AT = apical tubule; MG = midgut; MV = microvilli; N = nucleus; RsC = rectal sac

cells; RsL = rectal sac lumen.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 5D

CxTp CxTpA CxTpB

Fig. 40. Eukoenenia spelaea (Peyerimhoff, 1902), coxal organ. A. Schematic drawing of the coxal organ based on serial cross-

section light micrographs and 3D-reconstruction. It consists of the saccule (orange), proximal tubule (brown) and distal tubule

(yellow). The proximal and distal segments of the tubule have a basal labyrinth (dark) and an apical cytoplasmic part (light).

The excretory duct is connected with the cytoplasmic part of the tubule cells. B. Light micrograph of a cross-section through the

coxal organ region at the level of the 1‘ leg of a female. The saccule is not attached to musculature. The podocytes stain light and

no lumen is visible. C. Light micrograph of a cross-section posterior to the saccule. The proximal segment of the tubule stains

lighter than the distal segment tubule allowing for a straightforward distinction in light microscopy. Within the distal tubule, the

nucleus is sometimes located in the basal region of the cells. D. Transmission electron micrograph of the saccule. It is completely

surrounded by hemolymph. The saccule has a small lumen. E. Light micrograph of a cross-section of the proximal tubule at the

level of the 3“ leg of a male. The basal part of the glandular cells contain dark staining secretion granules. F. Transmission electron

micrograph of the proximal tubule. The basal labyrinth is filled with electron-dense material. Lateral membrane folds of the tubule

cells interdigitae thus connecting the apical parts of the tubule cells (arrows). Abbreviations: BL = basal labyrinth; C4 = cheliceral

muscle 4; CxS = coxal organ saccule; CxSL = coxal organ saccule lumen; CxTd = coxal organ distal tubule; CxTdA = distal tubular

cell apical region; CxTdB = distal tubular cell basal region; CxTp = coxal organ proximal tubule; CxTpA = proximal tubular cell

apical region; CxTpB = proximal tubular cell basal region; ED = excretory duct; EP = excretory pore; HLy = hemolymph; N =

nucleus; PC = podocyte; RER = rough endoplasmic reticulum.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

56 Sandra Franz-Guess & J. Matthias Starck

CxTdB CxTdA yy neni

Fig. 41. Eukoenenia spelaea (Peyerimhoff, 1902), coxal organ. A. Transmission electron micrograph of the distal segment of

the tubule. A large number of electron-translucent (almost empty) vesicles occurs within the basal labyrinth. The cells are tightly

connected by membrane interdigitations (arrows). B. Close-up transmission electron micrograph of the apical cell region in A,

where septate junctions connect the cells apically (arrows). There is no visible lumen due to the tight connection of the cells, only

a thin layer of extra-cellular matrix. C. Light micrograph of a cross-section of the anterior loop of the distal segment of the tubule.

Both arms of the tubule are closely neighboring each other. D. Light micrograph of a slightly oblique longitudinal section of the

excretory duct. The duct originates between the apical parts of the tubule cells. It is lined with a thin cuticle intima. The excretory

pore has a thicker cuticle. Abbreviations: BL = basal labyrinth; CxTd = coxal organ distal tubule; CxTdA = distal tubular cell apical

region; CxTdB = distal tubular cell basal region; ECM = extra-cellular matrix; ED = excretory duct; EP = excretory pore; N =

nucleus.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 57

is approximately twice as thick as the endocuticle and

shows regions of increased electron density (Fig. 37B).

The epicuticle is a thin layer covering the exocuticle and

appears to be electron-dense.

Esophagus

The esophagus extends from the posterior end of the

pharynx to the region of the 2™ leg (Fig. 34). Its lumen

has the shape of a stylized X. It is (partially) surrounded

by the connectives between the supra- and subesopha-

geal ganglia, and is separated from the neuropil by the

neurilemma. The esophagus is surrounded by a few, ir-

regular placed thin fibers of longitudinal musculature

followed by an outer ring of thin circular musculature

(CMC, IMC; Fig. 37C). The circular musculature is a

syncytium (Fig. 37C). The outer cell membrane of the

circular muscle layer is invaginated at the Z-lines, similar

to what we described for the ultrastructure of the heart

muscle. The paired nerve supplying the esophagus 1s lo-

cated ventrolateral between the circular musculature and

the epithelial cells (NF; Fig. 37C). The epithelium of the

esophagus consists of about five cells in cross-section

(Fig. 37C). These cells have irregularly shaped nuclei,

perinuclear cytoplasm, and few mitochondria. The lu-

men of the esophagus is covered by cuticle with a thick

electron-translucent layer of endocuticle, a thin electron-

dense layer of exocuticle, and a thin electron-translucent

layer of epicuticle (EnC, EpC, ExC; Fig. 37D).

Midgut

The prosomal midgut is tube-shaped and forms two later-

al diverticula in the region of the 3 leg (MGD; Fig. 34).

The diverticula are simple evaginations of the midgut

tube. The midgut and its diverticula are surrounded by

few strands of longitudinal and incomplete circular mus-

culature, which consists of one myofibril per muscle cell

(IMC, cMC; Fig. 38C—D). The lumen of the midgut and

diverticula is narrow. The epithelium is pseudo-stratified

and is identical in both the midgut tube and the divertic-

ula. It consists of two distinct types of cells, digestive

cells and secretory cells (DC, SC; Figs 38A—B). Diges-

tive cells are more numerous than secretory cells, they

are high prismatic and have a basal nucleus. Numerous

lipid vesicles can be seen in the lightly stained cytoplasm

of the digestive cells (LV; Fig. 38B). Secretory cells are

fewer in number, but are easily recognized in LM due

to the dark staining cytoplasm and the high number of

intensively staining secretory vesicles (*, SC; Fig. 38A—

B). Like in the digestive cells, their nuclei are located at

the base of the cells. Transmission electron micrographs

show a microvilli border in both cell types. The microvil-

li are numerous and extend into the small midgut lumen

(MV; Fig. 38B). Apical tubuli are more abundant in di-

gestive cells than in secretory cells (AT; Fig. 38B).

Bonn zoological Bulletin Suppl. 65: 1-125

The opisthosomal midgut (Fig. 38A) is a large sac with

constrictions caused by the segmental dorso-ventral mus-

culature (Fig. 34A). The epithelium of the opisthosomal

midgut is the same like that of the prosomal midgut and

a cytological distinction between midgut and midgut di-

verticula 1s not possible.

Rectal sac

The rectal sac is located in the metasoma, segments 1 5—

18 (Rs; Fig. 34). It is differentiated from the midgut by a

single-layered epithelium consisting of large, high-pris-

matic cells (RsC; Fig. 39A—B) with more or less basal

nuclei of the rectal sac cells. The cytoplasm stains inten-

sively in light microscopy. Small (secretory?) granules

and larger lipid vesicles are located mostly in the apical

cell region (Fig. 39A—B). Transmission electron micro-

graphs show an apical microvilli border with a higher

microvilli density than in the midgut epithelium. The

epithelial cells of the rectal sac are rich in apical tubuli

(AT; Fig. 39C). — It should be noted that no cuticle lining

was found and the rectal sac continues directly into the

anus, which is located ventral to the flagellum within the

membrane of segment 18 (A; Figs 34, 39A). The mem-

brane surrounding the anal opening is arranged in folds

(Fig. 13B).

Excretory organ

Eukoenenia spelaea has one pair of coxal organs as the

only excretory organs, Malpighian tubes are not present.

The coxal organ consists of a saccule, a proximal and a

distal tubule, an excretory duct, and an excretory pore

(CxS, CxTd, CxTp, ED, EP; Figs 34, 40-41). The proxi-

mal and the distal segment of the tubule both have long,

blind ending appendices, that reach from the forth seg-

ment in a posterior direction. The appendix of the proxi-

mal tubule reaches into the 9" segment.

The saccule is located in the region of the 1* leg, just

posterior to the insertion of the basal article of the first

leg on the prosoma. In total, it consists of seven to eight

podocytes (PC; Fig. 40B, D). The pedicels of the podo-

cytes are oriented towards the surrounding hemolymph.

The center of the saccule has a narrow lumen (CxSL;

Fig. 40D). The nuclei of the podocytes are oblong and

central in the cytoplasm. The cytoplasm is rich in rough

endoplasmic reticulum, free ribosomes and glycogen

granules, but has few electron-dense droplets. The sac-

cule connects to the most anterior end of the glandular

section (Fig. 40A). A distinct collecting tubule was not

identified.

The proximal segment of the tubule has a long, blind

ending extension, reaching straight along the lateral body

wall into segment 9 (Fig. 34). At the anterior, where the

proximal tubule connects with the saccule and the distal

tubule, it makes a sharp U-turn (Fig. 40A). At the pos-

©ZFMK

58 Sandra Franz-Guess & J. Matthias Starck

Uin Vex AGR AG RS OVD Egg

Fig. 42. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of the female reproductive organs based on serial cross-

section light micrographs and 3D-reconstruction. A. Dorsal view. The unpaired ovary (brown) extends from segment 10 to segment

13. The eggs are positioned in an anterior position within the ovary. The accessory gland (orange) is located anterior to the ovary.

The reservoirs of the accessory gland are located anterior to the club-shaped receptaculum seminis (dark blue). The accessory gland

and the ovarian ducts (red) are the only paired internal structure of the female reproductive system. The ovarian ducts connect to

the uterus interna (light blue) in segment 9. The uterus externa (yellow) opens toward the outside. B. Sagittal view. The ovarian

ducts originate from a middle position along the ovary in segment 12. The accessory gland is located within the genital operculum

and the genital lobes. The receptaculum seminis opens halfway between the genital opening and the posterior end of the genital

operculum. Arabic numbers indicate the segments. Abbreviations: AG = accessory gland; AGR = accessory gland reservoir; OV =

ovary; OVD = ovarian duct; RS = receptaculum seminis; Uex = uterus externa; Uin = uterus interna.

terior, it bends ventral and slightly towards the median

line before ending blind. The proximal segment of the

tubule has a prismatic epithelium with an apical cyto-

plasmic part and a basal labyrinth (CxTpA, CxTpB, BL;

Fig. 40C, E-F). Cross sections show only three cells

(Fig. 40F). Their heterochromatin-rich nuclei are located

in the cytoplasmic part of the cells. The lateral walls of

the cytoplasmic part of neighboring tubule cells interdig-

itate by lateral folds of the cell membrane; a thin layer of

extra cellular matrix (arrows, Fig. 40F) is found between

their cell membranes. Secretion granules are also present.

The basal labyrinth contains vesicles of different electron

density. The proximal segment has no open lumen. This

appearance is uniform throughout, from the anterior to

the posterior end.

Bonn zoological Bulletin Suppl. 65: 1-125

The distal segment of the tubule is located within the

basal article of leg 1 (Fig. 34). It originates from the

proximal segment in close neighborhood to the connec-

tion with the saccule. The distal tubule forms a hairpin

turn before it connects to the excretory duct (Figs 40A,

41C). The distal segment of the tubule also has a long

extension that parallels closely the proximal tubule to the

region of the 2! leg where it ends blind. The epitheli-

um of the distal tubule consists of prismatic cells with

an extensive basal labyrinth (Fig. 41A). A minimum of

three cells is found to build the tubule in cross-section

(Fig. 41A). The nuclei are located mostly apical, how-

ever, they can also be found within the basal labyrinth

(N; Fig. 41C). The cells of the basal labyrinth contain

various electron-dense and electron-translucent vesicles.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 59

Like in the proximal segment of the tubule, the apical cy- __ ticle-lined excretory duct (ED; Fig. 41D). The excretory

toplasmic parts of the cells are connected by membrane __ pore 1s internally covered by thick cuticle and opens pos-

interdigitations and septate junctions. The tubule has no _ teroventral to the basal article of leg 1, at the transition

lumen (arrows; Fig. 41B). The tubule connects to the cu- —_ region between basal article and prosoma.

20pm

Fig. 43. Eukoenenia spelaea (Peyerimhoff, 1902), light micrographs of cross-sections through the female reproductive organs.

A. Area of the genital operculum in segment 9. The cells of the paired accessory glands (orange line) contain numerous secretion

vesicles. The uterus externa (yellow line) has numerous infoldings and a thin cuticle intima. B. Area of the receptaculum seminis

in segment 10. The accessory gland extends into the genital operculum. The cuticle next to the reservoirs (arrows) differs from the

neighboring cuticle. The receptaculum seminis has a thick cuticle lining. C. Area of the ovary (brown) in segment 11. The lumen

of the ovarian duct (red line) is narrow and barely visible. The eggs next to the oocytes vary in size. Abbreviations: AG = accessory

gland; AGR = accessory gland reservoir; GO = genital operculum; MG = midgut; OC = oocyte; OV = ovary; OVD = ovarian duct;

RS = receptaculum seminis; SoC = somatic cell; Uex = uterus externa.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

60 Sandra Franz-Guess & J. Matthias Starck

i 2

™ tet Cia

4 7

=: 4

tikes ated aM OW rd ar i=

Fig. 44. Eukoenenia spelaea (Peyerimhoff, 1902), transmission electron micrographs of female reproductive organs. A. Cross-

section through the ovary (yellow line) in segment 11. The large roundish oocytes (orange line) are nested between the irregularly

shaped somatic cells (green line). The somatic cells are located at the wall of the ovary. B. Cross-section of the right genital lobe in

segment 10. The epithelial cells of the accessory gland are high-prismatic with a basal nucleus. The gland extends into the genital

lobe. Abbreviations: AGC = accessory gland cells; GL = genital lobe; N = nucleus; OC = oocyte; OV = ovary; SoC = somatic cell.

Reproductive organs

Details of the reproductive organs of Eukoenenia spelaea

are based on light microscopic (LM) observations and

transmission electron microscopy (TEM) of the female

ovary and accessory gland. Due to material limitations,

male structures were described using LM serial sections

only.

Female

The female reproductive organs consist of the ovary,

the ovarian ducts, the uterus interna, the uterus externa,

the accessory gland, and the receptaculum seminis. The

genital opening is located between the posterior genital

operculum on segment 9 and the anterior pair of genital

lobes on segment 10.

The ovary is a median, sac-shaped, unpaired organ and

located in segments 10-13 (OV; Fig. 42). It consists of

somatic cells, eggs of different sizes, oocytes as well as a

lipid-rich secretion in larger specimens (Figs 12C, 43C).

No musculature was found associated with the ovary

wall. The somatic cells are flattened, irregularly shaped

cells which attach directly to the ovarian wall (Figs 43C,

44A). The nuclei are also irregularly shaped. The oocytes

are nested between the somatic cells. They have a charac-

teristic large, heterochromatin-poor nucleus (Fig. 44A).

Oocytes are found at the apex of the ovary while devel-

Bonn zoological Bulletin Suppl. 65: 1-125

ie gk

oped eggs were found close to the opening to the oviduct.

Two of the studied females carried 6 and 12 eggs, re-

spectively. In the larger specimen, the eggs were between

15 um and 60 um in diameter. The eggs were located an-

terior in the ovary while the posterior region of the ovary

was filled with of lipid-rich secretions (Fig. 12C). In the

smallest specimen, the eggs were between 15 um and

25 um diameter, probably representing an earlier devel-

opmental stage. In this specimen, the eggs were located

posteriorly within the ovary and no secretion was found

in the lumen of the ovary.

One pair of ovarian ducts originates lateral from

the ovary at the border of segments 11 and 12 (OVD;

Fig. 42). The ducts extend anterior into segment 9, where

they connect to the unpaired uterus interna. The lumen of

the ovarian ducts is narrow (Fig. 43). The uterus interna

is located in the anterior part of segment 9 (Uin; Fig. 42).

It is flat, sac-shaped, and has a thin squamous epithelium.

It continues into the uterus externa, which is also locat-

ed in segment 9 but posterior to the uterus interna (Uex;

Fig. 42). Like the uterus interna, the uterus externa has a

squamous epithelium, however, it displays many infold-

ings and it is lined with a thin cuticle intima (Fig. 43A).

The nuclei are located basally (Fig. 43A).

The receptaculum seminis is an unpaired structure lo-

cated in segment 10, medioposterior to the genital open-

ing (RS; Fig. 42). The overall shape of the receptaculum

is club-shaped in a dorsal-ventral axis, with the opening

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 61

FSD GA aAGpAG VD

te%, Prk .

ate are

on ony’

eh) eS

eae 38!

* «

a5 *

aAGR

“

a*e, “ ete * te 7

rh kL.

» he

ae eT

ea? @

]

sete *

——

————

Fig. 45. Eukoenenia spelaea (Peyerimhoff, 1902), schematic drawing of the male reproductive organs based on serial cross-section

light micrographs and 3D-reconstruction. A. Dorsal view. The paired testes (brown) continue into the winding deferent ducts which

fuse anteriorly before terminating at the genital atrium (yellow). The paired anterior accessory gland (orange) is located ventral

and lateral to the genital atrium and deferent ducts. The unpaired posterior accessory gland (red) is dorsal to the genital atrium and

extends far into segment 10. B. Reconstruction of a spermatozoon. The large vacuole includes five dark staining spherical vesicles

surrounded by several smaller light staining vesicles (asterisk). Two oblong structures are located at the apex of the spermatozoon

(arrows). C. Sagittal view with a large number of spermatozoa contained in the vas deferens. Two fusule ducts originate from an

anterior accessory gland reservoir and extend into the genital lobe 1. The posterior accessory gland has extensions into genital

lobe 3. Arabic numbers indicate the segments. Abbreviations: aAG = anterior accessory gland; aAGR = anterior accessory gland

reservoir; pAG = posterior accessory gland; FSD = fusule duct; GA = genital atrium; SZ = spermatozoon; T = testes; VD = vas

deferens.

ventral in the gap between the genital operculum and the

base of the genital lobes (Figs 42B; 43B). The epithelium

is flattened and barely distinguishable in light microsco-

py. The lumen of the receptaculum is covered by a thick

cuticle intima (Fig. 43B). Dorsal, the cuticle is arranged

in two wrinkled lobes, originating medial and extending

towards lateral (*; Fig. 43B). These lobes build a closed

tube anterior, but are open posterior. No spermatozoa

were found in the receptaculum.

The paired accessory glands extend from the junction

of uterus interna and uterus externa to just anterior to the

ovary in segments 9 and 10 (AG; Fig. 42). The gland

is lobed, and extends into the genital operculum as well

as the genital lobes (Figs 42B, 43B, 44B). The epithe-

lium consists of high prismatic cells, which are filled

with numerous secretory vesicles (Fig. 43B). Two lateral

reservoirs are located within the accessory glands. They

extend anterior from the genital operculum to both sides

Bonn zoological Bulletin Suppl. 65: 1-125

of the receptaculum seminis posterior. Both reservoirs

are oriented towards the uterus externa and the genital

opening (AGR; Figs 42, 43B). A glandular opening could

not be identified, however, in regions where the reservoir

touches the body wall, 1.e., just past the genital opening,

the epidermis and the cuticle are thin (arrows; Fig. 43B).

Male

The male reproductive organs consist of the testes, the

vas deferens, a paired anterior accessory gland, an un-

paired posterior accessory gland, and the genital atrium.

The genital opening is located between the second and

third pair of genital lobes between segments 9 and 10.

The testes are paired and located lateroventrally in seg-

ments 10—13 (T; Fig. 45). They extend lateral and parallel

to the ventral musculature. No musculature was found

associated with the testes wall. The epithelium consists

©ZFMK

62 Sandra Franz-Guess & J. Matthias Starck

10um

* @ FSD a FSD

Fig. 46. Eukoenenia spelaea (Peyerimhoff, 1902), light micrograph of a cross-sections of the male reproductive organs. A. Anterior

region of left and right vas deferens (brown line) and the paired anterior accessory gland (orange line) in segment 9. In the middle

is the fused part of the vas deferens. The vas deferens has a flat squamous epithelium (arrows) and is filled with fully developed

spermatozoa. The posterior end of the subesophageal ganglion is located dorsal to the vas deferens. The secretory vesicles of the

anterior accessory gland epithelial cells stain differently. The fusule ducts originate at the anterior accessory gland’s reservoir

(inset). B. Genital atrium (yellow line) in segment 9. The spherical enclosures of the spermatozoan vacuole have lighter outer and

darker inner vesicles (asterisk, inset). The spermatozoa have paired structures located in their apex (arrows, inset). The genital

atrium is lined with a thick layer of cuticle. The fusule ducts are located inside the genital lobe 1. Abbreviations: aAG = anterior

accessory gland; aAGR = anterior accessory gland reservoir; FSD = fusule duct; GA = genital atrium; GL1 = genital lobe 1; MG =

midgut; SubEG = subesophageal ganglion; SZ = spermatozoon; VD = vas deferens.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 63

of squamous cells and is barely discernible in light mi-

croscopy (Fig. 47B). Two cell types are present, somatic

cells and germ cells. The somatic cells are small, of equal

shape and size, and the cytoplasm stains bluish (SoC;

Fig. 47B). They are irregularly dispersed between the

germ cells. Germ cells vary in size and the cytoplasm

stains purple in light microscopy (GC; Fig. 47B). Sper-

matozoa of different developmental stages, 1.e., sper-

matogonia, spermatocytes, and spermatids, are found

throughout the entire length of the testes (SpC, SZ;

Figs 46-47). The largest spermatozoa are 12-15 um in

diameter and have a large vacuole (diameter 10—14 um)

with five spherical enclosures (diameter 4.5—5 um) each

(*in inset; Figs 45B, 46B). These enclosures contain

several small droplets. The droplets in the periphery of

an enclosure stain light in LM, the droplets in the cen-

10pm

Fig. 47. Eukoenenia spelaea (Peyerimhoff, 1902), light micrographs of cross-sections through the male reproductive organs.

A. Section through the anterior part of the second genital lobe in segment 9. The epithelial cells of the posterior section of the

anterior accessory gland (orange line) have only smaller and darker staining secretory vesicles. The unpaired posterior accessory

gland (red line) has prismatic cells with nuclei located basally. B. Section through the testes (brown line) at the border of segments

11 and 12. The number of fully developed spermatozoa is reduced. The posterior section of the testes is filled with different

developmental stages of spermatozoa. Abbreviations: aAG = anterior accessory gland; pAG = posterior accessory gland; FSD =

fusule duct; GC = germ cell; GL1/2 = genital lobe 1/2; MG = midgut; SoC = somatic cell; SoC = spermatocyte; SZ = spermatozoon;

T = testes; VD = vas deferens.

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

64 Sandra Franz-Guess & J. Matthias Starck

Fig. 48. Eukoenenia spelaea (Peyerimhoff, 1902), flagellum. A. Light micrograph of a longitudinal section of flagellar articles 2—5.

The flagellum has no intrinsic musculature and is mostly filled with hemolymph. The cuticle is thinner where the flagellar articles

connect (arrowheads). Secretory vesicles are located lateral in the hemolymph space (arrows). Numbers indicate flagellar articles.

B. Light microscopic cross-section of a flagellar article. The hemolymph space is surrounded by large vesicles (asterisk). Secretory

vesicles are only found towards one side of the flagellar article (arrows). C. Transmission electron micrographic cross-section of a

flagellar article with a pair of lateral nerve fibers (yellow). Vesicles of varying sizes are found adjacent to the nerves. The secrete

vesicles appear to be located within the hemolymph (arrows). Abbreviations: EC = epidermal cell; HLy = hemolymph; N = nucleus;

NF = nerve fibers; SS = sensory seta; Sp = spike.

ter of the enclosure stain intensively (Figs 45B, 46). The

nucleus of the large spermatozoa is located basal and is

difficult to diagnose in LM because it 1s comparatively

small and oblong. Two small, dark staining, oblong struc-

tures are located at the apex of the spermatozoa (arrows;

Figs 45B, 46B). The nature of these structures is unclear.

During development of the spermatozoa, the vacuole

with the spherical enclosures increases in size, the nucle-

us changes shape from round to oblong, and the paired

apical structure is still missing.

The vasa deferentes extend from the anterior end of the

testes in segment 10 into segment 8, where they merge

into a short single tube before opening into the genital

atrium in segment 9 (VD; Fig. 45). The course of the vas

deferens is not straight but winding (Fig. 45). It is locat-

ed laterally of the dorso-ventral musculature of segments

9 and 10 (DV2, DV3; Fig. 19B). In segment 9, the vas

deferens is oriented towards the median line, in segment

Bonn zoological Bulletin Suppl. 65: 1-125

10, it is oriented towards the body wall. The tubes are

filled with spermatozoa. The epithelium consists of flat

squamous epithelial cells (Figs 46, 47A).

The paired anterior accessory gland 1s located ventral

to the vas deferens in segments 9 and 10 (aAG; Fig. 45).

The two glandular sacs have anterior extensions, which

form an anterior loop. The extensions are oriented toward

posterior, just anterior to the genital atrium. The sacs ex-

tend posterior, and are located close to the body wall and

lateral to the dorso-ventral muscle DV2. The epithelium

of the anterior accessory gland consists of high prismat-

ic cells with the nuclei located basally (Figs 46, 47A).

The cytoplasm is filled with secretory vesicles of differ-

ent composition, large and lightly stained, and small and

darker stained in light microscopy (LM). The larger vesi-

cles are found within cells oriented towards the median

line and closely associated with the reservoirs (Fig. 46).

The smaller vesicles, however, are found in cells oriented

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 65

towards the body wall and towards posterior in the glan-

dular sac (Figs 46, 47A). Anterior within the loop, the

anterior accessory gland has large reservoirs, which stain

lightly in LM (aAGR; Figs 45—46). Connected to these

reservoirs are the fusule ducts. There are two fusule ducts

per gland. These cuticle lined tubules extend through the

first genital lobes to the fusules, two per genital lobe

(FSD, GL1; Figs 14D-—F, 45-46).

The posterior accessory gland is unpaired, sac-like

in appearance, and is located in segments 9 and 10. It

lies posteriorly to the genital atrium and has extensions

into the third pair of genital lobes. Posteriorly it extends

mid-section in segment 10 (pAG; Fig. 45). The posterior

accessory gland consists of prismatic cells filled with two

types of secretory vesicles, one small type staining dark

and one larger type staining light (Fig. 47A). The nuclei

are located basally within the cells. No secretory duct

was found, but a region with thin epidermis and cuticle

just posterior to the genital opening.

The genital atrium is located in the posterior half of

segment 9 (GA; Fig. 45). It is a flattened sac with an-

terodorsal/posteroventral orientation and terminates at

the genital opening between genital lobes 2 and 3. In

cross-section, the lumen of the atrium has the shape of

a stylized W in the anterior region (Fig. 46B). Towards

the genital opening, the lumen is flattened. The genital

atrium is internally covered by a squamous epithelium

which secretes a thick layer of cuticle into the lumen. The

cuticle stains heterogeneous and is darker in the dorsal

and ventrolateral parts of the genital atrium (Fig. 46B).

Flagellum

The terminal flagellum is a prominent feature of Fu-

koenenia spelaea. It has a sclerotized basal ring-shaped

socket that connects it to the last opisthosomal segment.

The cuticle between flagellar articles is thinner than on

the articles (arrowheads; Fig. 48A). The flagellum has no

intrinsic musculature (Fig. 48). On each article, a super-

ficial cuticular groove surrounds the article separating a

proximal part with a group of sensory setae from a distal

part which carries cuticular spikes on articles 1-3, 5, 7

and 9 (Sp; Figs 3B, 4B, 48A). The epidermis of the fla-

gellum is a squamous epithelium with oblong nuclei. The

epidermal cells contain light staining vesicles of different

sizes (Fig. 48). A pair of nerves is located lateral in the

flagellum (Fig. 48C). The central lumen of the flagellum

is filled with hemolymph, which makes up approx. 50%

of the total flagellar volume. Within the hemolymph,

small dark staining vesicles can be found. These vesicles

are clustered and located adjacent to the epidermis. The

nature of the vesicles is unclear.

Bonn zoological Bulletin Suppl. 65: 1-125

Phylogenetic analysis

Phylogenetic position of Eukoenenia spelaea

Not surprisingly, when adding Eukoenenia spelaea to

Shultz’s (2007a) original character matrices either for ex-

tant or for both, extant and fossil taxa (Appendix I), the

two unweighted analyses result in consensus trees similar

to Shultz’s (2007a) original analysis.

Changes in character states and new characters in the

data matrix

Based on our results, we had to modify some of the

existing character states in the matrix and also created

new character codes, thus augmenting the data matrix of

Shultz (2007a; Appendix I: Tab. 6).

The first modified character state (6) is the division of

the carapace with distinct pro-, meso- or metapeltidial

sclerites. Our analysis of the musculature of Ewkoene-

nia spelaea showed that the middle sclerites (“meso-

peltidia’) are not tergites of a segment, as previously

assumed. Palpigrades have only two peltidia in a strict

sense. Therefore, the character coding was changed to

“absent”, “two peltidia” and “three” peltidia). The coding

was adjusted for E. spelaea, Palpigradi, Schizomida, and

Solifugae (Appendix I: Tab. 6).

The first new character is the prosternum (12a). Our

analysis showed that contrary to previous interpretations,

the anterior-most sternum of Eukoenenia spelaea incor-

porates the sterna of segments 2-4. It was previously as-

sumed that the sternum includes the sterna of segments

3 and 4 only. The coding was adjusted in E. spelaea to

character state 1 = present, and in the other palpigrade

groups to ? = unknown. All other taxa were coded 0 =

absent (Appendix I: Tab. 6).

The original character state involving the rostrosoma

(32) took only structures into account that included the

pedipalp coxae and anterior elements of the prosoma.

However, the rostrosoma of Eukoenenia spelaea is an

independent structure that does not involve the pedipalp

coxae or prosomal structures and would, therefore, be

coded as absent. To accommodate the specific morpholo-

gy of the palpigrade rostrosoma without pedipalpal con-

tributions, the character state was rephrased in a more

neutral way and the coding was adjusted for E. spelaea

and Palpigradi (Appendix I: Tab. 6).

Character (61) coding for a trochanter-femur joint with

a dorsal hinge or pivot operated by flexor muscles only,

was deleted. It was an assumed autapomorphy for Palpi-

gradi, however, our analysis revealed that an antagonistic

pair of muscles is present in legs 2 and 3 (Figs 19A, 20C).

The number of metasomal sclerites (116) lacked an ap-

propriate number for Eukoenenia spelaea. The original

coding included only zero, two, three, five and nine scleri-

tes. However, palpigrades have four metasomal sclerites.

©ZFMK

66 Sandra Franz-Guess & J. Matthias Starck

| Xiphosurida Xiphosurida

iy ; P

_Stomothecata Opiliones 78/1 Eurypterida s.lat.

57/1 aaa | Avail

COPIES Stomoalhecata BONES

18/3

ri Sollfugae Scorpiones

Haplocnemata |

34/0 14/2

Pseudoscorpiones —— E, spelaea

E. spelaea 98/4 Eukoenenia

76/3

100/0 Eukoenenia Lz Prokoenenia

400/29 85/2 } Arachnida or :

Arachnide Prokoenenia 6/1 —— Ricinulei

Acaromorphia Acaromorpha___| Anaetinotrichida

1710 Ricinulei vn { Opilioacariformes

Anactinotrichida 35/1

Opilioacariformes 16/1 Parasitiformes

39/0| = | .

25/0 ‘Parasitiformes 6/2 Acariformes

Acariformes Solifugae

Haplocnemata

Wee aan 28/2

Ma a Araneae Pseudoscorpiones

¥_ | Pedipalr : Pantetrapulmonata . P

561 | ———— Amblypygi on}' — Trigonotarbida’

Uropyal . Tetrapulmonata ' .

99/14)" Thelyphonida at | Araneae (incl. C. yingi)

gg C] ly

unweighted — Schizomida Th —— Haptopoda™

Pedipalpi ;

B — Amblypyai

Xiphosurida Uropya)

Stomothecata 88/3)" 1 — Thelyphonida

Tetrapulmonata unweighted Schizomida

Ricinulei

E. spelaea

Eukoenenia

Prokoenenia

IWA (k = 1-6) Acari

Fig. 49. Hypotheses about the phylogenetic relationship of Palpigradi. A. Minimal-length topology of the unweighted analysis of

extant taxa. Numbers below internodes indicate bootstrap percentages/Bremer support values. Deepest relationships within Arachnida

are unresolved. Palpigradi are placed as sister group to Acaromorpha. B. Implied weights topology of extant taxa. The implied

weights analysis (IWA) of the matrix of extant taxa with k = 1 (1 tree, best score = 56.72381), k =2 (1 tree, best score = 42.10714),

k = 3 (1 tree, best score = 33.75952), k = 4 (1 tree, best score = 28.27460), k = 5 (1 tree, best score = 24.36688), and k = 6 (1

tree, best score = 21.43009) all resulted in the same major group topology. Palpigradi are sister group of the monophyletic Acari.

Ricinulei are placed as sister group to this clade. C. Minimal-length topology of the unweighted analysis of extant and fossil

taxa. Numbers below internodes indicate bootstrap percentages/Bremer support values. Major group relationships are resolved.

Palpigradi are placed as sister group to Acaromorpha, the same result as in the analysis of extant taxa only. Implied weights analysis

resulted in topologies identical to the unweighted analysis. Conflicts were limited to relationships between terminal taxa. Extinct

taxa are marked by an asterisk.

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 67

Thus, the character coding was adjusted to include four

sclerites (Appendix I: Tab. 6).

Our analysis of the segmental musculature in the pro-

soma of Eukoenenia spelaea revealed the presence of

anterior oblique muscles. In the original data matrix of

Shultz (2007a), such a character was not available. To

accommodate the presence of this muscle type in E. spe-

laea, we created the new character (128a) “anterior

oblique muscles of BTAMS anterior to postoral somite

VI (state 1 only in E. spelaea)” (Appendix I: Tab. 6). The

character coding was adjusted for all taxa.

The suboral suspensor was originally included in the

matrix (Shultz 2007a) with the character state “a tendon

that arises from the BTAMS and inserts on the ventral

surface of the oral cavity via muscle” (131). We showed

that in Eukoenenia spelaea muscle E8 (Fig. 21; Tab 5)

does not insert on the oral cavity but posteriorly on the

pharynx. In order to include this result, the term “oral

cavity” was replaced with “foregut” and the character

coding was adjusted to include the options “ventral on

oral cavity” and “ventral and posterior on pharynx” (Ap-

pendix I: Tab. 6).

Character (135a) coding for the presence of the arcu-

ate body in the protocerebrum was added to the analysis.

This was done to accommodate the lack of this particular

brain structure in Eukoenenia spelaea as well as Acari.

The coding was changed accordingly (Appendix I:

Tab. 6).

A new character (143a) was introduced to account for

the (so far) unique morphology of the trichobothria in

Eukoenenia spelaea. We showed that in contrast to the

known morphology of trichobothrial dendrites of other

euchelicerates, the dendrites of E. spe/aea reach into the

shaft of the trichobothrium, possibly adding a second

sense modality to the trichobothrium, which has never

been described in Euchelicerata. The character coding

was adjusted to reflect this (Appendix I: Tab. 6).

The frontal organ of Eukoenenia spelaea is located

dorsal to the chelicerae. Therefore, character (148) was

rephrased from “intercheliceral” to “supracheliceral” to

accommodate that fact (Appendix I: Tab. 6).

The lateral organ (148a) of Eukoenenia spelaea as well

as Palpigradi was added to the analysis (Appendix I:

Tab. 6). The coding for all taxa was adjusted accordingly

(present in E. spelaea and Palpigradi, absent in all other

taxa).

The coxal organ of Eukoenenia spelaea includes a

morphologically different proximal and distal section of

the tubule. Such morphology of the tubule can also be

found in some Acari. To allow for a more complete phy-

logenetic analysis, we added the character state “coxal

organ with tubule differentiated into proximal and dis-

tal section” (178a). The coding was adjusted for Acari,

E. spelaea, and Palpigradi (Appendix I: Tab. 6).

The ventral plate with its unique morphology has pre-

viously never been described for euchelicerates or any

Bonn zoological Bulletin Suppl. 65: 1-125

palpigrade. It has been added as character 179a for Eu-

koenenia spelaea. The coding was adjusted in E. spelaea

to character state 1 = present. All other taxa were coded 0

= absent (Appendix I: Tab. 6).

The character state involving the dilator muscle of

the precerebral pharynx and/or preoral cavity (194) was

described in the original data matrix (Shultz 2007a) as

“attaching to ventral surface of prosoma”. However, in

Eukoenenia spelaea, these dilator muscles attach ven-

trolateral on the rostrosoma. The character state was

expanded to “attaching to ventral surface of prosoma or

rostrosoma’” and the coding was adjusted accordingly for

E. spelaea and Palpigradi (Appendix I: Tab. 6).

The hindgut with a cuticular lining (203) is part of the

ground pattern of euchelicerates. It is, however, missing

in Eukoenenia spelaea. Thus, the matrix was adjusted to

accommodate this result (Appendix I: Tab. 6).

Results of the analysis of the updated character matrix

including data for Eukoenenia spelaea

The unweighted analysis of the adjusted matrix (Ap-

pendix I: Tab. 6, Al) for the extant taxa produced eight

minimal-length trees (length 426, CI 0.560). The deep-

est interordinal relationships within Arachnida are un-

resolved (Fig. 49A). Five monophyletic groups with

a bootstrap percentage above 80 were recovered, 1.e.,

Arachnida (BP 100), Uropygi (Schizomida + Thely-

phonida, BP 99), Pedipalpi (Uropygi + Amblypygi,

BP 99), Tetrapulmonata (Pedipalpi + Araneae, BP 91),

and Palpigradi (Eukoenenia + Prokoenenia, BP 85). Aca-

romorpha (Acariformes, Anactinotrichida, and Ricinulei)

were reconstructed as with a bootstrap percentage of 25

and placed as sister group to Palpigradi. The placement

of Eukoenenia spelaea with Palpigradi showed a nodal

support of BP 100, all other groups had nodal support

below BP 60. The weighted analysis of extant taxa placed

Palpigradi as sister group to Acari (Fig. 49B). Ricinulei

are the sister group to this relationship.

The unweighted analysis of the matrix containing ex-

tant and fossil taxa resulted in 12 minimal-length trees

(length 475, CI 0.536; Fig. 49C). Only two monophy-

letic groups with bootstrap percentage above 80 were

recovered, i.e., Uropygi (Schizomida + Thelyphonida,

BP 87), and Pedipalpi (Uropygi + Amblypygi, BP 91).

Arachnida showed a nodal support of BP 72. Acari were

reconstructed as diphyletic with Anactinotrichida (Opil-

ioacariformes + Parasitiformes) as sister group to Rici-

nulei. Palpigradi were recovered as sister group to Ac-

aromorpha, however, with relatively weak support (BP

6). The placement of Eukoenenia spelaea with Palpigradi

showed a nodal support of BP 98. Palpigradi (Eukoene-

nia + Prokoenenia) showed a nodal support of BP 76.

All other groups showed nodal support below BP 40

(Fig. 49C).

©ZFMK

68 Sandra Franz-Guess & J. Matthias Starck

Opisthosama

| Mesosoma | Melasoma

Prosoma

Fig. 50. Eukoenenia spelaea (Peyerimhoff, 1902), schematized representation of the segmental axial musculature. In the prosoma,

dorsal suspensors are found in association with the pedipalp, 1°‘, 2™4, 3"*, and 4" leg. Three posterior oblique suspensors are associated

with segments five, six and seven. From the second dorsal suspensor an anterior oblique suspensor arises. The posterior oblique

suspensor at the posterior end of the endosternite inserts at the pleural membrane of segment 8. Ventral suspensor muscles are

present in the first three segments. Muscle E13 originates in the region of leg 2 and inserts in the opisthosoma just anterior of where

the ventral longitudinal musculature inserts. Lateral suspensor muscles are present in the region associated with leg 2 (E10) and

leg 4 (E16, E18, E19). Two dorsal longitudinal muscles in the prosoma might be extensions of the dorsal longitudinal musculature

of the opisthosoma. The first opisthosomal segment is free of dorsoventral and posterior oblique musculature. The following five

opisthosomal segments follow the BTAMS but lack posterior oblique muscles. The last mesosomal segment lacks the dorsoventral

musculature. Stars indicate the attachment sites of extrinsic leg musculature originating at the endosternite associated with legs

1—4. Abbreviations: aos = anterior oblique suspensor; Ch = chelicera; dlm = dorsal longitudinal muscle; ds = dorsal suspensor; dv

= dorsoventral muscle; es = endosternite; Im = leg musculature; Is = lateral suspensor, MtPIt = metapeltidium; PP = pedipalp; PrPlt

= propeltidium; Po = posterior oblique muscle; pos = posterior oblique suspensor; vlm = ventral longitudinal muscle; vs = ventral

suspensor.

DISCUSSION

Body tagmatization

The arthropod body 1s organized as an array of segments.

Blocks of segments may be integrated morphologically

and/or functionally, forming tagmata. As reviewed by

Fusco & Minelli (2013), the terms “segment” and “tag-

ma” are descriptive, and the application of the terms is

conceptually variable and differs between authors. Clear-

ly, [...] “their value as developmental units or units of

evolutionary change should not be uncritically assumed”

[...] (Fusco & Minelli 2013, p. 218). The term “somite” is

occasionally used instead of “segment”. Recently, Dun-

lop & Lamsdell (2017) suggested differentiating between

“somite” and “segment”, with the “somite” representing

a developmental blueprint and the “segment” being the

externally expressed, morphologically recognizable ex-

pression of the “somite”. However, this suggested dis-

tinction is potentially confusing for three reasons: (1)

“somite” denominates the serial mesodermal rudiments

of the dorsal musculature in chordate embryos. (2) As

suggested, it has an inherent component of idealistic

morphology, 1.e., assumes an archetypic blueprint of seg-

mental organization from which “real” animals deviate.

(3) The genetic layout of segmentation is conceptualized

by “parasegments”, which have been documented for all

Bonn zoological Bulletin Suppl. 65: 1-125

arthropods, including chelicerates (Damen 2002, 2007;

Deutsch 2004; Peel 2004; Peel et al. 2005).

Fusion may affect the whole segment or only the dorsal

or ventral part of it. Lamsdell (2013) reviews concepts

of “tagmosis”’, suggesting that true tagmata should be

defined by functional differences related to appendag-

es. While this approach conjures a functional perspec-

tive, it remains fundamentally descriptive and typolog-

ical, because most frequently, function is induced from

topographic morphology. We follow Fusco & Minelli

(2013) in their pragmatic approach, using “segment” and

“tagma” as descriptive terms of modular organization of

arthropods, and do not imply a priori homology when re-

ferring to segment numbers or tagmata. Because segment

borders and tagmata can vary according to methods used,

life stages, and phylogenetic relationship, they need to

be determined explicitly for each taxon and each life

stage. However, by using internal landmarks like serial

axial musculature, we provide a powerful interpretative

framework for segment homology.

The body of euchelicerates is divided into prosoma and

opisthosoma. The border between prosoma and opistho-

soma varies along the longitudinal axis and may even

differ between the dorsal and the ventral side of the body

(e.g., Xiphosurida, Scorpiones, Schizomida, Thelyphoni-

da, Ricinulei; Dunlop & Lamsdell 2017). In some taxa,

the opisthosoma is subdivided into meso- and metasoma

(e.g., Amblypygi, Thelyphonida, Schizomida, Ricinulei,

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 69

Table 7. Comparison of the endosternal musculature associated with the box-truss axial muscle system (BTAMS) by Shultz (2001,

2007b). The complete set of segmental, axial prosoma muscles occurring in the supposed euarthropod ground pattern is given in the

top row and mirrored as the grid system underlying the empirically observed/described muscles in various groups of euchelicerates.

We included the original muscle terminology in each cell of the table so that reference to the original literature is straightforward.

The identification of the muscles of a specific suspensor and segment is based on origin and insertion points. Due to the unclear

segment borders in the prosoma, assignment of suspensor muscles is additionally based on directionality of the muscles. The orig-

inal terminology of the previous authors was used to aid recognition. Question marks indicate that the association with a segment

is unclear. Color codes are as follows: orange = dorsal suspensors, purple = anterior and posterior oblique suspensors, red = lateral

suspensors, blue = ventral suspensors. Abbreviations: aos = anterior oblique suspensor, ds = dorsal suspensor, Is = lateral suspensor,

pos = posterior oblique suspensor, vs = ventral suspensor.

Segment Segment Segment Segment Segment Segment Segment Rapsreneas

1 2 3 4 S| 6 -

vs :

Euarthropod Is Firstman (1973),

BTAMS Shultz (2001, 2007b)

aos

pos

Limulus sp. Is Shultz (2001)

_14

ee Shultz (2001, 2007b)

aos

pos

. VS

Eukoenenia is this study

spelaea

Eukoenenia

mirabilis Millot (1943)

Prokoenenia

wheeleri

Firstman (1973)

Opiliones Is Shultz (2000)

pos

Scorpiones Is Shultz (2007b)

aos

OS EP2. EpP4

Amblypygi Is

aos ;

pos 19 19 19

He TE mer 0004) Fist

Schizomida Js. man (1973)

OS e2? h? 10?

Thelyphonida Is Shultz (1993)

aos

pes 14. 14. 14

Pseudo- ts Firstman (1973),

scorpiones “aos. Mehinert et al. (2018)

Ticks Is Firstman (1973)

aos

pos

Shultz (1999)

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

70 Sandra Franz-Guess & J. Matthias Starck

Scorpiones). It requires analyzing independent morpho-

logical landmarks like origin and insertion of the serial

axial musculature to assign segments to certain tagmata

(van der Hammen 1986; Shultz 1993, 2007b; Mehnert

et al. 2018).

Prosoma dorsum

In the (eu)chelicerate ground pattern, the prosoma con-

sists of seven segments (Weygoldt & Paulus 1979b; Dun-

lop & Lamsdell 2017) that form a single dorsal shield and

carry Six appendages on the ventral side. Indeed, most

euchelicerate groups, including fossil (e.g., Eurypterida)

and basal extant groups (e.g., Xiphosurida) have a single

prosomal shield covering the prosoma (Beier 1931; Ger-

hardt 1931; Gerhardt & Kastner 1931; Kastner 1931b, c,

d; Vitzthum 1931; Dunlop & Lamsdell 2017). Despite

branching-off basal from the euchelicerate phylogeny,

the prosomal shield of Limulus sp. includes derived fea-

tures, 1.e., the large lateral wings, and it is merged with

parts of the first opisthosoma segment (Chilaria seg-

ment). Actually, the prosoma of Limulus is a carapace

comprising seven prosomal segments and (parts of) the

first opisthosomal segment.

Assuming that a single prosomal shield (seven seg-

ments) represents the euchelicerate ground pattern (Wey-

goldt & Paulus 1979b; Dunlop & Lamsdell 2017), the

prosomal morphology has been modified in Palpigradi,

Schizomida, Solifugae and Acari. In these taxa, the pro-

somal shield is dorsally divided into three sclerites, 1.e.,

pro-, meso-, and metapeltidium. The propeltidium 1s as-

sociated with the chelicerae, the pedipalps, and the first

two pairs of walking legs. The meso- and the metapelti-

dium are associated with segments of walking leg 3 and

4, respectively (Kastner 193le). The mesopeltidia of

Schizomida are paired, dorsolateral sclerites, but Solifu-

gae have one unpaired medial sclerite (Kastner 1931e, f).

The morphological reorganization of the prosoma, actu-

ally the entire body, is even more substantial in Opiliones

and Acari (Alberti & Coons 1999). In actinotrichid mites

(Acariformes) the sejugal furrow divides the prosoma

into a proterosoma containing the anterior 4 extremities,

which, by segment numbers, is equivalent to the segments

covered by the propeltidium, and a hysterosoma contain-

ing the posterior two pairs of legs and the opisthosoma.

Pycnogonids also show a body tagmatization into an

anterior cephalosoma with four limb-bearing segments

followed by a number of trunk segments with legs. The

obvious external morphological tagmatization of the pro-

soma into an anterior tagma (proterosoma, cephalosoma)

with four pairs of extremities followed by two posterior

segments has therefore (at least historically) considered

representing an ancestral chelicerate condition (Kraus

1976; see Dunlop & Arango 2005; Ortega-Hernandez

et al. 2017 for a recent discussion).

Bonn zoological Bulletin Suppl. 65: 1-125

Indeed, at first glimpse, the tripartite morphology of the

prosoma of palpigrades appears similar to that of Schizo-

mida and Solifugae. However, in this study we provide

morphological evidence to assign sclerites to segments

by comparing associations of serial axial muscles with

the cuticular sclerites. The (presumptive) ground pattern

of musculature in the prosoma of euchelicerates recog-

nizes segmental dorsal and ventral suspensor muscles for

each of the six post-ocular segments (Shultz 2007b), and

a dorsal longitudinal muscle system in the opisthosoma

that reaches into segments 6 and 7 of the prosoma. Based

on this pattern, we would expect four dorsal suspensor

muscles connecting the endosternite and the propeltidi-

um (for the segments of the chelicerae, pedipalps, and the

first two pairs of walking legs), and one pair of muscles

for each, the mesopeltidium and the metapeltidium. We

would also expect that the extensions of the dorsal longi-

tudinal muscle system of the opisthosoma that reach into

the prosoma, connect to dorsal sclerites associated with

segments 7 (metapeltidium) and 6 (“mesopeltidium’”).

The association of muscles in Eukoenenia spelaea

differs from that expectation in an admittedly complex

manner. Indeed, four dorsal suspensor muscles are asso-

ciated with the propeltidium (E3, E5, E11, E14; Figs 21,

50; Tabs 5, 7). Based on their origin and insertion these

muscles must be assigned to prosomal segments 3—6 with

the second prosomal segment missing a dorsal suspen-

sor muscle. No musculature is associated with the me-

sopeltidium, but one dorsal suspensor muscle with the

metapeltidium (E17; Figs 21, 50; Tabs 5, 7). The mus-

cle assignment, of course, depends on the correct diag-

nosis of the dorsal suspensor muscles. However, this is

straightforward because Palpigradi (like Limulus poly-

phemus) maintain the most complete set of segmental

axial muscles (BTAMS; Fig. 50; Tab. 7). Like in most

other euchelicerates, the first dorsal suspensor muscle

is reduced, but, the ventral suspensor muscles are main-

tained in the anterior segments 2-4 (Figs 21, 50); ventral

suspensor muscles are only missing in the more posterior

segments of the prosoma. Together, dorsal and ventral

suspensor muscles provide a complete set of landmarks

from the first to the last prosomal segment, and their in-

sertion on the dorsal sclerites provides direct evidence

that the propeltidium is the common shield of segments

1—6. Because the mesopeltidium has no muscle insertion

(except P9, which is an intrinsic prosomal muscle) we

suggest it 1s not a segmental tergite, but a dorso late-

ral sclerotization of the pleural fold. Additional support

comes from the dorsal longitudinal muscle system (mus-

cles P11, P12) that connect the propeltidium with the

metapeltidium, and the metapeltidium with the opistho-

soma, respectively, thus skipping the “mesopeltidium”’.

Comparisons with reports on Eukoenenia mirabilis and

Prokoenenia wheeleri reveal differences in the endoste-

rnal muscles associated with the peltidia. Millot (1943)

reported four dorsal, five lateral, and six ventral suspen-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 71

sors in E. mirabilis (Tab. 7). The dorsal suspensor of seg-

ment 7 and parts of the dorsal suspensor of segment 5

(presumably dorsal oblique suspensors) attach to the pos-

terior border of the propeltidium, where they insert in the

same position. There is no mention of suspensor muscles

associated with the meso- and metapeltidium. Thus, fol-

lowing the author’s description of muscle topography, the

propeltidium of EF. mirabilis would span segments 1-7.

A similar endosternal suspensor muscle topography was

described by Firstman (1973) for P. wheeleri. However,

the most posterior dorsal suspensor muscle was associat-

ed with segment 6 by the author, therefore, assigning the

propeltidium to segments 1—6 (Tab. 7). An insertion of

dorsal suspensor muscle to the meso- and metapeltidium

was not described.

While the published results for the three species of Pal-

pigradi differ in some details of muscle topography, they

all converge in the observation that the “mesopeltidium”

has no attachment of dorsal suspensor muscles. They also

converge in the observation that the first prosomal seg-

ment has no suspensor axial muscles and a variable mor-

phology of dorsal and/or ventral suspensor muscles in the

second prosomal segment. Even if these studies present

slightly different observations, they do not report any

suspensor muscle attaching to the “mesopeltidium” thus

support our interpretation of the “mesopeltidium” being a

sclerotization of the lateral pleural membrane rather than

a tergite of the 6" segment. Weygoldt & Paulus (1979b)

already noted that no muscles attached to the “meso-

peltidia”, and discussed that the “mesopeltidia” should

not be considered derived from prosomal tergites. They

also suggested that the division of the prosoma in Pal-

pigradi, Schizomida and Solifugae might have evolved

independently.

Although the external morphology of the dorsal scler-

ites of Solifuges, Schizomida, and Palpigradi appears to

be similar we have now provided evidence (above) that,

indeed, Palpigradi differ from the two other groups in the

tagmatization of the dorsal prosoma. Supposing the ex-

isting descriptions for Solifugae and Schizomida are cor-

rect, 1.e., the propeltidium covers the dorsum of segments

1—5, the mesopeltidium segment 6 and the metapeltidium

segment 7, Palpigradi are different because the propelti-

dium covers segments 1-6. This observation and the ob-

vious phylogenetic distance of Palpigradi from the other

taxa, suggests that the dorsal subdivision of the prosoma

into a propeltidium and metapeltidium has evolved inde-

pendently.

The suggested interpretation of the dorsal subdivision

of the prosoma of Palpigradi has further implications

for our understanding of the evolutionary history of the

chelicerate prosoma. In some publications (Lauterbach

1973; Kraus 1976; Scholtz 1998; Waloszek & Miller

1998; Waloszek et al. 2005; Dunlop & Alberti 2008), a

division of the prosoma behind the second pair of walk-

ing legs was interpreted as a landmark of the ancestral ar-

Bonn zoological Bulletin Suppl. 65: 1-125

thropod head containing the ocular segment followed by

4 segments with extremities. According to this idea, pyc-

nogonids, schizomids, solpugids and Acariformes retain

an ancestral, five-segmented head region (see fig. 5 in

Dunlop & Arango 2005) with the propeltidium (protero-

soma, cephalosoma) as a landmark of a “retained head”.

Traditionally, Palpigradi had been assigned to that group

of chelicerates. However, with the new evidence, now

assigning the propeltidium to segments 1-6, Palpigradi

show a clearly derived pattern that must be considered an

autapomorphy of the group rather than symplesiomorphy

on an ancestral arthropod level.

Prosoma ventrum

The ventral side of the prosoma of arachnids is morpho-

logically variable. Distinct sclerites are present in many

groups, but they differ in number, position and morpho-

logical origin (Moritz 1993; Shultz 1993, 1999). Thus,

an a priori assignment of ventromedial sclerites as “ster-

nites” is a terminological simplification that potentially

creates confusion, in particular because the ancestral

chelicerate had no prosomal sternites, but a ventral food

groove occupying the space between the legs (e.g., Lau-

terbach 1973; Weygoldt & Paulus 1979b; Dunlop & AI-

berti 2008). As a sternite in a strict sense, we consider

the ventral sclerotization of a segment, as compared to

the tergite on the dorsal side. Ventral sclerites (1.¢., ster-

na) may have evolved in the context of terrestrialization.

Given the lack of sterna in Xiphosurida and the obvi-

ous differences in number, position, and origin of ster-

na in terrestrial groups of euchelicerates, they may have

evolved independently in various groups of arachnids.

In scorpions, a single sternum is located between the

coxae of legs 3 and 4. It possibly incorporates parts of the

sternite of the first opisthosomal segment (Farley 1999,

2005; Shultz 2007b). Schizomida and Thelyphonida have

three sterna. The anterior sternum is associated with legs

1 and 2, the middle sternum with leg 3, and the posterior

sternum occupies the region between the last pair of legs

(Borner 1904; Millot 1949c, Moritz 1993). Shultz (1993)

suggested that this posterior sternum is derived from the

sternite of the first opisthosomal segment. In Amblypy-

gi, ventral prosomal sclerites may form one single ster-

num spanning the entire region between legs 1—4 (Millot

1949d, Shultz 1999). However, Shultz (1999) recognized

a separate sclerite located between the last pair of legs

(“metasternum’”) as derived from the first opisthosomal

sternite. Some species of Pseudoscorpiones have a rudi-

mentary sclerotized tubercle located between legs 3 and

A others are missing ventral sclerites (Weygoldt 1969).

The tubercle has been interpreted as a residual sterni-

te (Vachon 1949; Moritz 1993), but its nature remains

vague. Ricinulei (Millot 1949f), and Solifugae (Millot &

Vachon 1949a) have no prosomal sterna. Acariformes

(Actinotrichida; Alberti & Coons 1999) and Palpigra-

©ZFMK

2 Sandra Franz-Guess & J. Matthias Starck

di possess podosomal sclerites that were interpreted as

merged “epimera” (i.e., sclerotizations of the ventrum of

segments 2—5; van der Hammen 1977b, 1982; Alberti &

Coons 1999). While the morphological origin of the “epi-

mera” remains obscure, van der Hammen (1977b) postu-

lated that the “epimera” represented the morphological

substratum from which the coxae evolved in more derived

groups of euchelicerates and suggested that Acariformes

and Palpigradi might be sister in a taxon “Epimerata”.

He based his reasoning on the position of the articula-

tions of the leg articles, the insertion points of intrinsic

leg muscles and the putative lack of a coxa in Palpigradi

and Acari. The value of the position of articulations be-

tween articles of a leg and insertion points of tendons as

phylogenetically informative character was questioned

early (Weygoldt & Paulus 1979b). Our material provid-

ed the opportunity to compare directly the articulations

and tendon insertions of all legs of Palpigradi in detail.

We could not even find a common pattern of articulations

when comparing the legs of individual specimens — each

leg showed a different pattern of articulations and tendon

insertion; none was consistent with the supposed pattern

of “epimerata”. — If “epimera” were the morphological

origin of coxae, we would also expect intrinsic leg mus-

culature emerging from the “epimera” as it does from

the coxa. However, they clearly do not. We are certainly

not the first to reject van der Hammen’s (1977b) idea of

“epimerata” (e.g., Weygoldt & Paulus 1979b, Dunlop &

Alberti 2008). However, by studying the fine structure of

the legs of Palpigradi we could provide additional and

direct evidence that there is no morphological support for

a taxon “epimerata” and that van der Hammen’s (1977b)

ideas are of mere historical interest.

In Eukoenenia spelaea, the ventral side of the prosoma

is covered by four distinct sclerites. Based on examina-

tion of the external morphology, the large anterior scler-

ite supposedly represents the fused sclerites associated

with the palpal segment and the segment of the first leg

(hence “deuto-tritosternum”’, van der Hammen 1982:

Moritz 1993; Alberti & Coons 1999). The three poste-

rior sclerites correspond with segments 5—7 (legs 2-4,

respectively). Again, the segmental musculature of the

prosoma provides independent morphological landmarks

for testing the existing hypotheses/interpretations. We

showed that the anterior sclerite is associated with the

ventral suspensor muscles (El, E2, E7/9), muscles that

are assigned to segments 2—4. Therefore, we suggest that

the anterior ventral sclerite of the prosoma 1s associated

with segments 2-4. This new evidence rejects the exist-

ing interpretation that the sclerite is a “deuto-tritosternum

(suggested alternative terminology “prosternum’”). Pro-

somal segments 5, 6 and 7 do not have ventral suspen-

sor muscles; however, a topographic association of these

sclerites with these three segments 1s straightforward be-

cause of their topographic association with the legs.

Bonn zoological Bulletin Suppl. 65: 1-125

Forming an anterior, large ventral sclerite that couples

the bases of the pedipalps and the first pair of legs sup-

ports the idea that the pedipalps and leg 1 together form

a functional unit. In contrast to the original idea that Pal-

pigradi were using the pedipalp as walking leg (hence

the name; Kastner 1931a), observations of live animals

showed that the first pair of walking legs and the pedi-

palps together are used as sensory appendages (Kovac

et al. 2002; Christian 2004). The exclusive occurrence

of trichobothria and high number of sensory setae on

leg 1, and the intensive innervation of that leg provide

additional morphological evidence that both pairs of ap-

pendages are used for sensing the environment. Similar

evolutionary transformations of the first pair of legs to

mechano- and chemo-receptive palps are known from

Uropygi, Amblypygi, Araneae, and Solifugae.

Chelicerae

The synapomorphic morphology of the chelicerate che-

licerae is tripartite (Weygoldt & Paulus 1979b; Shultz

2007a). The fixed digit and movable digit form a chela.

This morphology has been modified in various taxa of

the euchelicerates by reducing the basal article, forming

a subchela, and/or by adding combs, teeth or other cutic-

ular structures to the surface of the chelicerae.

The chelicerae of Eukoenenia spelaea consist of three

articles. The basal article 1s as long as the chela. Com-

pared with the body size, the chelicerae of E. spelaea are

large, as they have approximately the same length as the

propeltidium. While the tripartite chelicerae of Palpigra-

di certainly reflect a plesiomorphic condition, the surface

of the chelicerae carries large teeth ornamented with fine

cuticular combs, which are a unique feature of Palpigra-

di. Large chelicerae and pincer-like structures are con-

ventionally associated with raptorial function (Kaneko

1988; Moritz 1993). However, the relatively weak che-

liceral musculature, that does not provide antagonistic

function for opening and closing the chelae and the fine

combs on the cheliceral teeth question this interpretation.

Recently, Smrz et al. (2013, 2015) studied the gut con-

tent of palpigrades and found predominantly heterotro-

phic cyanobacteria. They suggested that palpigrades use

their cheliceral teeth and combs to scratch cyanobacteria

from the substrate. Indeed, the spacing between the fine

teeth of the cheliceral combs is 0.05—0.1 um and would

certainly be suitable to scratch cyanobacteria from the

sediment.

Pedipalps and legs

Euchelicerate appendages have been described as con-

sisting of seven articles, but the total number of leg arti-

cles may differ between legs. In the specialized antenni-

form legs of Amblypygi, the tibia can consist of up to 43

articles and the tarsus up to 105 (Weygoldt 1996). In The-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 73

Table 8. Comparison of leg article terminology of Palpigrad.

Article 1 Article 2

Shultz (1989) Coxa Trochanter

van der Hammen (1982) Trochanter Femur 1

Millot (1949/) “eoxa* Trochanter

Article3 Article4 Article5 Article6 Article 7-11

Femur Patella Tibia Basitarsus Telotarsus

Femur2 Genu Tibia Tarsus 1 Tarsus 2—7

Me Basitarsus Tarsus

Femur Patella Tibia (1-4) (1-3)

' Millot uses the phrase “hanche” in his publications.

lyphonida, leg 1 consists of 14 articles in total (Grams

et al. 2018). A reduction to six articles can be observed

in the pedipalps of Amblypygi, Araneae (Moritz 1993),

Opiliones (Pinto-da-Rocha et al. 2007), Pseudoscorpio-

nes (Weygoldt 1969), Ricinulei (Millot 1949f), Schizo-

mida (Moritz 1993), Scorpiones (Polis 1990), Solifugae

(Punzo 2012), and Thelyphonida (Grams et al. 2018).

Only two pedipalpal articles may be found in some Acari

(Moritz 1993). Not surprisingly, the homologization and

terminology of the leg articles differs among authors

(e.g., van der Hammen 1977a; Shultz 1989; Tab. 8).

Extremities of Eukoenenia spelaea differ in number

of articles and muscle topography according to their po-

sition. The pedipalp has nine articles and leg 1 has 11

articles. Legs 2 and 3 have seven articles, but leg 4 has

eight articles. What we describe as article 6 of leg 1 has

been documented as two separate articles in other species

(Kastner 1931a; Millot 1949a). In E. spe/aea, this article

shows a superficial cuticular groove in the middle, but it

has no cuticular joint structure. Also, muscle (LI8) and

tendon (LI9t; Figs 19A, 20C) span the entire article and

show no attachment points of muscles/tendons adjacent

to the circular cuticular groove located medially on the

article. Therefore, we cannot clarify, whether this article

is the result of fusion of two separate articles or whether

it is a single article.

Van der Hammen (1977a) compared articulations

points and muscle/tendon insertion in articles of leg IV

among groups of euchelicerates and concluded that Pal-

pigradi had no coxa, but the first article of the walking

leg was a trochanter. Based on this observation and the

occurrence of ventral prosomal sclerites he proposed his

idea that the coxae were ancestrally missing in Palpigradi

(and some Acariformes). Coxae would only later evolve

from the ventral sclerites, which he termed “epimera”

(thus epimerata). Our results provide the opportunity to

compare his data (e.g., see van der Hammen 1977; Ta-

ble 1) with our results (e.g., Fig. 11 A—B). There is not a

single article on the pedipalp, leg II and leg HI that would

compare to his documentation. It is obvious that legs dif-

fer in musculature, position of articulations and insertion

points of muscles, so we do not question his presentation

of the pattern for leg IV. However, the obvious morpho-

Bonn zoological Bulletin Suppl. 65: 1-125

logical variability of these structures on sequential ap-

pendages, suggests that his interpretations (based on leg

IV only) were too far reaching and not supported by the

morphology of other appendages. We think that his ideas

were stimulating and to a certain degree provocative, but

they must be considered of exclusively historical interest

(see also above where we have provided other reasons

why we consider the “Epimerata” unsupported).

Dunlop & Alberti (2008) correctly argued that if the

evolution of the coxa was supposed to represent an apo-

morphic state in other arachnid lineages, Epimerata were

united by a plesiomorphic character (no coxae but epi-

mera) — thus not supported as a taxon. Boxshall (2004)

and Waloszek et al. (2005) compared the morphology

of early arthropod fossils and showed that a coxa (L.e.,

protopodite sensu Boxshall; basipodid sensu Waloszek)

were part of the euarthropod ground pattern. Despite

variations in terminology, a coxa should be present in eu-

arthropods; palpigrades and mites included.

Opisthosoma

The opisthosoma of Arachnida has 12(13) segments with

segmental musculature and ganglia (Fage 1949; Millot

1949a; Shultz 2001, 2007b; Dunlop & Lambsdell 2017).

The heart and gonads occupy the anterior segments of

the opisthosoma (Fage 1949; Millot 1949b; Alberti

et al. 2007). In some taxa, the opisthosoma is divided in

two morphologically distinct regions, 1.e., a meso- and

a metasoma. In those groups (1.e., Scorpiones [Kastner

1931b, Polis 1990], Ricinulei [Millot 1949e], Schizo-

mida, Thelyphonida [Kastner 1931f], and the extinct

Aranea Chimerarachne yingi [Wang et al. 2018]), the

mesosomal segments carry a dorsal tergite and a ven-

tral sternite connected by lateral pleural folds, while the

metasomal segments have sclerite rings without pleural

folds. The number of mesosomal and metasomal seg-

ments differs among these groups (Tab. 9; Millot 1949e;

Talarico et al. 2011; Fusco & Minelli 2013; Wang et al.

2018). All arachnids with a metasoma also have a termi-

nal flagellum or sting (Fusco & Minelli 2013; Wang et al.

2018) except Ricinulei that lack a terminal structure on

the metasoma (Millot 1949f; Talarico et al. 2011).

©ZFMK

74 Sandra Franz-Guess & J. Matthias Starck

Table 9. Morphological characters of the mesosoma (yellow) and metasoma (green) in relevant arachnid taxa. The presence of the

structure 1s indicated by color fill of the fields. Where information on the character is missing, a lighter version of the respective

color or question mark was used. Abbreviations: DV = dorsoventral muscle; I = intersegmental muscle including dorsal, ventral,

transversal muscles.

Segment 8 9 10 11 12

Muscles

Ganglia

Heart

Gonads

Book

Scorpiones' *

Muscles

Ganglia

Heart

Gonads

Book

lungs

Muscles

E. spelaea

Ganglia

Ricinulei? Heart

Gonads

Book

Muscles

Ganglia

Heart

Gonads

Book

lungs

Muscles

Schizomida?

Ganglia

Thelyphonida* Heart

Gonads

Book

13 14 15 16 17 19 20 20

References: 'Scorpiones: Millot and Vachon (1949b), Alberti et al. (2007), Shultz (2007b), Wirkner and Prendini (2007), Snod-

grass (1965). *Ricinulei: Millot (1949e), Talarico et al. (2008, 2011). 3Schizomida: Bérner (1904), Millot (1949b). *Thelyphonida:

Borner (1904), Millot (1949b), Shultz (1993). “Segment #8 (first opisthosoma segment, of scorpions is incorporated into the

prosoma, and its musculature is incorporated in the formation of the diaphragm, its sternite is incorporated in the formation of the

sternum (Farley 2001; Schultz 2007b). Because of segmental compression, the segment is not externally visible (Shultz 2007b). It

has been source for considerable debate. For comparative purpose, we think it is important to refer to this segment here.

The opisthosoma of Eukoenenia spelaea is divided

into a mesosoma (seven segments) and metasoma (four

segments; Tab. 9). The mesosoma carries externally

separate sclerites dorsal and ventral, which are connect-

ed by the pleural membrane. The metasoma is charac-

terized by the presence of sclerite rings. Internally, the

free opisthosomal ganglia, and the heart are restricted to

the mesosomal segments 8—14 (mesosoma; Tab. 9). The

segmental dorso-ventral and transversal musculature, is

located in segments 9-13, thus, lacks in the first and the

last mesosomal segment. The gonads are also restrict-

ed to segments 9-13. Serial intersegmental muscles are

Bonn zoological Bulletin Suppl. 65: 1-125

present throughout mesosoma and metasoma. The

metasoma contains the rectal sac as prominent structure.

There is substantial variation in the external and inter-

nal morphology of the mesosoma and metasoma among

arachnid groups and topographic shifts or organ systems

are frequent (Tab. 9). However, such morphological vari-

ability is also evident in other arachnid groups without

an opisthosomal subdivision (André 1949; Berland 1949;

Millot 1949d, e; Millot & Vachon 1949a; Alberti et al.

2007) and therefore may be independent of the subdi-

visioning of the opisthosoma. All comparative morpho-

logical evidence (number of segments, position of organ

systems; Tab. 9) and phylogenetic relationship among

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 75

clades suggest that the metasoma evolved independently

in several lines of euchelicerates. The standard paradig-

matic explanation (and probably most plausible explana-

tion) proposes that the metasoma increases the degrees

of freedom for movements of a terminal appendage. For

example, Scorpiones carry a sting and poison gland on

the last segment of the metasoma, which is used for over-

powering prey, but also used during courtship behavior

(Stahnke 1966; Polis & Farley 1979; Polis 1990). Thely-

phonida use their metasomal flagellum as feeler (Moritz

1993; Alberti et al. 2007). Schizomida have a sexually

dimorph flagellum, suggesting that movements of the

flagellum are somehow relevant during courtship or mat-

ing (Hansen & Sorensen 1905; Moritz 1993; de Armas &

Teruel 2002; Teruel & de Armas 2002; Pinto-da-Rocha

et al. 2016). Eukoenenia spelaea has been observed rais-

ing their flagellum (and possibly metasoma) when irri-

tated (Kovaé et al. 2002). The presence of sensory setae

along the flagellum suggests a sensory function (Ferreira

and Souza 2012; Souza & Ferreira 2010b). The function

of the small metasoma in Ricinulei is unclear because a

terminal appendage is missing.

Cuticle

The cuticle of arthropods consists of a thick procuticle

covered by a thin epicuticle. Depending on topograph-

ic and functional specializations, the procuticle can be

differentiated into an inner endocuticle and an outer,

sclerotized exocuticle (Hackman 1984; Neville 1984).

Sclerotized cuticle can be either dark in color or colorless

depending on its chemical composition and mode of scle-

rotization (Hackman 1984). The epicuticle is deposited

on the cuticular surface through pore canals that pene-

trate the cuticle in large numbers. Soft cuticular mem-

branes connect sclerotized regions (sclerites), allowing

for movements and size changes, e.g. when feeding or

during gestation. The cuticle of Eukoenenia spelaea 1s

mostly unsclerotized, thus, lacks a distinct differentia-

tion into endo- and exocuticle. This might be due to their

small body size (Polilov 2015a).

Cutaneous respiration

Arachnids typically breathe with book lungs and/or

tracheae. Respiratory organs may be reduced with de-

creasing body size, and cutaneous respiration has been

reported for numerous mites (Levi 1967; Alberti &

Coons 1999). The theoretical size limit of the effective

dimension an organism can obtain for cutaneous respi-

ration depends on shape, oxygen partial pressure, diffu-

sion distance, metabolic rate, and the diffusion constant

for oxygen (and carbon dioxide, of course). A maximum

diameter of 1 mm for a spherical animal was estimat-

ed (Graham 1988). Eukoenenia spelaea is well below

that size limit of diffusive respiration, given a maximum

Bonn zoological Bulletin Suppl. 65: 1-125

opisthosoma diameter of approx. 300 um (left-right;

180 um dorso-ventral), and a prosoma diameter of ap-

prox. 280 um (left-right; 150 um dorso-ventral). Also,

the soft cuticle of E. spelaea is only 0.5 um thick, and the

underlying epidermis is single layered and flat, so that the

overall diffusion barrier through the integument is less

than 1 um. The hemolymph space 1s limited, thus, gas ex-

change may occur directly across the cells of the organs

in close contact to each other and across the body wall.

For comparison, Prokoenenia wheeleri (Rucker, 1901)

which is larger (2-3 mm; Rucker 1903) than E. spelaea,

has ventral sacks which are considered to function as re-

Spiratory organs (but histological evidence is lacking).

Surface structures

Cuticular surface structures are common in arthropods,

representing a plethora of forms and functions (Gorb

2001a). For example, the hair density in spiders was as-

sociated with water repellence (Suter et al. 2004; Bush

et al. 2007). Eukoenenia spelaea displays an extensive

pubescence on most parts of its body consisting of short

cuticular protrusions (2—3 um length). It 1s, however, not

clear whether this dense pubescence can act as a water

repellant surface 1n palpigrades.

Grooming behavior has been discussed for spiders as

well as several coleopterans in association with main-

taining their hydrophobic character (Kovac & Maschwitz

2000; Suter et al. 2004). Such behavior has also been re-

ported for palpigrades (Christian 2004; Ferreira & Souza

2012; Souza & Ferreira 2010). Different from spiders

and coleopterans, palpigrades do not use their legs for

grooming, but their chelicerae. Specialized cuticular

structures can be found on the fixed and movable digits

of the chelicerae. These display serrated cuticular teeth.

The distance between the teeth is approx. 1—1.5 um. Be-

tween the teeth of the serration, the distance is approx.

0.05—0.1 um. These structures match the thickness of the

setae and trichobothria, the thickness of their spikes as

well as the pubescence. Thus, one function of the cheli-

cerae might be removing particles.

Ventral plate and underlying structure

A previously undocumented structure of Eukoene-

nia spelaea is the ventral plate with its cuticular teeth

and the underlying epidermis. The cuticle of the ventral

plate has enlarged pore canals in which microvilli reach

from the underlying epidermal cells. The epidermis cells

under the ventral plate are large, loaded with glycogen

granules, possess a basal labyrinth and have numerous

apical microvilli. These are typical cytological features

of the cuticle and epidermis, usually found in association

with a type | transport epithelium (Noirot & Quennedey

1974; Conte 1984). Of course, we could not directly test

specific functions of these cells, but a comparison to a

©ZFMK

76 Sandra Franz-Guess & J. Matthias Starck

similar cellular morphology with known functions pro-

vides at least comparative evidence for the functioning

of the ventral plate. Cellular polarization and an exten-

sive brush border of the epidermal cells has been found

in the ventral vesicles of collembolans, which is the main

place for sodium uptake (Noble-Nesbitt 1963; Eisenbeis

1974). A similar morphology has also been reported for

the nuchal organ (cephalothoracic organ or salt organ) of

Eucrustacea, which is involved in salt excretion (Conte

1984; Lowy & Conte 1985). In eucrustaceans, the epi-

thelial cells also store glycogen granules, similar to our

findings in EF. spelaea (Hootman & Conte 1975; Lowy &

Conte 1985). Thus, comparative cytology suggests that

the ventral plate and its associated epithelium in E. spe-

laea might likely be involved in osmoregulatory process-

es.

Musculature

In the first part of this section, we discuss the segmental

axial musculature of Eukoenenia spelaea in relation to

the ancestral pattern of muscle anatomy as suggested by

the euarthropod and euchelicerate box-truss axial muscle

system (BTAMS; Shultz 1993, 1999, 2001, 2007b). This

will be followed by a comparative discussion of other

muscle systems (e.g., pharyngeal musculature, append-

ages) in the second part of this section. Our comparative

discussion is to some degree “opportunistic” because

it depends on presence, availability, completeness, and

quality of the published record.

Suspensor muscles originating from the prosomal

endosternite

The BTAMS as suggested by Shultz (1993, 1999, 2001,

2007b) was based on dissections of Xiphosurida, Scorpi-

ones, Amblypygi, Thelyphonida, and comparisons with

published record from other euarthropod taxa. The euar-

thropod box-truss axial muscle system assumes dorsal,

ventral, and lateral (transverse connectors in terminology

of Shultz [2007b]) suspensor muscles as well as anteri-

or and posterior oblique muscles (Shultz 2007b). In the

prosoma, all these muscles originate serially from the

endosternite, i.e., each prosomal segment, except the first

segment, carries a complete set of muscles. According

to Shultz (2001, 2007) the chelicerate BTAMS ground

pattern is identical to the euarthropod ground pattern.

It was reconstructed on the assumption of serial muscle

homology of the opisthosomal dorso-ventral muscula-

ture with the dorsal and ventral suspensor muscles of

the prosoma, and the observation that Xiphosurida have

anterior oblique muscles in the opisthosoma, while they

are missing in all arachnids. For BTAMS in arachnids,

Shultz (2001, 2007b) proposed that the anterior oblique

suspensors were completely reduced. For the posterior

Bonn zoological Bulletin Suppl. 65: 1-125

oblique muscles, he proposed a shift of insertion from the

tergites to the lateral pleural folds.

Table 7 gives an overview on the occurrence and

published evidence of the prosomal axial musculature

among euchelicerates. All studied euchelicerates deviate

from BTAMS by missing some muscles of the suppos-

edly ancestral pattern. However, Eukoenenia spelaea

deviates from the arachnid pattern by possessing an

anterior oblique suspensor muscle in segment 4, which

occurs only in the euarthropod ground pattern (Tab. 7,

Figs 21, 50). Muscle E13, which originates in segment 6

and inserts posteriorly in segment 8 might be interpret-

ed as ventral suspensor that, untypically, spans several

segments (but see discussion below). Lateral suspensors

were described for segments 5 and 7. Our description of

five pairs of dorsal suspensor muscles (Figs 21, 50) dif-

fers from that of Borner (1904), Millot (1943, 1949a) and

Firstman (1973) who explicitly state that Eukoenenia mi-

rabilis and Prokoenenia wheeleri have only four dor-

sal suspensor muscles, respectively. However, because

our study is the only study based on serial sections and

a complete reconstruction of the ground pattern of the

musculature, while the others were based on dissections

or cleared specimens, we respectfully consider our data

as more complete.

We found four pairs of muscle that were assigned ven-

tral suspensor muscles (with muscles E7/E9 assigned to

the third pair of ventral suspensors and E13 to the last

prosomal segment; Figs 21, 50), while earlier descrip-

tions of Eukoenenia mirabilis and Prokoenenia wheeleri

reported the full set of six ventral suspensor muscles. Pal-

pigrades with six ventral suspensor muscles would repre-

sent the plesiomorphic condition of the arachnid BTAMS

(Shultz 2007b). The lateral suspensor system shows a

similar pattern, with the full set of segmental muscles de-

scribed for FE. mirabilis and P. wheeleri, while E. spelaea

has only two lateral suspensor muscles (Figs 21, 50).

Interpretations of the axial muscle system in Palpigradi

are straightforward. The prosomal axial muscle system

is not only close to the arachnid ground pattern, but, be-

cause of the occurrence of an anterior oblique muscle in

segment 4 (Tab. 7), contains elements of the euarthro-

pod (!) BTAMS.

This plesiomorphic feature that is more ancestral than

the euchelicerate ground pattern calls for explanation. —

This reoccurrence of plesiomorphic morphology may be

explained as resulting from paedomorphic development,

i.e., developmental truncation. Under the assumption of

anagenetic evolution, paedomorphosis by developmental

truncation (Alberch et al. 1979) may create pattern of “re-

verse recapitulation”, 1.e., the adults of a species showing

ancestral features of a stem group. The evolutionary im-

plications of paedomorphosis will be discussed in more

detail below.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) fei

Other prosomal muscles of the box truss axial muscle

system

According to the euarthropod BTAMS (Shultz 2001),

paired dorsal longitudinal musculature extends from the

posterior prosoma segments (6 and 7) into the opistho-

soma. These muscles have been documented for Limu-

lus polyphemus (Shultz 2001), Amblypygi, Thelyphoni-

da, and Eukoenenia mirabilis (Borner 1904). In the latter,

these muscles also connect propeltidium and metapelti-

dium.

In Eukoenenia spelaea, we described two muscles

(P11, P12; Figs 19B—C, 20, 50; Appendix I: Tab. 5) that

represent anterior extension of the dorsal longitudinal

muscle system and extend into segments 7 and 6 of the

prosoma. Importantly, muscle P11 connects the propelti-

dium with the metapeltidium (thus skipping the “me-

sopeltidium”). If the mesopeltidium were a true tergite

of segment 6, we would expect the dorsal longitudinal

muscles connecting to this element. The described mor-

phology where the muscles “skip” the “mesopeltidium”

supports our interpretation that the mesopeltidium is not

a tergite, but a sclerotization of the pleural fold. Muscle

P12 is largely reduced in females, but in males connects

the metapeltidium with the first opisthosomal segment,

thus representing the ancestral condition of the eucheli-

cerate BTAMS (Shultz 2001).

Muscles of the pharynx

In the arachnid ground pattern, two muscular pharyngeal

pumps are present, a precerebral and a postcerebral pump

(Snodgrass 1948). The presence of both muscular pumps

varies among arachnids. The precerebral pharyngeal

pump has dorsal and lateral dilator muscles and circular

constrictor musculature. The dorsal dilator is part of the

epipharyngeal complex, which, again, consists of an an-

terior and a posterior component (Shultz 1993). Dorsal

dilator muscles of the anterior complex span between the

pharynx and the intercheliceral septum, the dorsal dilator

muscles of the posterior component attach to the proso-

mal shield. Variation of the pharyngeal muscles occurs in

all components of the pre- and postcerebral pump as well

as the anterior and posterior components of the precere-

bral pump.

The postcerebral pharyngeal pump Is missing in palpi-

grades (Millot 1942). As shown here for Eukoenenia spe-

laea, the precerebral pharyngeal pump is simplified, 1.e.,

it consists of the anterior component with a medio-dorsal

dilator muscle (P3) spanning between the pharynx and

the intercheliceral septum/sclerite (1ChS; Figs 29, 37A).

The lateral dilator muscles (P4) span between the later-

al wall of the pharynx and the ventrolateral wall of the

rostrosoma. Eukoenenia spelaea has a muscle (E8) that

originates from the anterior margin of the endosternite

and inserts at the posterior end of the pharynx. The mus-

Bonn zoological Bulletin Suppl. 65: 1-125

cular topography of E. spelaea largely resembles that of

Eukoenenia mirabilis (Millot 1942). However, Millot

(1942, 1943) described two muscles attaching in a ven-

tral and anterior position of the pharynx (m2, m3) while

we find only muscle E8 attaching in a ventral and rather

posterior position of the pharynx. He also described an

additional muscle (m4) spanning between the lower lip

and the transition between mouth opening and pharynx.

We could not find such muscle in E. spelaea. His rostro-

somal muscles of the upper lip (L. sup., fig. 2 in Millot

1942) are probably equivalent to our muscles P1 and P2.

Shultz (1993) stated that the epipharyngeal complex of

Eukoenenia mirabilis consists of a smaller anterior and a

larger posterior component. His interpretation was based

on data reported in the studies by Roewer (1934), and

Millot (1942). However, in our view the medio-dorsal

dilator of the pharynx in E. mirabilis (m1; Millot 1942)

represents the anterior component of the epipharyngeal

complex, while the posterior component is missing. The

small anterior muscle(s) associated with the anterior end

of the pharynx (L. sup., Fig. 2 in Millot 1942; Pl, P2

in our nomenclature) insert(s) in the upper lip and thus,

are not part of the epipharyngeal complex as defined by

Shultz (1993).

Axial musculature of the opisthosoma

For the opisthosoma of euarthropods, Shultz (2001) pro-

posed longitudinal dorsal and ventral muscles, segmental

dorso-ventral muscles, as well as anterior and posterior

oblique muscles. According to Shultz (2001), the anterior

oblique muscles are reduced, except in Limulus, where

a highly modified pattern of anterior oblique muscles is

maintained. The posterior oblique muscles insert on the

lateral pleural membrane in all arachnids. The dorsal and

ventral longitudinal muscle systems attach in each seg-

ment. The dorsal muscle system extends into the proso-

ma where muscles attach to the sclerites of segments 6

and 7.

The dorso-ventral musculature of the first opisthoso-

mal segment requires special discussion. The morpho-

logy of the first opisthosomal segment is modified in

many arachnid taxa, e.g., in Scorpiones (Shultz 2007b)

it is integrated in the diaphragm, in Araneae (Gerhardt &

K4stner 1931) it forms the pedicel, in Amblypygi, Schi-

zomida, and Thelyphonida (Borner 1904; Kastner 1931f)

the ventral parts of the first opisthosomal segment are

supposedly transformed into the sternite of the proso-

ma. In other groups, the dorso-ventral musculature is

completely missing in the first opisthosomal segment,

e.g., Pseudoscorpiones (Mehnert et al. 2018) and Solif-

ugae (Kastner 1931e). In our documentation of the axial

musculature of palpigrades, dorso-ventral muscles are

completely missing in the first opisthosomal segment.

Firstman (1973) mentioned opisthosomal dorso-ven-

©ZFMK

78 Sandra Franz-Guess & J. Matthias Starck

tral musculature, but does not provide necessary details

about the segmental topography.

The posterior oblique musculature is completely ab-

send from the opisthosoma. Muscle E20 that originates

from the posterior end of the endosternite and inserts lat-

eral on the pleural membrane of the first opisthosomal

segment actually is the posterior oblique muscle of the

last prosomal segment. This interpretation is in contrast

to, but more parsimonious than that of Borner (1904) and

Kastner (1931a), who suggested that muscle E20 is the

dorso-ventral muscle of the 1‘ opisthosomal segment that

moved its ventral attachment from the sternite to the pos-

terior end of the endosternite and its dorsal attachment

from the tergite to the pleural membrane.

The dorsal and ventral longitudinal muscles of Eu-

koenenia spelaea have a single point of origin and inser-

tion, respectively. Unusual are the two lateral branches of

the ventral longitudinal muscles. Similarly, noteworthy is

the topography of dorsal longitudinal muscle D1, which

originates in the prosoma from the metapeltidium and in-

serts posterior to the origin of dorsal longitudinal muscle

D2 in segment 10. This overlap of the dorsal longitudi-

nal muscles is similar to that of the dorsal longitudinal

muscles in Amblypygi, Thelyphonida, and Xiphosurida.

However, the lack of segmental attachment of the dor-

sal and ventral longitudinal musculature appears to be

unique for E. spelaea.

Borner (1904) and Kastner (1931la) suggested that

muscle E13 is an anterior extension of the ventral lon-

gitudinal muscle inserting on the posterior region of the

endosternite. Borner’s (1904) and Kastner’s (1931a) in-

terpretation, assuming a shift of the attachment from the

ventral sclerite to the endosternite, is equally parsimoni-

ous to our interpretation, that E13 is a ventral suspensor

that moved the insertion from the ventral sclerite of the

last prosomal segment to that of the first opisthosomal

segment.

The 2™ and 4" opisthosomal segment (segments 9

and 11) show two pairs of peculiar muscles that might

be either interpreted as lateral branches of the ventral

longitudinal muscle system, or as posterior and anteri-

or oblique muscles of opisthosomal segments 2 and 4.

Both situations would be unusual, because (1) the ventral

longitudinal muscle system usually does not branch in

arachnids, and (2) anterior and posterior oblique muscles

are intersegmental, but the muscle of the 2™ opistho-

somal segment is intrasegmental and the muscle of the

4 opisthosomal segment bridges two segments. Also,

anterior oblique muscles are absent from the arachnid

ground plan, thus the description of these muscles as an-

terior oblique requires the assumption of reoccurrence of

plesiomorphic characters (but see below).

Bonn zoological Bulletin Suppl. 65: 1-125

Other axial muscles (non-BTAMS)

We identified several additional muscles in the opistho-

soma that do not belong to the BTAMS. Intertergal and

intersternal muscles, i.e., muscles connecting tergites and

sternites, can be found in Amblypygi (Shultz 1999), Ara-

neae (Whitehead & Rempel 1959), Scorpiones (Shultz

20076), and Thelyphonida (Shultz 1993). An incomplete

set of such muscles was described for Opiliones (Shultz

2000). Borner’s (1904) report of Eukoenenia mirabilis

mentioned one pair of intersegmental muscles in each

segment from segment 10—15. Additionally, muscles as-

sociated with the last opisthosomal segment and its ap-

pendage were reported in Thelyphonida (Shultz 1993)

and Xiphosurida (Shultz 2001).

Eukoenenia spelaea has up to six pairs of intersegmen-

tal muscles in each segment 9-17. Except for segment 8,

these intersegmental muscles are more or less homoge-

nously formed between all opisthosoma segments. How-

ever, in segment 9 we described intrasegmental muscles

(JI1—J14; Figs 19C, 20A) that closely resemble in topog-

raphy and number the intersegmental muscles (JU-—JIX)

of the following segments, but, muscles JI1—JI4 do not

extend into the preceding segment 8. A straightforward

interpretation is that these muscles were originally in-

tersegmental muscles that lost their origin on segment 8.

In the last opisthosomal segment, two paired and one

unpaired strong longitudinal muscles attach to the basis

of the flagellum. The large number of small intersegmen-

tal muscles is in contrast to earlier reports of Eukoene-

nia mirabilis.

Musculature of the appendages

Chelicerae

The musculature associated with the chelicerae varies

within the euchelicerates. In groups with tripartite che-

licerae, a variable number of extrinsic muscles with an-

tagonistic function (Tab. 10) moves the basal article. A

varying number of extrinsic and antagonistic muscles

(Tab. 10) also moves bipartite chelicerae. The chela

opens and closes by an antagonistic pair of muscles/ten-

dons in both tripartite and bipartite chelicerae, which at-

taches to the movable digit only (Millot & Vachon 1949a;

Steinbach 1952; Whitehead & Rempel 1959; van der

Hammen 1966, 1967; Dubale & Vyas 1968; Vyas 1974:

van der Hammen 1982; Shultz 1993; Alberti & Coons

1999; Shultz 1999; 2000, 2001; Meijden et al. 2012). The

depressor muscle of the movable digit is generally more

prominent than the levator muscle. The fixed digit is free

of musculature.

Three extrinsic and ten intrinsic muscles move the tri-

partite chelicerae of Eukoenenia spelaea. Based on the

topography of origin and insertion of these muscles, we

suggest that muscles Cl, C3 (Tab. 10) possibly cause

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 79

Table 10. Musculature of chelicerae of Eukoenenia spelaea in comparison with other euchelicerate groups (where data is avail-

able). The muscles are color coded according to their insertion points on the articles of the chelicera. Grey color indicates extrinsic

prosoma muscles that insert on the basal article of the chelicera; orange codes muscles in the basal article that insert on the fixed

finger, and purple codes muscles originating the fixed finger and inserting on the movable finger. Line drawings visualize a sche-

matized ground pattern of antagonist muscles in the tripartite (upper part) and bipartite (lower part) chelicerae, with chelae and

subchelae, respectively. Antagonist functions of depressor — levator muscles refer to opening / closing of the chela or lowering /

lifting of cheliceral articles. Of course, the precise movement depends on the degrees of freedom provided by the joint morphology

which is not known for most taxa. We therefore present a simplified scheme based on muscle topography only. Generalized antag-

onist functions “depressor—levator”, include lateral (abduction, adduction) and rotational movements which, however, cannot be

deduced from muscle topography.

Depressor

Tripartite chelicerae

Xiphosurida

(Shultz 2001)

Scorpiones |

E. mirabilis (van der

Hammen 1982)

Solifugae (Millot &

Vachon 1949a, Meijden

et al. 2012)

Araneae (Steinbach

1952, Whitehead &

Rempel 1959)

downward movements of the chelicerae, while muscle

C4 (Tab. 10) moves the chelicerae upward. Muscles C5,

C7, and C8 originate in the first article of the chelicerae

and share a common insertion point on the lower edge of

the chela, probably causing a sidewards and downward

movement of the entire chela (Tab. 10). The function of

the small intrinsic muscle C2 is not clear. Different from

other euchelicerates, a muscular antagonist is missing,

because origin and insertion of muscle C6 rather sug-

gest a median rotation. Upward movement of the chela

might be achieved through hemolymph pressure. The

prominent closing musculature of the fixed digit (C9-13;

Tab. 10) has no muscular antagonist. Thus, we find no

muscles that actually open the chela. We therefore as-

Bonn zoological Bulletin Suppl. 65: 1-125

Depressor

Levator Levator

Depressor

sume that hemolymph pressure opens the chelicera. Eu-

koenenia spelaea appears to be the only euchelicerate,

which differs from the ground pattern of euchelicerate

musculature and has hydraulic functions that operate the

chelicerae.

Pedipalps and legs

In the ground pattern of arachnids, walking legs consist

of seven articles. The basal article (= “coxa’”’) was prob-

ably moved by nine extrinsic muscles. Of these muscles,

five originated from the dorsal shield and four originated

from the endosternite (Shultz 1991). However, van der

Hammen (1982) proposed for Palpigradi (and Parasiti-

©ZFMK

80 Sandra Franz-Guess & J. Matthias Starck

Table 11. Musculature of leg 4 of Eukoenenia spelaea in comparison with the proposed euchelicerate ground pattern and other

euchelicerate groups for leg 4 (Shultz 1989). For better distinguishability of the separate muscles, the ground pattern was divided

into two schematics. Muscle color codes in the table are identical to the schematic in the header. Light color code for E. spelaea

indicates muscles which are not a good fit to the proposed ground pattern. Muscle 8 is coded as one muscle, but it is divided into

several smaller muscles in most euchelicerate.

Muscles

oe a4

L414 LVI-3)—OLIV10 Ss LIVS LIVE =o LINT LIVE

— = es

‘

= - = ace

3 2 1

Leg article 7(-8) 6 5 4

Ground pattern a ;

Xiphosurida ——

Opiliones

Scorpiones ———£==_——

Araneae y

Amblypygi

LU = ss

Thelyphonida S=

——————

Pseudoscorpiones Twa

Solifugae

eee

Ricinulei re

____ ee

Acariformes —_— i.

Parasitiformes A ————_

E. mirabilis SSS

EEE

E. spelaea ee

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 81

Table 12. Eukoenenia spelaea, comparison of musculature in pedipalps and legs. Color coding is identical to table 11. Light color

code indicates muscles which are not a good fit to the proposed ground pattern. The muscles are coded according to their presence

in one or more leg articles (the additional articles of pedipalp and leg 1 are assumed to follow article 7).

Legarticle 11 10 9 8 7 6 5 4 3 2 1

| PP1-2 |

Pedipalp

Leg 1

Leg 4

Bonn zoological Bulletin Suppl. 65: 1-125 ©ZFMK

82 Sandra Franz-Guess & J. Matthias Starck

formes) that the first article is not a coxa, but a trochanter.

He based his interpretation on the pattern of muscle in-

sertion and the position of articulations found in the first

leg article of the forth leg of Eukoenenia mirabilis. Ac-

cording to his study, a pair of muscles/tendons inserted

laterodorsal and lateroventral, the articulation is located

ventral. This view was already rejected by Shultz (1989),

who reasoned that the number of extrinsic muscles in Eu-

koenenia sp., 1S consistent with the muscle arrangement

of the coxa in most euchelicerates. — While there are a

number of other reasons for rejecting the “Epimerata

hypothesis” by van der Hammen (1977a, 1982), we will

briefly discuss the topographic pattern of leg muscles be-

cause it is at the core of that hypothesis.

Extrinsic musculature is found in all extremities of

Eukoenenia spelaea. However, the number and origin of

these muscles varies between legs. Legs 1-3 have one

extrinsic, endosternal muscle each, but leg 4 has three

endosternal muscles. One extrinsic muscle originating on

the dorsal shield is present in the pedipalp, and legs 1-3.

Leg 4 has no extrinsic musculature originating from the

dorsal shield.

The muscle/tendon configuration proposed by van der

Hammen (1982) for the first article in Eukoenenia mira-

bilis was only found in part in leg 4 of E. spelaea. Mus-

cle LIV4 fits the description, however, LIV5 does not.

For the other leg joints, only few muscles matched the

suggested configuration. The muscle configuration re-

ported by Shultz (1989) for the coxa in Eukoenenia sp.

can be partially found in legs 2-4. As mentioned above,

in E. spelaea, deviations from the proposed ground pat-

tern are the reduced number of muscles originating from

the dorsal shield and the endosternite. However, despite

these variations, the presence of endosternal musculature

inserting in the first article rather supports the idea that

the first article is a coxa than a trochanter. — Also, there is

no single match between muscle insertions and articula-

tion points between the descriptions by van der Hammen

(1977a, Tab. 1) and our observations (Fig. 11). This con-

cerns not only the number of articles, muscles and posi-

tion of joints, but also the topography between pedipalps

and all legs which varies considerably (Tab. 12, Fig. 11).

The intrinsic locomotor musculature of Euchelicera-

ta was described by Shultz (1989, 2001). The proposed

ground pattern for Arachnida consists of 13 muscles

(Tab. 11). Extensors in articles 3—7 are present in Opil-

iones, Pseudoscorpiones, Scorpiones, Solifugae (Shultz

1989), and Xiphosurida (Shultz 1989, 2001). For Eu-

koenenia mirabilis, the analysis of leg 4 shows only mus-

cles 1-3, 6—8, and 11 of the arachnid ground pattern and

no extensors in articles 3—7 (Shultz 1989).

It is apparent, that the number of muscles as well as

their overall origin and insertion varies between legs in

Eukoenenia spelaea (Tabs 5, 12). The terminal articles

of the pedipalp and leg 1 contained a single tendon each

(PP6t—PP10t) attached to the joints with a pulley. This

Bonn zoological Bulletin Suppl. 65: 1-125

differs from the free tendon (1) in the ground pattern of

euchelicerates. Legs 2-4 do not have a muscle or ten-

don that could be considered homologous to muscle 1 of

Shultz (1989).

Muscle 2 of the proposed euchelicerate ground pattern

might be homologous with muscles in the pedipalp and

legs 1-4 (PPS, LI8, LII9, LIII10, LIV14; Tab. 12). In leg

4, however, this muscle (LIV14) originates ventral in ar-

ticle and does not insert at or near the tarsal claw. This

topography deviates from the proposed ground pattern;

therefore, its origin and insertion point either moved or

it cannot be homologized with muscle 2 (Figs 19A, 20C;

Tab. 12). — Muscle 3 is missing in the pedipalp. In leg 1,

muscle LI7 is not a clear match with muscle 3, because

its point of origin is lateral, not dorsal. Homologization

of LI7 with muscle 3 requires the assumption that the

point of origin has shifted. Muscles LII8 (leg 2), LHI9

(leg 3), and LIV11—13 (leg 4) can be identified as muscle

3 of the ground pattern (Tab. 12).

Similar to Eukoenenia mirabilis, Acariformes, Parasit-

iformes and Pseudoscorpiones, muscles 4 and 5 are miss-

ing in the pedipalp and the four walking legs of E. spe-

laea (Tab. 12). Muscle 6 of the ground pattern is present

in the pedipalp and legs 1-4, however, in the pedipalp

and leg 1, the muscle (PP4, LI6) is a side branch of a

larger muscle. In legs 2 and 3, the muscle (LII7, LIII8)

continues into the following article, and in leg 4 (LIV10)

the point of origin is ventral, not dorsal (Figs 19A, 20C).

Thus, a clear match to muscle 6 is not possible (Tab. 12).

A similar situation can be found with muscle LIV9 of

leg 4. It also has the point of origin shifted to ventral and

is, thus, not a clear match with muscle 7 of the ground

pattern (Figs 19A, 20C; Tab. 12). Homologues of muscle

7 are missing in pedipalps and legs 1-3. Poor matches

for muscle 8 are present in legs 1-4 (LIS, LII6, LIII6/7,

LIV8; Tab. 12). Discrepancies are present in the point of

origin and insertion (Figs 19A, 20C). The pedipalp lacks

this muscle. Muscle 9 of the ground pattern was only ob-

served in leg 4 (LIV7). It is missing in the pedipalp and

legs 1-3. This is similar to leg 4 in Amblypygi, Araneae,

and Xiphosurida.

The lack of muscle 10 in pedipalp and all legs is similar

to E. mirabilis, Parasitiformes, and Opiliones (Tab. 12).

Muscle 11 is only present in the pedipalp (PP3) and leg

4 (LIV6) in E. spelaea. However, there 1s a shift in point

of origin and insertion which makes a match with the

ground pattern difficult (Figs 19A, 20C; Tab. 12). A pos-

sible homologue of muscle 12 can be found in legs 1-3

(LI4, LIS, LIWS) but shifts in points of origin/insertion

as well as muscle LIIS spanning three articles does not

match the ground pattern (Figs 19A, 20C; Tab. 12). Ad-

ditional possible matches are muscles LI4 and LII4 of

legs 2 and 3 with muscle 13, however, shifts in points of

origin and insertion are apparent. Muscle 13 is missing in

the pedipalp, leg 1, and leg 4 (Figs 19A, 20C; Tab. 12).

While the comparison of the muscle topography is te-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 83

dious and probably obscured by different degrees of

precision and differences between methods used, it is

evident that the leg musculature of Eukoenenia spelaea,

although variable between legs, shows most similarities

to Acariformes and Parasitiformes (Tab. 11). The simi-

larity roots primarily in reduction of same muscles from

the arachnid ground pattern, and as such, 1s a relatively

weak character. However, together with additional fea-

tures it places Palpigradi in phylogenetic relationship to

Acariformes.

Nervous system

Ground pattern

According to recent interpretations, the prosomal synce-

rebrum (supraesophageal ganglion) of euchelicerates

comprises of the proto- (ocular segment), deuto- (cheli-

ceral segment), and tritocerebrum (pedipalpal segment:

Damen et al. 1998; Telford & Thomas 1998; Mittmann &

Scholtz 2003; Harzsch et al. 2005; Scholtz & Edgecombe

2006; Loesel et al. 2013; Wolf 2016; Ortega-Hernandez

et al. 2017). Developmental studies of Limulus poly-

phemus also suggest that the stomodaeum is enveloped

by the deutocerebrum, and not, as previously assumed,

by the tritocerebrum; supposedly this is a feature of the

euarthropod ground pattern (Harzsch et al. 2005; see

also Ortega-Hernandez et al. 2017). However, this view

is somewhat contentious and there is considerable dis-

cussion about the segmental nature of the syncerebrum,

i.e., whether a deutocerebrum is present or absent (Babu

1965; Weygoldt 1985; Wegerhoff & Breidbach 1995;

Bitsch & Bitsch 2007). — The syncerebrum contains the

arcuate body, 1.e., a large unpaired neuropil at the poste-

rior margin of the brain. The arcuate body is a protoce-

rebral visual integration center that has been described

for all chelicerate taxa (Strausfeld et al. 2006; Doeffinger

et al. 2010; Lehmann et al. 2012; Loesel et al. 2013) ex-

cept Acari that lack this neuropil.

The subesophageal ganglion complex contains the

pedal ganglia and a variable number of opisthosomal

ganglia (Gottlieb 1926; Hanstrom 1928; Beier 1931;

Kastner 1931b, d, e; Babu & Barth 1984: Wegerhoff &

Breidbach 1995). Syncerebrum and subesophageal gan-

glion are connected by a pair of circumesophageal con-

nectives (Horn & Achaval 2002) and, together, form the

synganglion.

Within the opisthosoma, the ancestral morphology

probably was a typical ladder nervous system with free

ganglia (Handlirsch 1926). This is still found in some

basal euchelicerate groups, though with modifications.

In Guvenile) Limulus sp. the first opisthosomal ganglion

is fused to the prosomal synganglion, but the following

seven pairs of ganglia form a typical ladder nervous sys-

tem. The opisthosomal ganglia merge only later during

ontogeny (Tanaka et al. 2013; Battelle 2017). Scorpions

Bonn zoological Bulletin Suppl. 65: 1-125

have three pairs of ganglia in the mesosoma and four

pairs in the metasoma. A small unpaired opisthosomal

ganglion (of variable position) can be found in Thely-

phonida, Schizomida and Solifugae. For Palpigradi we

described four pairs of ganglia in opisthosomal segments

11-14. All other arachnids lack opisthosomal ganglia

(Millot 1949b; Babu 1985).

The brain of Eukoenenia spelaea

The brain of Eukoenenia spelaea is proportionally large

and shows a high degree of fusion. We have no quan-

titative data comparing brain volume to body size, but

the fact that the synganglion basically fills the entire pro-

soma (Figs 22—24) with the perikarya layer next to the

epidermis is indicative of a proportionally large brain.

— We documented three commissures, two supraesoph-

ageal, i.e., the protocerebral and cheliceral commissure,

and one subesophageal commissure associated with the

pedipalps (Figs 24—25). Based on that topographic evi-

dence, the cheliceral commissure 1s second thus can be

associated with the deutocerebrum; the post-esophageal

commissure of the pedipalps is third and thus associated

with the tritocerebrum. This topography is coherent with

the general arthropod ground pattern of the tritocerebral

commissure running behind the esophagus. Supposing

these topographic relationships are correct, the esopha-

gus 1s enveloped by the part of the supraesophageal gan-

glion associated with the chelicerae (Fig. 25), thus the

deutocerebrum. This would be in agreement with the

arthropod ground pattern as suggested by Harzsch et al.

(2005).

A similar morphology of the synganglion was de-

scribed for larval Limulus polyphemus, where a tripar-

tite brain was recognized (Mittmann & Scholtz 2003;

Harzsch et al. 2005). Similar to the larval syncerebrum of

L. polyphemus, the cheliceral commissure of £. spelaea

is located supraesophageally and the pedipalpal commis-

sure 1S Subesophageal. The protocerebrum is small and a

visual center (arcuate body) is lacking in E. spelaea.

The first three opisthosomal segments of Eukoene-

nia spelaea have no free ganglia thus, they are probably

fused to the subesophageal ganglion which reaches far

into the second opisthosomal segment. Fusion of opistho-

somal ganglia to the subesophageal ganglion 1s the default

morphology with the few exceptions mentioned above

(Babu 1985). In Eukoenenia, opisthosomal segments 4

through 7 show individual small ganglia. The number of

neurons within the opisthosomal ganglia of E. spelaea

is approx. 25, 1.e., an exceedingly small number that is

usually found in arthropod embryos (10-90 neurons per

ganglion; Gerberding & Scholtz 2001; Harzsch 2003),

but not in adults where neuron numbers vary between

approx. 110 neurons in the fused opisthosomal gangli-

on of the mite Ornithodoros parkeri (Pound & Oliver Jr.

1982) to more than 4000 neurons per ganglion in L. poly-

©ZFMK

84 Sandra Franz-Guess & J. Matthias Starck

phemus (Bursey 1973). Whereas free opisthosomal

ganglia represent a plesiomorphic condition, the small

number of neurons in those ganglia must be considered

derived, probably in association with the paedomorphic

(developmental truncation) morphology of the Palpi-

gradi. If, indeed, Palpigradi evolved by developmental

truncation, plesiomorphic features like the opisthosomal

ganglia can be explained as “reversed recapitulation”

(Alberch et al. 1979) thus, representing a unique and de-

rived phylogenetic condition of palpigrades despite their

plesiomorphic morphology.

The ground pattern of euchelicerates has a perineural

vascular sheath, which surrounds the synganglion and

(probably) supplies it with oxygen and nutrients (First-

man 1973; Alberti & Coons 1999; Coons & Alberti

1999; KluBmann-Fricke et al. 2012; Wirkner & Huck-

storf 2013; Gopel & Wirkner 2015; KluBmann-Fricke &

Wirkner 2016). In some euchelicerates, the perineural

vascular sheath has been transformed into a network of

arteries and capillaries. In other groups, e.g., tracheate

arachnids, it has been reduced (e.g., solifuges [Klann

2009] and mites [Alberti & Coons 1999]).

Firstman (1973) documented a perineural vascular

sheath in Prokoenenia wheeleri. However, despite using

TEM, we found no evidence or any residue of it in Eu-

koenenia spelaea. Instead, the synganglion 1s surround-

ed by a thin and probably incomplete perineurium (glia

cells surrounding the perikarya layer). The perineurium

produces a thin extracellular matrix that might represent

a neural lamella. — The lack of the perineural vascular

sheath may be related to the small size of the animals and

the fact that they are typical diffusion animals, 1.e., diffu-

sion 1s sufficient to ensure continuous oxygen supply to

the prosomal ganglia.

Frontal Organ

The frontal organ of Eukoenenia spelaea consists of two

modified setae that share a common base, thus, forming

a sensory unit. Each seta of the frontal organ has its own

set of enveloping cells. The surface of the setae is cov-

ered with numerous cuticular grooves. The topographic

position of the frontal organ and its construction from

two setae on a common basis makes it a unique structure

among arthropods.

While the topographic anatomy is unique, its ultra-

structure characterizes it as a sensory unit and allows rec-

ognizing features typical for sense modalities in terrestri-

al arthropods, 1.e., hygroreception, thermoreception and

chemoreception. Different sense modalities commonly

combine in sensory units of terrestrial arthropods, 1.e.,

hygro- and thermoreceptors, or chemo-/thermo-/hygrore-

ceptors. All sensory cells are embedded in dense materi-

al/receptor lymph. A hygroreceptive unit usually consists

of two cells, a moist cell and a dry cell, but single cell

hygroreceptors have also been reported (Anton & Tichy

Bonn zoological Bulletin Suppl. 65: 1-125

1994; Tichy & Loftus 1996; Barth 2002; Gainett et al.

2017). Their ultrastructure is characterized by branched

dendrites. Thermoreceptors contain lamellate dendrites

(Davis & Sokolove 1975; Altner et al. 1978; Yokohar1i

1999). Chemoreceptors contain branched or unbranched

dendrites (Steinbrecht 1969; Altner & Prillinger 1980;

Foelix 1985; Tichy & Barth 1992; Tichy & Loftus 1996)

in combination with one pore at the tip of the hair for

contact chemoreception, or numerous pores evenly dis-

tributed along the hair shaft for non-contact chemorecep-

tion (Altner & Prillinger 1980; Foelix & Hebets 2001;

Barth 2002).

Thus, ultrastructural analysis can, to some degree,

provide comparative evidence of the receptive modali-

ties of sensory hairs, 1.e., (two) branching dendrites in

combination with a smooth cuticle surface usually are

associated with hygroreception, branched dendrites in

combination of cuticle pores are associated with chemo-

reception (contact/non-contact), and lamellate dendrites

are associated with thermoreception. This ultrastructural

distinction describes a general pattern, however, some

arachnids have thermo- and/or hygroreceptors in combi-

nation with pored sensory setae (Foelix & Axtell 1972:

Foelix & Chu-Wang 1973; Foelix 1985; Anton & Tichy

1994: Tichy & Loftus 1996), thus, morphological evi-

dence should rather be considered as suggestive of sense

modalities, but explicit determination of sense modalities

requires experimental testing.

Among terrestrial arthropods, combined thermo- and

hygroreceptors are common (Waldow 1970; Altner et al.

1973, 1978, 1983; Loftus 1976; Altner 1977). Thermo-/

hygroreceptors or combination chemo-/thermo-/hygrore-

ceptors were described in Acari (Foelix & Axtell 1972;

Hess & Loftus 1984), Araneae (Foelix & Chu-Wang

1973; Ehn & Tichy 1994) and Opiliones (Gainett et al.

2017). They were specifically found on legs of arachnids

(Anton & Tichy 1994; Gainett et al. 2017). The tarsal or-

gan in some arachnid groups (Foelix & Chu-Wang 1973;

Foelix & Schabronath 1983; Talarico et al. 2005) and

Haller’s organ in Parasitiformes (Alberti & Coons 1999;

Foelix & Axtell 1972) act as sensory unit consisting of

several closely neighboring sensory structures, thus,

possibly improving sensory resolution (Anton & Tichy

1994).

The frontal organ of Eukoenenia spelaea is unusual for

terrestrial arthropods because of its asymmetry, 1.e., left

and right setae contain dendrites with obviously different

sense modalities. The base of the frontal organ contains

two pairs of sensory cells that reach into the left and right

seta. The left seta receives two cylindrically branching

dendrites. In combination with a smooth surface this

would be indicative of hygroreception, in combination

with pore(s) it would suggest (non-)contact chemore-

ception. However, it is not that easy in palpigrades: as

described above, the surface of the seta has cuticular

grooves. These grooves are shallow and do not pene-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

Table 13. Number of chemo- and mechanoreceptive dendrites within sensory setae in Arachnida.

Chemoreceptive Mechanoreceptive

dendrites dendrites

Acari 3-8 2

Amblypygi 9-12 2

Araneae ~20 2

Opiliones ~16 unknown

Pseudoscorpiones 3-5 unknown

Scorpiones ~20 4

Solifugae 12 4-7

E. spelaea 2 5?

References

Foelix and Chu-Wang (1972), Chu-Wang and Axtell (1973),

Hess and Vlimant (1982)

Foelix et al. (1975),

Foelix and Hebets (2001)

Foelix and Chu-Wang (1973),

Harris and Mill (1973)

Foelix (1976), Gainett et al. (20175)

Foelix (1985)

Foelix and Schabronath (1983),

Cushing et al. (2014)

Haupt (1982)

this study

trate the entire procuticle but the cuticle is extremely thin

(< 0.2 um) in these positions. An extremely thin cuticle

reduces the diffusion barrier, but based on the ultrastruc-

tural analysis it is impossible to decide if the two branch-

ing dendrites in the left seta in combination with the cu-

ticular grooves stand for chemo- or hygroreception. The

right seta of the frontal organ contains one cylindrically

branching dendrite and a lamellate dendrite. Its surface

is sculptured with the same honeycomb pattern like on

the left seta. Therefore, the branching dendrite could be

either a non-contact chemoreceptor or a hygroreceptor,

while the lamellate dendrite 1s possibly a thermoreceptor.

— What is unusual for the frontal organ as a sense organ 1s

the asymmetrical organization, with both setae showing a

different ultrastructure and probably providing different

sense modalities.

For lamellate receptors, a correlation exists between

the degree of lamellation and the range of the operating

temperature. A lower range of the operating temperature

is correlated with a high number of dendritic lamellae

(Loftus & Corbiere-Tichané 1981; Corbiere-Tichané &

Loftus 1983; Altner & Loftus 1985). Such receptors are

often found in small arachnids such as Opiliones, for

which temperature and humidity are an integral part of

their lives (Todd 1949; Wiens & Donoghue 2004; Cur-

tis & Machado 2007). The lamellation within the pro-

posed thermoreceptive dendrite in Eukoenenia spelaea

is extensive. The environment in which these animals

live is cool and moist. The temperature within the cave

is stable between +7.9 and +10.7 C, and the humidity is

always around 97% (Kovaé et al. 2002, 2014). One could

speculate that the intensive lamellation of the dendrites

might be indicative to the functional range of a thermore-

ceptor at a relatively low temperature and high humidity.

Bonn zoological Bulletin Suppl. 65: 1-125

Lateral organ

The paired lateral organ of Eukoenenia spelaea is a

sensory organ unique for palpigrades. Each consists of

four modified sensory hairs. Each sensory hair has its

own base, unlike the common socket of the sensory se-

tae of the frontal organ. The ultrastructure of all setae is

the same as that of the left seta of the frontal organ, L.e.,

branching dendrites in combination with the numerous

cuticular grooves. Based on this ultrastructure, we sug-

gest that the lateral organs function as non-contact che-

moreceptors and/or hygroreceptors.

Sensory setae

The combination of chemo- and mechanoreceptive

sensory cells in setae 1s common in arthropods (Chu-

Wang & Axtell 1973; Foelix & Chu-Wang 1973; Har-

ris & Mill 1973; Ozaki & Tominaga 1999). Especially

eyeless soil arthropods possess such combined sensory

organs (Eisenbeis & Wichard 1987).

In contrast to the modified setae of the frontal and the

lateral organ, the dendrites in chemoreceptive sensory

setae of arthropods are unbranched. Few pores on the tip

of the seta are usually interpreted as indicative of contact

chemoreception while many pores along a large portion

of the shaft of the seta suggest non-contact chemorecep-

tion. The cuticular wall of the seta can be single or double

with two or three canals. The dendrites are located in the

inner canal whereas the outer canal(s) is either filled with

dense material/receptor lymph or it is empty. The outer

canal is typically crescent-shaped in cross-section (Fo-

elix & Chu-Wang 1973; Foelix et al. 1975). Mechano-

receptive cells typically end in a tubular body at a cu-

ticular socket at the basis of the hair (Foelix et al. 1975;

©ZFMK

86 Sandra Franz-Guess & J. Matthias Starck

Gaffal et al. 1975; McIver 1975; Keil 1997; Barth et al.

2004; Dechant et al. 2006). Thus, ultrastructural fea-

tures of combined chemo-mechanoreceptors are distinct,

i.e., pored surface in combination with one or few un-

branched dendrites, and a cuticular socket with a tubular

body at the base of the sense hair.

Numerous examples of combined chemo-mechano-

receptors representing this ultrastructural organization,

together with the taxon specific variations, have been

published (Scorpiones: 4 dendrites; Tab. 13 [Foelix &

Schabronath 1983; Cushing et al. 2014], and Solifugae:

4—7 dendrites [Haupt, 1982]). In Acari (Foelix & Chu-

Wang 1972; Chu-Wang & Axtell 1973; Hess & Vlimant

1982), Amblypygi (Foelix et al. 1975; Foelix & Hebets

2001), and Araneae (Foelix & Chu-Wang 1973; Harris &

Mill 1973), the number is reduced to two mechanorecep-

tive dendrites per seta (Tab. 13).

In Eukoenenia spelaea, the sensory setae of the body

and the flagellum have few cuticular pores at their tip and

the seta is double walled with an empty outer canal. At

least two dendrites reach to the tip of the hair shaft. All

sensory setae have a cuticular socket with tubular bodies

at which five dendrites terminate. These ultrastructural

features suggest that these hairs function as combined

(contact)chemo-mechanoreceptors. — Among eucheli-

cerates, there seems to be a correlation between the num-

ber of chemoreceptive dendrites and body size (Tab. 13).

While most larger-bodied groups have 10—20 dendrites,

taxa with small body size like Acari or Pseudoscorpiones

have only 3—8 or 3—5 dendrites, respectively. FE. spelaea

is among the smallest euchelicerates and has only two

chemoreceptive dendrites.

Typically, double-walled contact-chemoreceptors of

arthropods have plugged pores in the outer wall; also the

outer canal is filled with dense material/receptor lymph

(Foelix & Chu-Wang 1973). Again, Eukoenenia spelaea

is special, because the outer pore on each side of the seta

is open and the outer canal is empty, but the inner pore is

plugged. Such a configuration might be associated with

receptor specificity, as chemicals pass differently through

dense material/receptor lymph and cuticular plugs (Alt-

ner et al. 1977).

Trichobothria

Trichobothria are common and characteristic mechano-

receptive sensory organs of terrestrial arthropods. The

typical structure of an arachnid trichobothrium includes

a thin hair, which is directly or indirectly connected to

sensory cells in a cuticular socket. This socket has either

bilateral symmetry as in spiders (Gorner 1965; Harris &

Mill 1977) or it is circular like in scorpions (Hoffmann

1967; Messlinger 1987). The socket has either smooth

ridges (Scorpiones [Messlinger 1987; Farley 1999], and

Araneae [Barth 2014]) or is ridged and teethed (orbatid

mites [Alberti et al. 1994]). The socked consists of an

Bonn zoological Bulletin Suppl. 65: 1-125

outer and inner cavity that are separated by a thin cuticu-

lar membrane. The inner cavity is an extension of the out-

er receptor lymph cavity and filled with receptor lymph.

Dendrites are surrounded by the dendritic sheath and ter-

minate in a tubular body at the base of the hair. Bending

of the hair may cause displacement of the cuticular hel-

met at the basis of the hair resulting in deformation of the

tubular body and ultimately in the creation of an action

potential in the sensory cell. The seta itself is e1ther sol-

id with only a short lumen at its proximal end (Alberti

et al. 1995), or hollow (Reiland & Gorner 1985), but it

does not contain dendrites. This overall fine structure of a

trichobothrium is widely found among arachnids.

The trichobothria of Eukoenenia spelaea follow this

general arachnid pattern. However, they differ in one im-

portant detail, 1.e., of the five dendrites associated with

the trichobothrium, four continue into the hair shaft, with

one extending into the distal section of the hair. In the

base of the hair, the dendrites retain their 9 x 2 + 0 mi-

crotubule configuration. We found a cuticular thickening

at the base of the hair, which 1s interpreted as the residue

of a cuticular helmet. A true helmet-like structure is not

developed because the dendrites reach into the hair. We

did not find tubular bodies. However, the material was

limited and the presence of tubular bodies cannot be en-

tirely ruled out. Dendrites inside the hair shaft of tricho-

bothria are unusual and indicate a second sense modality

in addition to mechanoreception. We could not find pores

along the hair shaft but the material was limited. A gener-

al similarity to a chemoreceptor is evident.

Similar unusual trichobothria were described for the

millipede Polyxenus. In this species, three unbranched

dendrites enter the hair and retain their 9 x 2 + 0 micro-

tubule configuration in the outer dendritic segments. A

cuticular helmet structure as well as tubular bodies were

not found (Tichy 1975). In addition, the trichobothrium

of Polyxenus has several plugged pores along its shaft

and, thus, its function was proposed to be chemorecep-

tive (Tichy 1975).

In Eukoenenia spelaea, the cuticular teeth of the socket

might serve three functions: (1) the teeth might prevent

sediment particles from entering the socket and, thus,

obstructing the movement of the hair. Similar retention

structures are known from mites (Alberti et al. 1994;

Gorb 2001b). (2) The teeth might prevent breakage of

the hair due to overextension while moving through sedi-

ment. The thin cuticle teeth might bend more easily when

the hair shaft gets pressed against them, thus, be more

flexible than a solid ridge. (3) The cuticular teeth might

prevent the seta from adhering to the side of the socket,

thus, impairing its flexibility.

Heart

In the ground pattern of arthropods, the heart is a dorsal

muscular tube surrounded by a pericardium and suspend-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 87

ed by musculo-elastic ligaments, which act as antagonist

to the circular muscles of the heart tube (Shear 1999). He-

molymph enters the heart through paired segmental ostia.

The heart of arachnids is typically innervated (Zwicky &

Hodgson 1965; Sherman et al. 1969; Bursey & Sher-

man 1970; Obenchain & Oliver Jr. 1975; Alberti & See-

man 2004). Pure myogenic hearts have been described

for some ticks (Schriefer et al. 1987; Coons & Alberti

1999). The muscle cells of the heart tube are striated

muscles, with clearly defined sarcomeres. The Z-line, A-

and I-band, T-tubular system as well as the sarcoplasmic

reticulum are well developed (Tjonneland et al. 1987).

The heart pumps the hemolymph through the body. The

hemolymph-vascular system of euchelicerates can be

morphologically complex in pulmonate euchelicerates

(Wirkner et al. 2013), but tends to be simplified in tra-

cheate arachnids (Crome 1953; Levi 1967), or reduced

in some miniaturized mites (Crome 1953; Levi 1967;

Wirkner et al. 2013).

During embryogenesis, the heart develops from paired

lateral coelomic cavities that merge in the dorsal midline

of the embryonic body. From this, it differentiates into

an initially closed tube (Strubell 1892; Kastner 1931f;

Scholl 1977; Rugendorff et al. 1994). Ostia and dilatator

muscles develop during later development stages, as has

been shown for Limulus (Scholl 1977) and Drosophila

(Rugendorff et al. 1994; Bodmer 1995; Molina & Cripps

2001).

The heart of Eukoenenia spelaea is a simple muscular

tube without ostia, without a pericardium, and without

dilator ligaments/muscles. Its structural simplicity and

similarity to the embryonic heart of other arthropods

suggests that it might be paedomorphic by developmen-

tal truncation. The paedomorphic morphology of the

adult heart is corroborated on the ultrastructural level.

The number of mitochondria is low, the sarcoplasmic

reticulum is poorly developed, myofilaments are irreg-

ularly placed throughout the myofibril, and Z-lines are

not clearly differentiated. Such cytological appearance

of myocardial cells has been described for the heart of

juvenile ticks (Coons & Alberti 1999: p. 360, fig. 67) and

the heart of larval Drosophila (Lehmacher et al. 2012).

The microscopic anatomy and the ultrastructure of the

heart of adult E. spelaea resembles that of juvenile or

larval stages of other arthropods. Together they support

the view that the heart of E. spelaea is paedomorphic.

The paedomorphic morphology of the heart suggests

a low degree of functionality, and, consequently, that it

is not involved in circulation of the hemolymph through

the body. This is not surprising because the overall hemo-

lymph space is extremely small or missing. Movement of

the residual hemolymph may be driven by muscle con-

traction of the body musculature in prosoma and opist-

hosoma. Reduction of the heart goes also along with the

lack of respiratory organs and dependence on diffusive

Bonn zoological Bulletin Suppl. 65: 1-125

gas exchange (discussed in 4.2.1; Rucker 1901; Kastner

193 1a).

Rostrosoma

All arachnids have a preoral cavity that is formed by var-

ious morphological contributions from the labrum, the

chelicerae, the pedipalps, or sternal elements (K4stner

1931g; Collatz 1987; Moritz 1993; Farley 2001). These

elements can be combined to form a more or less com-

plex rostrosoma that may surround the preoral cavity, the

mouth and parts of the pharynx. The preoral cavity and

the mouth may be equipped with cuticular teeth, ridges

or other filtering structures that prevent larger particles

from entering the pharynx. In some taxa, external cuticu-

lar surface structures on the lower lip might be involved

in guiding secretions from salivary glands that open ven-

tral on the body to the mouth. Obviously, the rostroso-

ma, preoral cavity and the various associated structures

evolved independently and parallel in many groups of

arachnids (Snodgrass 1948; Dunlop 2000).

The rostrosoma of Eukoenenia spelaea is a simple

cuticular tube that forms the upper and the lower lip at

its anterior part, surrounds the preoral cavity, the mouth

and the anterior part of the pharynx. It is a distinct mor-

phological structure that does not involve parts from

chelicerae, pedipalps or any other appendage, as in oth-

er arachnids (Kastner 1931g; Snodgrass 1948; Collatz

1987; Moritz 1993; Dunlop 2000; Farley 2001). At the

first glimpse, the simple morphology of the rostrosoma

compares to that of the pycnogonid rostrum. However,

it clearly differs by the internal mouth opening, the lat-

eral lips, and the topography of the precerebral suction

pump that partially inserts at the intercheliceral septum.

Indeed, the rostrosoma seems to be a simple cuticular

tube formed on the precheliceral segment. Based on the

muscle morphology we showed that a ventral sclerite of

the cheliceral element is part of the prosternum and thus

cannot contribute to the formation of the rostrosoma.

Historically, the lower lip has been viewed as derived

from a protosternite (Borner 1902a, 1903). However,

such structure has never been confirmed in either adult

or developmental stages of euchelicerates. In addition,

our observations show that segmental musculature of the

cheliceral segment attaches to the prosternum. Being so,

Snodgrass’ (1948, p. 21) conclusion “/.../ If therefore,

the mouth of the palpigrades lies between the labrum and

the sternum of the first postoral somite, we see here an

embryonic condition retained in no other modern adult

arthropod [... |” appears to be of merely historical inter-

est.

Millot (1942) reported glandular structures in the low-

er lip of Eukoenenia mirabilis and postulated the secre-

tion of saliva. However, we could not detect cells with an

unequivocal secretory character. Some cells with large

nuclei in the lower lip (Fig. 7C) might correspond to

©ZFMK

88 Sandra Franz-Guess & J. Matthias Starck

what Millot (1943) described as glandular cells, but we

did not see vesicles or structures that would indicate se-

cretory activity.

Palpigrades feeding on a food source has not been ob-

served and their actual food is a matter of speculation.

Like in all other arachnids, the cuticular ridges on the

inside of the upper and lower lip most likely function as

filter mechanism to prevent large particles from enter-

ing the alimentary system (e.g., Dunlop 1994). Wheel-

er (1900) and Rucker (1901) suggested that palpigrades

feed on arthropod eggs because they observed vitellin

vesicles in the gut. This was rejected by Millot (1942),

who found that the reported vitellin vesicles were in fact

not inside the midgut, but in the surrounding storage tis-

sue. Smrz et al. (2013, 2015) suggested that Eukoene-

nia spelaea feeds on heterotrophic cyanobacteria (Ch-

roococcidiopsis) which, indeed, have been identified in

the gut of E. spelaea. The serrated cuticular teeth of the

chelicerae might be used as a comb to graze cyanobac-

teria off the surrounding soil. The spacing of the teeth

(1-1.5 um) is considerably smaller than the diameter of

most cyanobacteria found within the gut (6-8 um; Smrz

et al. 2013, 2015) and thus appears suitable for raking

cyanobacteria from sediment.

Digestive tract

The digestive tract of euchelicerates can be divided in an

ectodermal foregut, comprising pharynx and esophagus,

a mesodermal midgut, and an ectodermal hindgut (Millot

1949b; Alberti & Coons 1999; Coons & Alberti 1999;

Farley 1999; Talarico et al. 2011). Along the foregut, di-

lator muscles attach to the precerebral and postcerebral

pharynx forming suction pumps. Pre- and postcerebral

suction pumps can be found in Amblypygi (K4stner

1931f), Araneae (Felgenhauer 1999), Ricinulei (Lud-

wig et al. 1994; Talarico et al. 2011), Scorpiones (Farley

1999), and Thelyphonida (K4stner 1931f). The posterior

suction pump is reduced in Acari (Alberti & Coons 1999;

Coons & Alberti 1999), Opiliones (Pinto-da-Rocha et al.

2007), Pseudoscorpiones (Weygoldt 1969), and Solifu-

gae (Klann & Alberti 2010).

Eukoenenia spelaea also lacks a postcerebral suction

pump. Rucker’s (1901) and Borner’s (1904) description

of a postcerebral suction pump in Prokoenenia wheeleri

is clearly a misinterpretation of the prosomal midgut di-

verticula (Millot 1942; Weygoldt & Paulus 1979b; Shultz

2007a; this study). The ectodermal esophagus of E. spe-

laea directly merges into the midgut tube and shows no

sign of additional musculature. The midgut is a straight

tube in the prosoma where it forms two diverticula. In

the opisthosoma, the midgut is rather sac-like with in-

dentations caused by the dorso-ventral musculature; dis-

tinct midgut diverticula are missing in the opisthosoma.

Similar sac-like midguts have been reported from instars

of Araneae (Gerhardt & K4stner 1931), Pseudoscorpi-

Bonn zoological Bulletin Suppl. 65: 1-125

ones (Weygoldt 1969), Thelyphonida (Kastner 1931f),

and Xiphosurida (Kimble et al. 2002). The morpholog-

ical simplicity of the midgut in E. spelaea, and its over-

all similarity with that of developmental stages of other

euchelicerate taxa is suggestive of a paedomorphic mor-

phology.

The epithelium of the midgut and prosomal midgut di-

verticula in Eukoenenia spelaea are identical and contain

secretory and digestive cells, which are characterized by

the numerous apical microvilli. It is a dimorphic epithe-

lium like in most arachnids (Polis 1990; Ludwig et al.

1994; Farley 1999; Klann & Alberti 2010; Talarico et al.

2011) except Acari (Alberti & Coons 1999; Coons & Al-

berti 1999), Araneae (Felgenhauer 1999), and Opiliones

(Becker & Peters 1985) in which additional cell types

like ferment cells, replacement cells, or excretory cells

are present.

Historically, the rectal sac has been considered part of

the hindgut (Kastner 1931a). However, because a cuti-

cle-lining of the rectal sac is missing in Eukoenenia spe-

laea, and because its epithelium consists of high prismat-

ic cells with long microvilli, we consider the rectal sac

part of the mesodermal midgut with absorptive function.

The rectal sac opens in a short, cuticle-lined section, 1.e.,

the anal opening, which appears to be the only residue of

an otherwise reduced ectodermal hindgut.

Excretory organ

The coxal organs of arachnids typically consists of sac-

cule, tubule (labyrinth), bladder (vesicle), excretory duct,

and excretory pore (Buxton 1913, 1917; Millot 1949b;

Moritz 1993). Muscle fibers may attach to the saccule,

e.g., in Acari (Alberti & Coons 1999) and Solifugae

(Buxton 1913; Alberti 1979b; Klann 2009), supposedly

allowing for extension of the saccule thus producing a

pressure gradient for ultrafiltration. Among arachnids,

the tubule varies in shape and morphological complexity

(Buxton 1913, 1917, Alberti & Coons 1999). In Solifu-

gae (Buxton 1913, 1917; Alberti 1979b; Klann 2009) a

glandular section is inserted between saccule and tubule.

In some Acari (Coons & Alberti 1999; Filimonova 2004,

2016, 2017), the tubule is cytologically differentiated in

a proximal and a distal segment; the distal segment may

contain glandular sections. The different position of the

glandular segments and the different details of cytology

in Solifugae and Acari suggest an independent evolution-

ary origin of these glandular elements of the coxal organ.

The location of the excretory pore varies among Arach-

nida. In Solifugae, the pore is located next to the pedipalp

(Buxton 1913; Alberti 1979b); in Acari on or adjacent to

leg 1; in Amblypygi, Araneae (Buxton 1913, 1917), Opil-

iones (Pinto-da-Rocha et al. 2007), and Thelyphonida

(Buxton 1913, 1917) next to leg 2; in Pseudoscorpiones

(Weygoldt 1969) and Xiphosurida (Shultz 1990) close to

leg 3, and in Scorpiones (Farley 1999) between leg 3 and

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 89

4. Some species of Amblypygi and Araneae have two ex-

cretory pores, one close to leg 2 and one close to leg 4

(Buxton 1913, 1917).

In Eukoenenia spelaea, the tubule of the coxal organ

is cytologically differentiated in a proximal and a distal

segment, each carrying a long blind ending extension.

Based on light microscopic descriptions provided by

Borner (1904) and Buxton (1917), the proximal part of

the tubular system was described as “glandular” and the

distal part as excretory. This interpretation was perpetuat-

ed in later publications, and, homology with the coxal or-

gans of solifuges was postulated (Buxton 1917; Kastner

1931a; Alberti 1979b) because the “glandular” part were

intercalated between the saccule and the tubule.

Our transmission electron micrographs of all parts of

the coxal organ of Eukoenenia spelaea allow updating

details of the microscopic anatomy of the coxal organ and

re-interpreting supposed similarities with ultrastructural

details of the coxal organs of solifuges (Alberti 1979b:;

Klann 2009). In Eukoenenia spelaea, the saccule is a

simple, small pouch with an epithelial wall of podocytes

and a narrow lumen. No muscles were found inserting on

the saccule, but several muscles were found passing near-

by. The coxal organ muscle inserting on the saccule in

Eukoenenia mirabilis as reported by Millot (1942) might

be one of those muscles in immediate neighborhood to

the saccule, and is probably not attached to the saccule;

however light microscopy on paraffin sections as used by

Millot (1942) did not provide the necessary optical reso-

lution. The microscopic anatomy of the saccule in E. spe-

laea equals the basic organization of the epithelium of

the saccule of euchelicerates with podocytes exposed to

the hemolymphatic space. The lumen is rather small, but

there is considerable variation in the size of the lumen of

the saccule, e.g., in some Acari (Alberti & Coons 1999;

Coons & Alberti 1999) and Thelyphonida (Buxton 1917)

the lumen is small. However, in other Acari (Alberti &

Coons 1999; Coons & Alberti 1999), Amblypygi (Bux-

ton 1913, 1917), Araneae (Buxton 1913, 1917; Lopez

1983), Scorpiones (Buxton 1913, 1917), Solifugae (Bux-

ton 1913, 1917; Alberti 1979b; Klann 2009), and Xipho-

surida (Fahrenbach 1999) the lumen can be extensive.

In Eukoenenia spelaea, an elongate tubular section

extends from the saccule into the second opisthosomal

segment. This proximal segment of the tubule has been

described as glandular segment (Borner 1904; Buxton

1917). However, already Millot (1942) questioned a

glandular function and described it as tubule with secre-

tory function. Transmission electron microscopy now

proves that the basal part of the cells posses an extensive

basal labyrinth (infoldings of the cell membrane) which

is typical for the transport epithelium and is associated

with excretory function of the tubule. The transport epi-

thelium of Eukoenenia clearly differs from that of the

glandular part of the tubule of Solifugae as shown by

light microscopic histology and the ultrastructure of the

Bonn zoological Bulletin Suppl. 65: 1-125

cells (Alberti 1979b; Klann 2009). In solifuges, the cells

have no basal labyrinth, but a folded basal lamina, cells

are large with large nuclei, possess numerous apical mi-

crovilli and are filled with secretory vesicles.

The distal segment of the tubule of Eukoenenia spelaea

is a simple, bent tube with an additional branch emerging

from a position where the proximal section fuses with the

tubule. It has not yet been reported in that detail for other

palpigrades. The ultrastructure of the epithelial cells is

similar to that of the proximal part of the tubule, but the

basal labyrinth is apparently less intensively developed.

Neither the proximal nor the distal part of the tubule

show an open lumen. The cells of the tubule in arachnids

typically have some form of apical microvilli, sometimes

varying in number and length along the course of the tu-

bule (Alberti 1979b; Coons & Alberti 1999; Fahrenbach

1999; Filimonova 2004, 2016, 2017). Due to the lack of

a lumen of the tubule, no microvilli are present in FE. spe-

laea, but apical folds of the epithelial cells may represent

their residues. A bladder is missing in EF. spelaea, which

differs from description of Eukoenenia mirabilis by Mil-

lot (1942).

The cytological details elaborated here for Eukoene-

nia spelaea reject the homologization of the coxal organ

of Palpigradi with that of Solifugae. Rather it appears

that the tubule is differentiated into a proximal and a

distal segment, both with excretory/secretory epithelia.

Such differentiation resembles that in Acari (Alberti &

Coons 1999; Coons & Alberti 1999; Filimonova 2004,

2016, 2017) where the tubule is differentiated in a prox-

imal and a distal segment. Of course, the microscopic

anatomical differentiation of the coxal organ in Acari

may show additional cellular differentiations or loss of

structures. However, the coxal organ of Palpigradi might

serve rather as a plesiomorphic template for the coxal or-

gan of Acari than as homologon of the coxal organ of

Solifugae.

Reproductive organs

Female reproductive organs

The female reproductive organs of arachnids consist of

the ovaries, oviducts, seminal receptacle, and genital

chamber. The detailed morphology of these structures

varies considerably between groups, and, with the ex-

ception of ladder-like gonad morphology found in Schi-

zomida, Scorpiones, and Thelyphonida (Shultz 2007a),

contains little phylogenetic information. The ovaries can

be paired or display various degrees of fusion (Kastner

1931f; Polis 1990; Moritz 1993; Michalik et al. 2005;

Klann 2009; Foelix 2010). The oviducts are typically

paired, originate anteriorly at the ovary, and vary in thick-

ness (Moritz 1993; Alberti & Michalik 2004). A seminal

receptacle may be found in groups with sperm transfer

with gonopods or a penis (Alberti & Michalik 2004), but

©ZFMK

90 Sandra Franz-Guess & J. Matthias Starck

it is lacking in Amblypygi (Borner 1902b) and Solifugae

(Millot & Vachon 1949a; Klann 2009), also groups with

morphological adaptations for sperm transfer. The genital

chamber is generally lined with cuticle (Borner 1902b;

Kastner 1931d; Millot & Vachon 1949b; Weygoldt 1969;

Alberti & Coons 1999; Coons & Alberti 1999; Felgen-

hauer 1999; Klann 2009). A receptaculum seminis was

described for Prokoenenia wheeleri by Rucker (1901),

but was later questioned by Borner (1902b). In Acari (AI-

berti & Coons 1999; Coons & Alberti 1999), Amblypygi

(Borner 1902b; Kastner 1931f), Pseudoscorpiones (Wey-

goldt 1969), Schizomida, and Thelyphonida (Borner

1902b; Kastner 1931f), an accessory gland is associated

with the reproductive organs.

The slightly posterior origin of the paired ovarian ducts

from the unpaired ovary of Eukoenenia spelaea appears

to be unique among arachnids. The diameter of the ducts

is small compared to the diameter of an egg. Although no

transmission electron micrographs of the ovarian ducts

are available, it can be assumed that the epithelium is

high prismatic and expandable to accommodate the eggs

prior to laying. Our description of the seminal recep-

tacle of E. spelaea confirms that by Rucker (1901) for

Prokoenenia wheeleri. The paired accessory gland has no

opening which indicates that the secretions collected in

the reservoir is secreted through pores in the thin layer

of cuticle in the region lateral to the genital opening. A

small number of relatively large eggs 1s characteristic for

small/miniaturized animals and suggests that only few

eggs are laid at a time.

Male reproductive organs

The male reproductive organs of arachnids typically con-

sist of the paired testes, vas deferens, and genital atrium

(Alberti et al. 2007). Like the female ovary, the morpho-

logy of the testes varies between groups. Unpaired testes

as well as testes in different stages of fusion have been de-

scribed (Weygoldt 1969; Polis 1990; Moritz 1993; Alber-

ti & Coons 1999; Coons & Alberti 1999; Pinto-da-Rocha

et al. 2007; Talarico et al. 2008; Klann 2009; Michalik

2009). The transfer of sperm can be coupled with the pro-

duction of a spermatophore. These sperm packages can

display a species-specific morphology (e.g., Amblypy-

gi [Weygoldt et al. 2010], Pseudoscorpiones [Weygoldt

1969], and Scorpiones [Polis 1990]). Spermatophores are

built in Acari (Alberti & Coons 1999; Coons & Alberti

1999), Pseudoscorpiones (Weygoldt 1969), Scorpiones

(Polis 1990), Solifugae (Klann 2009), Amblypygi, Schi-

zomida, and Thelyphonida (Moritz 1993).

The male reproductive system of Eukoenenia spelaea

consists of paired testes, vas deferens, a genital atrium,

and two accessory glands. The unpaired posterior gland

has no secretory duct, but, similar to the accessory gland

in females, possibly secretes through pores in the cuti-

cle (Bereiter-Hahn et al. 1984). This gland corresponds

Bonn zoological Bulletin Suppl. 65: 1-125

to the paired accessory gland described for Prokoene-

nia wheeleri (Rucker 1901). The paired anterior acces-

sory gland is larger than the posterior accessory gland

and is associated with the glandular fusules of the first

genital lobes. The general morphology of E. spelaea’s

male reproductive organs is similar to Amblypygi, Ara-

neae, Ricinulei, Schizomida, and Thelyphonida. Howev-

er, the morphology of the accessory glands differs greatly

from these groups. The posterior accessory gland has no

recognizable opening into the genital atrium. The secre-

tory ducts of the anterior accessory gland appear to be a

unique character of E. spelaea.

Sperm morphology

Sperm morphology has been used in phylogenetic anal-

yses of euchelicerates/arachnids (Alberti 1995). The

sperm of euchelicerates show a variety of shapes and

complexity (Alberti 1995; Alberti & Michalik 2004; Pit-

nick et al. 2009). Xiphosurida have plesiomorphic sperm,

i.e., a spherical head containing the acrosomal complex

and nucleus with condensed chromatin, a middle piece

containing a few relatively large mitochondria, and an

elongate sperm tail representing the flagellum. Arachnids

have either filiform-flagellate sperm (scorpions), coiled

flagellate sperm (Pseudoscorpiones, Uropygi, Ambly-

pygi, Araneae, Ricinulei) or aflagellate sperm (Acari,

Opiliones, Solifugae, Palpigradi; Alberti 1995; Alberti &

Michalik 2004). Vacuolated sperm occur in different taxa

of the arachnids, with flagellate and aflagellate sperm.

However, anactinotrichid mites have highly complex

vacuolated sperm that obviously evolved independently

(Alberti 1995, 2000; Alberti & Michalik 2004). In this

type of sperm, the large central vacuole develops through

fusion of multiple peripheral vacuoles and the fully de-

veloped sperm is later turned inside out (Alberti & Mi-

chalik 2004). Sperm of Opiliones and anactinotrichid

mites may have reduced the acrosomal filament, howev-

er, this appears to be variable among taxa.

The sperm of Palpigradi have been described for

Prokoenenia wheeleri (Alberti 1979a). They have aflagel-

late sperm that are characterized by a large vacuole that

contains dense vesicles. The nucleus of P. wheeleri sperm

is coiled several times around the spermatozoon (Alberti

1979a; Alberti & Michalik 2004). The acrosomal com-

plex lacks the acrosomal filament, like in some opilionid

and acarine taxa (Alberti 1979a; 1995).

The spermatozoa of Eukoenenia spelaea are similar

to the spermatozoa of Prokoenenia wheeleri. They con-

tain a large vacuole, with a constant number of spherical

vesicles. The development of the vacuole appears to be

identical to that in P. wheeleri. The most advanced sperm

we found closely resembles an “almost mature sperm” as

described by Alberti (1979a; Fig. 2a). Unfortunately, no

transmission electron microscopic images could be ob-

tained from spermatozoa in E. spelaea.

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 91

The unique morphology of the sperm of Palpigradi

makes it difficult to discuss their phylogenetic relation-

ship. The aflagellate sperm morphology places them to-

gether with Opiliones, Solifugae and Acari. The lack of

an acrosomal filament has also been reported for some

Acari and Opiliones, however with variable distribution

among taxa of these groups. Large vacuolated sperm

characterize anactinotrichid mites, but the fine structure

of the vacuole and their development apparently differ

from that in Palpigradi. Also, various vacuolated sperm

have been reported from other arachnid taxa. Overall, the

relevant traits are either unique (1.e., coiled nucleus), re-

duced characters, or subject to multiple and parallel evo-

lutionary appearance among arachnids. Therefore, sperm

characters appear of limited value for a phylogenetic

analysis of Palpigradi — despite more optimistic claims

by Alberti (1995).

Phylogenetic analysis

Autapomorphies of Eukoenenia spelaea

The current study of Eukoenenia spelaea recognized sev-

eral characters that are unique for Palpigradi and there-

fore should be considered autapomorphies (of course,

supposing that Eukoenenia is representative for the

group). Some of these characters have been known for a

long time, but our study provides explicit morphological

evidence for a new interpretation of these characters now

presenting them as unique features of Palpigradi.

(1) The prosoma of Palpigradi is dorsally divided in

only two peltidia (propeltidium, metapeltidium), not

three, as traditionally assumed (character 6; Appendix I:

Tab. 6, Al). Assuming that this new interpretation holds

for Palpigradi and that the published tagmatization (..e.,

pro-, meso- and metapeltidium) of Schizomida and Solif-

ugae remains valid, the division of the prosoma into two

sclerites is a unique character of Palpigradi.

(2) On the ventral side of the prosoma, the anterior

sclerite is now assigned to three anterior segments (_.e.,

segments 2-4) now termed “prosternum”. The term

“deuto-tritosternum” that refers to only segments 3 and

4 should be abandoned (character 12a).

(3) The frontal organ (character 148; Appendix I:

Tab. 6, Al) is found only in Palpigradi. The ultrastruc-

ture of the setae of the frontal organ with presumably dif-

ferent sense modalities for left and right setae 1s unique

among Euchelicerata.

(4) The lateral organ (character 148a) is found only in

Palpigradi.

(5) Preoral cavity, mouth, and pharynx of Eukoene-

nia spelaea are inside a simple cuticular cone shaped

rostrosoma (character 32; Appendix I: Tab. 6, Al). The

morphology of the rostrosoma differs from the rostroso-

ma described for Pseudoscorpiones and Solifugae (Shul-

tz 2007a) where it includes material of pedipalps or legs.

Bonn zoological Bulletin Suppl. 65: 1-125

Despite being a simple cuticular cone, it also differs from

the ancestral rostrum of the pycnogonids by the forma-

tion of a preoral part of the rostrosoma, and differences

in the muscle attachment of the suction pump. The dila-

tor muscles attach to the pharynx and the cuticular wall

of the rostrosoma (character 198; Appendix I: Tab. 6,

A1). In Araneae (Whitehead & Rempel 1959), Solifugae

(Roewer 1934), und Xiphosurida (Shultz 2001) these

muscles insert ventral on the prosoma. — We consider the

rostrosoma as a complex character that differs from other

euchelicerates described so far.

(6) Eukoenenia spelaea \acks a hindgut (character

203). Previous studies of palpigrades misinterpreted the

rectal sac as rectum/hindgut (Kastner 193 1a).

We describe several morphological features that have

not been reported before in any euchelicerate.

(7) The ventral plate has a unique morphology (1.e.,

cuticular teeth, specialized epidermal cells; character

179a). Since none of the earlier studies used TEM and

SEM, we assume that it was overlooked previously and

we describe it as an autapomorphy for Palpigradi.

(8) The ultrastructure of the trichobothria, with den-

drites reaching into the hair shaft, is unique among eu-

chelicerates (character 143a).

(9) Anterior oblique suspensor muscle (muscle E6)

in segment four. Although an ancestral element of the

ground pattern of arthropods, the re-occurrence of this

muscle must be considered autapomorphic (character

128a).

Suggested synapomorphies with Acaromorpha

Palpigradi were occasionally placed as sister group

to Acaromorpha (Regier et al. 2010) or sister group to

Parasitiformes (Sharma et al. 2014). We also recovered

Palpigradi as sister to Acaromorpha (Fig. 49A, C). This

sister group relationship is indeed supported by several

morphological synapomorphies:

(1) The opening of the coxal organ (character 180; Ap-

pendix IT). In Palpigradi (Millot 1942) and Acaromorpha

(Legg 1976; Alberti & Coons 1999) the excretory pore

of the coxal organ opens on or near the basal article of

appendage III (= leg 1). This character possibly evolved

in parallel in (Pan)Tetrapulmonata.

(2) Palpigradi and Acaromorpha lack a postcerebral

pharynx (199; Appendix II; Shultz 2007a). Loss of the

postcerebral pharynx must have occurred then 1n parallel

in Opiliones and Pseudoscorpiones.

(3) Aflagellate sperm that lack an acrosomal filament

have been documented in Palpigradi, Acari and Opilion-

es. Although sperm morphology appears to be a rather

weak character, it supports a sister group relationship of

Palpigradi and Acaromorpha (167, Appendix IJ; Alberti

1979a, 1995; Alberti & Michalik 2004). — However, Rici-

nulei have coiled sperm and this character does clearly

not support a close relationship to Palpigradi and Acari.

©ZFMK

92 Sandra Franz-Guess & J. Matthias Starck

(4) The topography of the musculature of leg 4 of

Acariformes, Parasitiformes, and Palpigradi is similar in

terms of reduction of muscles (Tab. 11; Shultz 1989).

(5) The tubule of the coxal organ is differentiated in a

proximal and a distal segment (178a; Appendix II). The

epithelia of both segments possess a distinct basal laby-

rinth thus are involved in excretory processes. The cel-

lular arrangement is also similar in mites and Eukoene-

nia spelaea. A tubule lumen is missing in both groups

and there are always three cells present in cross-section

(Alberti & Coons 1999). Because of considerable differ-

ences in the ultrastructure, we do not support the previ-

ously postulated putative homology with the coxal organ

of Solifugae (Buxton 1913, 1917; Alberti 1979b; Klann

2009).

(6) The presence of a myogenic heart in some ticks

(Schriefer et al. 1987; Coons & Alberti 1999) is also sim-

ilar to the proposed myogenic heart of Eukoenenia spe-

laea.

(7) Lack of the arcuate body in the syncerebrum (135a;

Appendix II). Acari and Palpigradi are the only groups

of euchelicerates that have no arcuate body. Since Palpi-

gradi and (many) Acari are eyeless; the lack of the visual

integration center may not be surprising. As a reduced

structure, the character 1s not strong but it fits the general

picture.

The phylogenetic analysis with TNT resulted in low

bootstrap percentage and Bremer support values, and

the unweighted analysis left the deep arachnid relation-

ships unresolved (Fig. 49A). In general, bootstrap per-

centages and Bremer support values were lower than in

the phylogenetic analysis by Shultz (2007a). While our

morphological analysis contributed a number of new

autapomorphic features for the Palpigradi, it was less

powerful in providing convincing arguments for a sister

group relationship with any of the Arachnida. Generally,

morphological characters seem to support a sister group

relationship between Palpigradi and Acaromorpha, but

most of the characters are reduction characters or subject

of multiple and independent evolution among the arach-

nids. The distinction of the coxal tubule in a proximal and

distal segment, appears to be one of the better characters

supporting a sister group relationship between Acaro-

morpha and Palpigradi.

While our morphological findings support a phyloge-

netic relationship between Palpigradi and Acaromorpha,

we, paradoxically, reject all morphological features that

were suggested by van der Hammen (1977b) as support-

ing his “Epimerata” (i.e., Palpigradi and Parasitiformes).

The articulations of the leg articles can by no means be

compared to the schematic of van der Hammen (1977b)

and the intrinsic musculature observed here shows a sub-

stantially different pattern (Figs 11A—C, 20C, Tabs 5, 11-

12). Furthermore, the origin of extrinsic leg muscles re-

jects the idea that the ventral plates of the prosoma were

“epimera” from which the coxae evolved. — As pointed

Bonn zoological Bulletin Suppl. 65: 1-125

out by others (Dunlop & Alberti 2007), van der Hammen

(1977b) used explicitly plesiomorphic traits to support

his “Epimerata” making his reasoning quite useless.

Miniaturization

Miniaturization is directional evolution toward small

adult body size. Thus, miniaturization can only be recog-

nized in a phylogenetic context when small taxa evolve

from proportionally larger ancestor(s). As pointed out by

Hanken & Wake (1993), the distinction between minia-

turized and non-miniaturized taxa is to a certain degree

arbitrary, and the range of size change depends on the

size of the ancestor. Miniaturization appears to be limited

by certain physiological and constructural/morphological

principles. In particular, neuron size, neuronal network

connectivity and consequently brain size seem to be lim-

ited by sensory and behavioral complexity resulting in

proportionally large brains of small animals. High lev-

els of fusion within the brain (Ioffe 1963; Wegerhoff &

Breidbach 1995), and displacement of parts of the brain

in other body regions (e.g., coxae, opisthosoma; Quesada

et al. 2011) are typical signs of morphological reorgani-

zation that come along with an allometrically enlarged

brain in miniaturized species (Peters 1983; Mares et al.

2005; Seid et al. 2011; Polilov & Makarova 2017). Thus,

miniaturization causes reorganization of the body design

to accommodate the disproportionately large brains, or

sacrifice on behavioral complexity and/or sensory inte-

gration (Eberhard & Wcislo 2011, 2012). Miniaturization

may result in structural simplification and reduction of

organ systems, but also lead to morphological novelty

and an increase of morphological variability (Hanken &

Wake 1993; Polilov 2016). Besides the negative brain-

size body-size allometry, several morphological features

have been reported that appear to be common tn miniatur-

ized arthropods (Polilov 2008, 2015a, b, 2016; Quesada

et al. 2011; Polilov & Shmakov 2016), 1.e.: (i) a mostly

undifferentiated gut, 1.e., no distinction between mid-

gut tube and midgut diverticula, lacks musculature; (11)

gonads are (often) unpaired and they contain a reduced

number of large eggs; (ii) the heart and major compo-

nents of the circulatory system are reduced; (iv) the neu-

rilemma is reduced, (v) respiratory organs are reduced or

missing; (vi) the cuticle lacks sclerotization and differen-

tiation into exo- and endocuticle; (vil) in some regions of

the body muscles are reduced, and (viii) the number of

Malpighian tubules is reduced or they are missing (Poli-

lov 2015a, b; Polilov & Shmakov 2016).

The constructural morphology of being small is one

aspect of miniaturization. Another aspect is how small

body size is achieved during evolution, i.e., which pro-

cesses result in small adults. Since Gould (1977) and Al-

berch et al. (1979), various evolutionary developmental

processes have been identified as potential mechanism

by which miniaturization is achieved through relatively

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 93

simple changes of developmental programs. According

to Gould (1977), progenesis (1.e., early maturation) may

result in small but paedomorphic adults. Alberch et al.

(1979) included growth rates in their model and provided

a more dynamic model of how development may affect

evolution. They considered neoteny, post-displacement,

and progenesis as evolutionary developmental processes

resulting in the same small and paedomorphic morpho-

logy of adults, and suggested measuring rates of develop-

ment to decide ultimately, which process was involved.

Several morphological features found in Eukoene-

nia spelaea indicate miniaturization: (1) there is a lack of

a distinct differentiation into endo- and exocuticle over

large regions of the body. (2) E. spelaea has no respira-

tory organs, but utilizes cutaneous respiration. (3) Sev-

eral muscles, e.g., dorso-ventral muscles in the posterior

opisthosoma and posterior oblique muscles are reduced

in all opisthosoma segments. (4) The prosomal ganglia

are proportionally large in comparison to the rest of the

body, fill almost the entire prosoma and display a high

level of fusion. Eberhard & Wceislo (2011) documented

that the (proportionally large) brain of small arthropods is

displaced into the coxae and the opisthosoma. (5) Neuron

size 18 small (approx. 2 um) and ranges at the minimum

size described for arthropods. According to Eberhard &

Weislo (2011), the minimum possible neuron size in ar-

thropods is 2 um. Recently, the range has been extended

and the smallest insects (e.g., the ptiliid beetle Nanosella)

shows a neuron size range from 1.19 um to 1.98 um (Po-

lilov 2016). (5) E. spelaea has only 14 trichobothria in

total. (6) The simplified structure of the coxal organ, L.e.,

the reduced number of cells and a simple tube instead of

a complex labyrinth, could be an indication for miniatur-

ization. (7) E. spelaea has no Malpighian tubules. (8) The

female gonads are an unpaired structure. (9) Within the

ovary, only few eggs appear to be fully developed.

For several organ systems of Eukoenenia spelaea, it

is difficult to decide whether their adult morphology is

shaped by miniaturization, paedomorphosis, or both: (1)

the portion of the syncerebrum associated with the che-

licerae (deutocerebrum) embraces the esophagus. This

morphology is similar to that of larval Limulus poly-

phemus — but it could also be result from displacement

of the central nervous system, or both together. (2) The

number of neurons within the opisthosomal ganglia of

E. spelaea is small. Again, this may be a result of min-

iaturization, or paedomorphosis, because a small number

of neurons is indicative of early developmental stages

and was documented in the opisthosomal ganglia of ar-

thropod embryos.

Finally, we described several adult organs of Eukoene-

nia spelaea that were clearly paedomorphotic: (1) the

heart of E. spelaea lacks ostia, a pericardium, and the

ultrastructure of the musculature (sarcomere structure)

is weakly developed. This morphology resembles that of

the hearts of early developmental stages of other arthro-

Bonn zoological Bulletin Suppl. 65: 1-125

pods. (2) The midgut is a simple sac with no epithelial

differentiation between the different regions. The mus-

culature associated with the midgut is poorly developed.

A similar morphology is found in juveniles of several

arachnid groups. (3) The reappearance of ancient plesi-

omorphies, e.g., (1) the anterior oblique muscle, (11) an

almost complete set of posterior oblique muscles in the

prosoma, (111) the deutocerebral connectives embracing

the esophagus, and (iv) the segmental chord of ganglia

in the opisthosoma, are features of the arthropod ground

pattern that may be interpreted as “reverse recapitula-

tion” sensu Alberch et al. (1979). Assuming anagenetic

evolution, paedomorphosis by developmental truncation

(neoteny, progenesis or post-displacement; Alberch et al.

1979) may result in patterns of “reverse recapitulation”,

or more correctly, in an array of plesiomorphic features.

The proof of evolutionary miniaturization requires

phylogenetic comparisons, 1.e., the last common ancestor

must have been large as compared to the crown group in-

vestigated. Such comparative analysis strongly depends

on the phylogeny used. In our phylogenetic analysis, Pal-

pigradi are hypothesized to be sister group to Acaromor-

pha (Fig. 49). Acaromorpha contain almost exclusively

small species with some of the smallest chelicerates at

all (Acari). Thus, the last common ancestor of Palpigradi

and Acaromorpha was probably already small, suggest-

ing that miniaturization occurred in the stem lineage

leading to the last common ancestor of Palpigradi and

Acaromorpha.

If we extend the comparison, we find considerable

variation of body size among euchelicerates (e.g., Eu-

rypterida: up to 1.8 m body length, Alberti et al. 2007;

Scorpiones: up to 210 mm body length, Polis 1990; Solif-

ugae: 10-70 mm body length, Punzo 2012; Xiphosurida:

up to 850 mm body length, Alberti et al. 2007) with some

groups showing a tendency towards small body size (e.g.,

Acari: up to 14 mm body length, Dunlop 2019; Opilion-

es: up to 22 mm body length, Pinto-da-Rocha et al. 2007;

Pseudoscorpiones: 1-7 mm body length, Weygoldt 1969;

Ricinulei: up to 10 mm body length, Alberti et al. 2007;

Schizomida: approx. 5 mm body length, Harvey 2003).

Thus, if we move down the phylogram, the sister group

to Palpigradi and Acaromorpha is Haplocnemata + (Pan)

Tetrapulmonata (Fig. 49C). Both taxa contain species

with large body size. Further down the tree, the sister

group to ((Haplocnemata + Tetrapulmonata) and (Palp-

igradi + Acaromorpha) are the Stomothecata (Fig. 49C),

again a group with large sized representatives (Scorpio-

nes). At the base of the euchelicerate phylogeny, we find

again two groups with large body size, 1.e., the extinct

Eurypterida and the recent Xiphosurida. Considering the

phylogeny presented here, we suggest that the last com-

mon ancestor of ((Haplocnemata + Tetrapulmonata) and

(Palpigradi + Acaromorpha)) was large, and that minia-

turization took place in the last common ancestor of (Pal-

pigradi + Acaromorpha). With their relatively simple and

©ZFMK

94 Sandra Franz-Guess & J. Matthias Starck

highly plesiomorphic morphology, one may speculate

that Palpigradi represent a morphologically unchanged

offshoot of this early lineage, while the lineage leading

to the Acaromorpha successfully diversified based on a

paedomorphic morphology.

Highlights

1. Analysis of the segmental axial musculature showed,

that the dorsal prosoma is divided in two peltidia, not

three as previously assumed. The traditionally recog-

nized ,,mesopeltidia“ are lateral sclerotizations of the

pleural fold

2. Analysis of the segmental axial musculature showed

that, the anterior ventral sclerite covers segments 2, 3

and 4, not 3 and 4 as traditionally assumed. Palpigra-

di have no ,,deuto-tritosternum“, but a large ,,proster-

num.

3. We find no evidence that the ventral sclerites (“sterna’’)

of the prosoma are homologous to coxae (sternocoxal

plates).

4. The metasoma is not a plesiomorphic structure but in-

dependently derived in various groups of arachnids.

5. The ventral side of the prosoma carries a so far unde-

scribed ventral plate. Ultrastructural analysis suggests

it functions as an osmoregulatory organ.

6. The reappearance of features of the arthropod ground

pattern result in a hyperplesiomorphic morphology,

e.g., (1) the anterior oblique muscle, (ii) an almost

complete set of posterior oblique muscles in the proso-

ma, (111) the deutocerebral connectives embracing the

esophagus, and (iv) the segmental chord of ganglia in

the opisthosoma. We discuss this pattern in the context

of models of evolutionary development and we suggest

that the hyperplesiomorphic adult morphology is result

of “reverse recapitulation’, 1.e., re-appearance of plesio-

morphic patterns through developmental truncation.

7. Structures of the syncerebrum can be interpreted as

tripartite thus supporting the more recent view that a

deutocerebrum is present in chelicerates. At the same

time, the esophagus is embraced by the deutocerebral

connectives, indicating an ancestral structure (or re-

verse recapitulation due to paedomorphosis).

8. We describe a most complete segmental chord of gan-

glia in the opisthosoma. This plesiomorphic feature of

the arthropod ground pattern may also be interpreted as

“reverse recapitulation”.

9. Left and right bristle of the frontal organ provide dif-

ferent sense modalities.

10. Trichobothria contain dendrites that reach into the

tip of the hair. This unusual morphology has so far not

been described for euchelicerates.

11. The heart is largely rudimentary suggesting a paedo-

morphic origin.

12. The rostrosoma is a unique cuticular tube. Chelicerae

or pedipalps are not involved.

Bonn zoological Bulletin Suppl. 65: 1-125

13. Palpigradi do not have an ectodermal hindgut.

14. We reject the putative homology of the coxal organ

of Palpigradi with that of Solifugae. Instead, we recog-

nize an overall similarity of the coxal organ with that

of Acari.

15. We suggest a phylogenetic position of the Palpigradi

as sister group to Acaromorpha.

16. Palpigradi are not miniaturized but most probably

derived from ancestors that were already small (minia-

turized). We recognize paedomorphosis as putative

mechanism that resulted in the small overall body size.

Acknowledgements. Specimen were collected, taxonomical-

ly diagnosed and made available for this study by Lubomir

Kovaé, P. J. Safarik University, Ko8ice, Slovakia. We grate-

fully acknowledge providing this rare material for this study.

We gratefully acknowledge discussions with Roland Melzer,

Zoological state collection, and Carolin Haug, Dept. Biology,

LMU, who provided valuable input. We thank Christine Dun-

kel and Antoinette von Sigriz-Pesch for their invaluable help in

the laboratory. SFG was supported by a PhD fellowship of the

Rosa-Luxemburg foundation, Berlin. We are grateful to four

reviewers (Erhard Christian and three anonymous reviewers)

for their careful and throughtful reviews of this long paper. We

gratefully acknowledge their time and efforts to help improving

this paper.

Author contributions. SFG prepared specimens for histology,

prepared all light and electron micrographs, drew all schemat-

ics, and prepared a draft of the manuscript. JMS conceived the

project, supervised all laboratory work, discussed all results,

and revised and rewrote an earlier draft of the manuscript.

Data availability. All animal material used in this project (in-

cluding unsectioned specimens) is stored at the Bavarian State

Collection of Zoology in Munich, Germany (project numbers

ZSMS820190030 — ZSMS20190051) from where it is accessible

upon request.

REFERENCES

Alberch P (1980) Ontogenesis and morphological diversifica-

tion. American Zoologist 20: 653-667

Alberch P, Gould SJ, Oster GF, Wake DB (1979). Size and

shape in ontogeny and phylogeny. Paleobiology 5: 296-317

Alberti G (1979a) Zur Feinstruktur der Spermien und Spermio-

cytogenese von Prokoenenia wheeleri (Rucker, 1901) (Palpi-

gradi, Arachnida). Zoomorphologie 94: 111-120

Alberti G (1979b) Licht- und elektronenmikroskopische Unter-

suchungen an Coxaldrtisen von Walzenspinnen (Arachnida:

Solifugae). Zoologischer Anzeiger 203: 48-64

Alberti G (1995) Comparative spermatology of Chelicerata: re-

view and perspective. Advances in spermatozoal Phylogeny

and Taxonomy. Mémoires du Muséum National d’Histoire

Naturelle 166: 203—230

Alberti G (2000) Chelicerata. Pp. 311-388 in: Jamieson BGM

(ed.) Reproductive Biology of Invertebrates, Vol. 9, part B.

John Wiley & Sons Ltd, Chichester, United Kingdom

Alberti G, Coons B (1999) Acari: Mites. Pp. 515-1265 in: Har-

rison F, Foelix RF (eds) Microscopic Anatomy of Inverte-

brates, Vol. 8C. Wiley-Liss, New York

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 95

Alberti G, Seeman O (2004) Ultrastructural observations on

Holothyrida (Acari: Anactinotrichida). Phytophaga XIV:

103-111

Alberti G, Michalik P (2004) Feinstrukturelle Aspekte der Fort-

pflanzungssysteme von Spinnentieren (Arachnida). Pp. 1-62

in: Biologiezentrum des Oberdsterreichischen Landesmuse-

ums (ed.) Diversitaét und Biologie von Webspinnen, Skorpi-

onen und anderen Spinnentieren, Neue Serie 14, Denisia 12

Alberti G, Moreno AI, Kratzmann M (1994) The fine struc-

ture of trichobothria in moss mites with special emphasis on

Acrogalumna longipluma (Berlese, 1904) (Oribatida, Acari,

Arachnida). Acta Zoologica 75: 57—74

Alberti G, Moreno AI, Kratzmann M (1995) Fine structure of

trichobothria in moss mites (Oribatida). Pp. 23-30 in: Kro-

pezynska D, Boczek J, Tomezyk A (eds) The Acari: Physio-

logical and Ecological Aspects of Acari-Host Relationships,

Vol. 2, Dabor, Warsaw

Alberti G, Thaler K, Weygoldt P (2007) Chelicerata. Pp. 479-

532 in: Westheide W, Rieger R (eds) Spezielle Zoologie. Teil

1: Einzeller und Wirbellose Tiere, 2nd ed. Spektrum Akade-

mischer Verlag, Mtinchen

Altner H (1977) Insect sensillum specificity and structure: an

approach to a new typology. Olfaction and Taste 6: 295-303

Altner H, Prillinger L (1980) Ultrastructure of invertebrate che-

mo-, thermo-, and hygroreceptors and its functional signifi-

cance. International Review of Cytology 67: 69-139

Altner H, Loftus R (1985) Ultrastructure and function of insect

thermo-and hygroreceptors. Annual Review of Entomology

30: 273-295

Altner H, Sass H, Altner I (1977) Relationship between struc-

ture and function of antennal chemo-, hygro-, and thermore-

ceptive sensilla in Periplaneta americana. Cell and Tissue

Research 176: 389-405

Altner H, Tichy H, Altner I (1978) Lamellated outer dendritic

segments of a sensory cell within a poreless thermo-and hy-

groreceptive sensillum of the insect Carausius morosus. Cell

and Tissue Research 191: 287-304

Altner H, Ernst KD, Kolnberger I, Loftus R (1973) Feinstruk-

tur und adaquater Reiz bei Insektensensillen mit Wandporen.

Verhandlungen der Deutschen Zoologischen Gesellschaft 66:

48-53

Altner H, Schaller-Selzer L, Stetter H, Wohlrab I (1983) Pore-

less sensilla with inflexible sockets. Cell and Tissue Research

234: 279-307

André M (1949) Ordre des Acariens. Pp. 794—892 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6, Masson et Cie., Paris

Anton S, Tichy H (1994) Hygro-and thermoreceptors in tip-

pore sensilla of the tarsal organ of the spider Cupiennius

salei: innervation and central projection. Cell and Tissue Re-

search 278: 399-407

Armas LF de, Teruel R (2002) Un género nuevo de Hubbardii-

dae (Arachnida: Schizomida) de las Antilles Mayores. Revis-

ta Ibérica de Aracnologia 6: 45-52

Babu KS (1965) Anatomy of the central nervous system of

arachnids. Zoologische Jahrbticher. Abteilung fiir Anatomie

und Ontogenie der Tiere 82: 1-154

Babu KS (1985) Patterns of arrangement and connectivity in

the central nervous system of arachnids. Pp. 3-19 in: Barth,

FG. (ed.) Neurobiology of Arachnids. Springer, Berlin / Hei-

delberg

Babu KS, Barth FG (1984) Neuroanatomy of the central ner-

vous system of the wandering spider, Cupiennius salei

(Arachnida, Araneida). Zoomorphology 104: 344-359

Bonn zoological Bulletin Suppl. 65: 1-125

Barranco P, Mayoral JG (2007) A new species of Eukoene-

nia (Palpigradi, Eukoeneniidae) from Morocco. Journal of

Arachnology 35: 318-324

Barranco P, Mayoral JG (2014) New palpigrades (Arachnida,

Eukoenentidae) from the Iberian Peninsula. Zootaxa 3826:

544-562

Barth FG (2002) A spider’s world: senses and behavior. Spring-

er Verlag, Berlin / Heidelberg

Barth FG (2014) The slightest whiff of air: airflow sensing in

arthropods. Pp. 169-196 in: Flow Sensing in Air and Water.

Springer, Berlin / Heidelberg

Barth FG, Németh SS, Friedrich OC (2004) Arthropod touch

reception: structure and mechanics of the basal part of a spi-

der tactile hair. Journal of Comparative Physiology A 190:

523-530

Battelle B-A (2017) Opsins and their expression patterns in the

xiphosuran Limulus polyphemus. The Biological Bulletin

233: 3-20

Becker A, Peters W (1985) Fine structure of the midgut gland

of Phalangium opilio (Chelicerata, Phalangida). Zoomor-

phology 105: 317-325

Beier M (1931) Ordnung Pseusoscorpionidae (Afterscorpione).

Pp.118—192 in: Krumbach T. (ed.) Handbook of Zoology On-

line, Vol. 2. De Gruyter, Berlin / Boston

Bereiter-Hahn J, Matoltsy AG, Richards KS (1984) Biology of

the Integument. 1 Invertebrates. In: Bereiter-Hahn J, Matolt-

sy AG, Richards KS (eds) Springer, Berlin / Heidelberg

Berland L (1949) Ordre des Opilions. Pp. 761—793 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Bitsch J, Bitsch C (2007) The segmental organization of the

head region in Chelicerata: a critical review of recent studies

and hypotheses. Acta Zoologica 88: 317—335

Blick T, Christian E (2004) Checkliste der Tasterlaufer Mit-

teleuropas. Checklist of the palpigrades of Central Europe

(Arachnida: Palpigradi). Version 1. Oktober 2004. Online at

http://www. AraGes.de/checklist.html #2004 Palpigradi [last

accessed 4 May 2016]

Bodmer R (1995) Heart development in Drosophila and its re-

lationship to vertebrates. Trends in Cardiovascular Medicine

5: 21-28

Borner C (1902a) Arachnologische Studien. II. und HI. Zoolo-

gischer Anzeiger 25: 433-466

Borner C (1902b) Arachnologische Studien. IV. Die Genitalor-

gane der Pedipalpen. Zoologischer Anzeiger 26: 81—92

Borner C (1903) Arachnologische Studien. V. Die Mundbil-

dung bei den Milben. Zoologischer Anzeiger 26: 99-109

Borner C (1904) Beitrage zur Morphologie der Arthropoden:

I. Ein Beitrag zur Kenntnis der Pedipalpen. Zoologica 16:

1-174

Boxshall GA (2004) The evolution of arthropod limbs. Biolog-

ical Reviews 79: 253-300

Bremer K (1994) Branch support and tree stability. Cladistics

10: 295-304

Bu Y, Souza MFVR, Ferreira RL (2019) A new locality of

Koeneniodes madecassus Remy, 1950 (Palpigradi: Eu-

koenentidae) in China, with the first complete redescrip-

tion of a Koeneniodes species. Zootaxa 4658 (3): 541-555.

https://do1.org/10.11646/zootaxa.4658.3.6

Bursey CR (1973) Microanatomy of the ventral cord ganglia of

the horseshoe crab, Limulus polyphemus (L.). Zeitschrift fir

Zellforschung und Mikroskopische Anatomie 137: 313-329

Bursey CR, Sherman RG (1970) Spider cardiac physiology I.

Structure and function of the cardiac ganglion. Comparative

and General Pharmacology 1: 160-170

©ZFMK

96 Sandra Franz-Guess & J. Matthias Starck

Bush JWM, Hu DL, Prakash M (2007) The Integument of wa-

ter-walking arthropods: form and function. Pp. 117—192 in:

Casas J, Simpson SJ (eds) Advances in Insect Physiology,

Vol. 34. Elsevier, Amsterdam, Netherlands

Buxton BH (1913) Coxal glands of the arachnids. Pp. 230-454

in: Spengel JW (ed.) Zoologische Jahrbticher, Supplement

14. Gustav Fischer Verlag, Jena

Buxton BH (1917) Notes on the anatomy of arachnids. Journal

of Morphology 29: 1-31

Christian E (2004) Palpigraden (Tasterlaufer-)Spinnentiere in

einer Welt ohne Licht. Pp. 473-483 in: Biologiezentrum des

Oberosterreichischen Landesmuseums (ed.) Diversitat und

Biologie von Webspinnen, Skorpionen und anderen Spinnen-

tieren, Neue Serie 14, Denisia 12

Christian E (2014) A new Eukoenenia species from the Cau-

casus bridges a gap in the known distribution of palpigrades

(Arachnida: Palpigradi). Biologia 69: 1701-1706

Christian E, Isaia M, Paschetta M, Bruckner A (2014) Differ-

entiation among cave populations of the Eukoenenia spelaea

species-complex (Arachnida: Palpigradi) in the southwestern

Alps. Zootaxa 3794: 52-86

Chu-Wang I-W, Axtell RC (1973) Comparative fine structure of

the claw sensilla of a soft tick, Argas (Persicargas) arboreus

Kaiser, Hoogstraal, and Kohls, and a hard tick, Amblyomma

americanum (L.). The Journal of Parasitology 59: 545—555

Collatz K-G (1987) Structure and function of the digestive

tract. Pp. 229-238 in: Nentwig W (ed.) Ecophysiology of

Spiders. Springer, Berlin / Heidelberg

Condé B (1996) Les Palpigrades, 1885-1995: acquisitions et

lacunes. Proceedings of the XIIIth Congress of Arachnolo-

gy : Geneva, 3—8 September 1995 (1): 87-106

Conte FP (1984) Structure and function of the crustacean larval

salt gland. International Review of Cytology 91: 45-106

Coons LB, Alberti G (1999) Acari: Ticks. Pp. 267-514 in: Har-

rison FW, Foelix RF (eds) Microscopic Anatomy of Inverte-

brates, Vol. 8B. Wiley-Liss, New York

Corbiere-Tichané G, Loftus R (1983) Antennal thermal recep-

tors of the cave beetle, Speophyes lucidulus Delar. Journal of

Comparative Physiology A 153: 343-351

Crome W (1953) Die Respirations-und Circulationsorgane der

Argyroneta aquatica Cl. (Araneae). Wissenschaftliche Zeit-

schrift Humboldt-Universitat Berlin 2: 53—83

Curtis DJ, Machado G (2007) Ecology. Pp. 280-308 in: Pin-

to-da-Rocha R, Machado G, Giribet G (eds), Harvestmen:

The Biology of Opiliones. Harvard University Press, London

/ Cambridge

Cushing PE, Casto P, Knowlton ED, Royer S, Laudier D, Gaffin

DD, et al. (2014) Comparative morphology and functional

significance of setae called papillae on the pedipalps of male

camel spiders (Arachnida: Solifugae). Annals of the Entomo-

logical Society of America 107: 510-520

Damen WG (2002) Parasegmental organization of the spider

embryo implies that the parasegment is an evolutionary con-

served entity in arthropod embryogenesis. Development 129:

1239-1250

Damen WG (2007) Evolutionary conservation and divergence

of the segmentation process in arthropods. Developmental

dynamics: an official publication of the American Associa-

tion of Anatomists 236: 1379-1391

Damen WG, Hausdorf M, Seyfarth, E-A, Tautz D. (1998) A

conserved mode of head segmentation in arthropods revealed

by the expression pattern of Hox genes in a spider. Proceed-

ings of the National Academy of Sciences 95: 10665-10670

Bonn zoological Bulletin Suppl. 65: 1-125

Davis EE, Sokolove PG (1975) Temperature responses of an-

tennal receptors of the mosquito, Aedes aegypti. Journal of

Comparative Physiology 96: 223-236.

Dechant H-E, H68l B, Rammerstorfer FG, Barth FG (2006)

Arthropod mechanoreceptive hairs: modeling the direction-

ality of the joint. Journal of Comparative Physiology A 192:

1271-1278

Deutsch JS (2004) Segments and parasegments in arthropods: a

functional perspective. BioEssays 26: 1117-1125

Doeffinger C, Hartenstein V, Stollewerk A (2010) Compart-

mentalization of the precheliceral neuroectoderm in the spi-

der Cupiennius salei: development of the arcuate body, optic

ganglia, and mushroom body. Journal of Comparative Neu-

rology 518: 2612—2632

Dubale MS, Vyas AB (1968) The structure of the chela of

Heterometrus sp. and its mode of operation. Bulletin of the

Southern California Academy of Sciences 67: 240-244

Dunlop JA (1994) Filtration mechanisms in the mouthparts of

tetrapulmonate arachnids (Trigonotarbida, Araneae, Ambly-

pygi, Uropygi, Schizomida). Bulletin of the British arachno-

logical Society 9: 73

Dunlop JA (2000) The epistomo-labral plate and lateral lips in

solifuges, pseudoscorpions and mites. Ekologia (Bratislava)

19: 67-78

Dunlop JA 2019. Miniaturisation in Chelicerata. Arthropod

Structure & Development: 20-34

Dunlop JA, Arango CP (2005) Pycnogonid affinities: a review.

Journal of Zoological Systematics and Evolutionary Re-

search 43: 8—21

Dunlop JA, Alberti G (2008) The affinities of mites and ticks: a

review. Journal of Zoological Systematics and Evolutionary

Research 46: 1-18

Dunlop JA, Lamsdell JC (2017) Segmentation and tagmosis in

Chelicerata. Arthropod Structure & Development 46: 395-

418

Eberhard WG, Wcislo WT (2011) Grade changes in brain-body

allometry: morphological and behavioural correlates of brain

size In miniature spiders, insects and other invertebrates. Ad-

vances in Insect Physiology 40: 155

Eberhard WG, Wcislo WT (2012) Plenty of room at the bot-

tom? Tiny animals solve problems of housing and maintain-

ing oversized brains, shedding new light on nervous-system

evolution. American Scientist 100: 226—233

Ehn R, Tichy, H (1994) Hygro-and thermoreceptive tarsal or-

gan in the spider Cupiennius salei. Journal of Comparative

Physiology A 174: 345-350

Eisenbeis G (1974) Licht-und elektronenmikroskopische Un-

tersuchungen zur Ultrastruktur des Transportepithels am

Ventraltubus arthropleoner Collembolen (Insecta). Cytobiol-

ogie 9: 180-202

Eisenbeis G, Wichard W (1987) Atlas on the biology of soil

arthropods. Springer Science & Business Media, Berlin /

Heidelberg

Engel MS, Breitkreuz LC, Cai C, Alvarado M, Azar D, Huang

D (2016) The first Mesozoic microwhip scorpion (Palpigra-

di): a new genus and species in mid-Cretaceous amber from

Myanmar. The Science of Nature 103: 1—7

Fage L (1949) Classe des Mérostomacés. Pp. 219-262 in:

Grassé P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie.,

Paris

Fahrenbach WH (1999) Merostomata. Pp. 21-115 in: Harrison

FW, Foelix RF (eds) Microscopic Anatomy of Invertebrates,

Vol. 8A. Wiley-Liss, New York

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 97

Farley RD (1999) Scorpiones. In: Harrison FW, Foelix, RF

(eds), Microscopic Anatomy of Invertebrates, Vol. 8A. Wi-

ley-Liss, New York, Pp. 117-222

Farley RD (2001) Development of segments and appendages

in embryos of the desert scorpion Paruroctonus mesaensis

(Scorpiones: Vaejovidae). Journal of Morphology 250: 70-88

Farley RD (2005) Developmental changes in the embryo,

pronymph, and first molt of the scorpion Centruroides vit-

tatus (Scorpiones: Buthidae). Journal of Morphology 265:

1-27

Felgenhauer BE (1999) Araneae. Pp. 223—266 in: Harrison FW,

Foelix RF (eds) Microscopic Anatomy of Invertebrates, Vol.

8A. Wiley-Liss, New York

Felsenstein J (1985) Confidence limits on phylogenies: an ap-

proach using the bootstrap. Evolution 39: 783-791

Ferreira RL, Souza MF VR (2012) Notes on the behavior of the

advances troglobite Eukoenenia maquinensis, Souza & Fer-

reira 2010 (Palpigradi: Eukoenentidae) and its conservation

status. Speleobiology Notes 2012 4: 17-23

Ferreira RL, Souza MFVR, Machado EO, Brescovit AD (2011)

Description of a new Eukoenenia (Palpigradi: Eukoenenti-

dae) and Metagonia (Araneae: Pholcidae) from Brazilian

caves, with notes on their ecological interactions. Journal of

Arachnology 39: 409-419

Filimonova SA (2004) The fine structure of the coxal glands in

Myobia murismusculi (Schrank) (Acari: Myobiidae). Arthro-

pod Structure & Development 33: 149-160

Filimonova SA (2016) Morpho-functional variety of the coxal

glands in cheyletoid mites (Prostigmata). I. Syringophilidae.

Arthropod Structure & Development 45: 356-367

Filimonova SA (2017) Morpho-functional variety of the coxal

glands in cheyletoid mites (Prostigmata). II. Cheyletidae. Ar-

thropod Structure & Development 46: 777—787

Firstman B (1973) The relationship of the chelicerate arterial

system to the evolution of the endosternite. Journal of Arach-

nology 1: 1-54

Foelix R (2010) Biology of spiders. 3rd ed. Oxford University

Press, New York

Foelix R, Hebets E (2001) Sensory biology of whip spiders

(Arachnida, Amblypygi). Eileen Hebets Publications 32

Foelix RF (1985) Mechano- and chemoreceptice sensilla. Pp.

118-137 in: Barth FG (ed.) Neurobiology of Arachnids.

Springer, Berlin / Heidelberg

Foelix RF, Axtell RC (1972) Ultrastructure of Haller’s organ in

the tick Amblyomma americanum (L.). Cell and Tissue Re-

search 124: 275-292

Foelix RF, Chu-Wang I-W (1972) Fine structural analysis of

palpal receptors in the tick Amblyomma americanum (L.).

Zeitschrift fiir Zellforschung und Mikroskopische Anatomie

129: 548-560

Foelix RF, Chu-Wang I-W (1973) The morphology of spider

sensilla II. Chemoreceptors. Tissue and Cell 5: 461-478

Foelix RF, Schabronath J (1983) The fine structure of scorpi-

on sensory organs. I. Tarsal sensilla. Bulletin of the British

Arachnological Society 6: 53-67

Foelix RF, Chu-Wang I-W, Beck L (1975) Fine structure of tar-

sal sensory organs in the whip spider Admetus pumilio (Am-

blypygi, Arachnida). Tissue and Cell 7: 331-346

Fusco G, Minelli A (2013) Arthropod segmentation and tagmo-

sis. Pp. 197-223 in: Minelli A, Boxshall G, Fusco G (eds)

Arthropod Biology and Evolution: Molecules, Development,

Morphology. Springer, Berlin / Heidelberg

Gaffal KP, Tichy H, TheiB J, Seelinger G (1975) Structural po-

larities in mechanosensitive sensilla and their influence on

Bonn zoological Bulletin Suppl. 65: 1-125

stimulus transmission (Arthropoda). Zoomorphology 82:

79-103

Gainett G, Michalik P, Miller CH, Giribet G, Talarico G,

Willemart RH (2017) Putative thermo-/hygroreceptive tar-

sal sensilla on the sensory legs of an armored harvestman

(Arachnida, Opiliones). Zoologischer Anzeiger 270: 81-97

Garwood RJ, Dunlop J (2014) Three-dimensional reconstruc-

tion and the phylogeny of extinct chelicerate orders. PeerJ 2:

e641. https://doi.org/10.7717/peer).641

Gerberding M, Scholtz G (2001) Neurons and glia in the mid-

line of the higher crustacean Orchestia cavimana are gen-

erated via an invariant cell lineage that comprises a median

neuroblast and glial progenitors. Developmental Biology

235: 397-409

Gerhardt U (1931) Ordnung Xiphosura/Poecilopoda

(Schwertschwanze). Pp. 47—96 in: Krumbach T (ed.) Hand-

book of Zoology Online, Vol. 1. De Gruyter, Berlin / Boston

Gerhardt U, Kastner A (1931) Ordnung Araneae (Echte Spin-

nen oder Webspinnen). Pp. 394-656 in: Krumbach T (ed.)

Handbook of Zoology Online, Vol. 2. De Gruyter, Berlin

Boston

Giribet G, Edgecombe GD, Wheeler WC, Babbitt C (2002)

Phylogeny and systematic position of opiliones: A combined

analysis of chelicerate relationships using morphological and

molecular data. Cladistics 18: 5—70

Giribet G, McIntyre E, Christian E, Espinasa L, Ferreira RL,

Francke OF, et al. (2014) The first phylogenetic analysis of

Palpigradi (Arachnida) — the most enigmatic arthropod order.

Invertebrate Systematics 28: 350-360

Goloboff PA (1993) Estimating character weights during tree

search. Cladistics 9: 83-91

Goloboff PA, Catalano SA (2016) TNT version 1.5, including a

full implementation of phylogenetic morphometrics. Cladis-

tics 32: 221-238

Goloboff PA, Farris JS, Nixon KC (2008) TNT, a free program

for phylogenetic analysis. Cladistics 24: 774-786

Gopel T, Wirkner CS (2015) An “ancient” complexity? Evolu-

tionary morphology of the circulatory system in Xiphosura.

Zoology 118: 221-238

Gorb S (2001a) Principles of cuticular attachment in Arthropo-

da. Pp. 37—76 in: Attachment Devices of Insect Cuticle. Klu-

wer Academic Publishers, Dordrecht

Gorb S (2001b) Cuticular protuberances of insects. Pp. 21-36

in: Attachment Devices of Insect Cuticle. Kluwer Academic

Publishers, Dordrecht

Gorner P (1965) A proposed transducing mechanism for a mul-

tiply-innervated mechanoreceptor (trichobothrium) in spi-

ders. Cold Spring Harbor Symposia on Quantitative Biology

30: 69-73

Gottlieb K (1926) Uber das Gehirn des Skorpions. Zeitschrift

fiir wissenschaftliche Zoologie 127: 195—243

Gould SJ (1977) Heterochrony and the parallel of ontogeny and

phylogeny. Pp. 209-266 in: Ontogeny and Phylogeny. Belk-

nap Press of Harvard University Press, Cambridge

Graham JB (1988) Ecological and evolutionary aspects of in-

tegumentary respiration: body size, diffusion, and the Inver-

tebrata. American Zoologist 28: 1031-1045

Grams M, Wirkner CS, Runge J (2018) Serial and special: com-

parison of podomeres and muscles in tactile vs walking legs

of whip scorpions (Arachnida, Uropygi). Zoologischer An-

zeiger 273: 75-101

Hackman RH (1984) VIII Arthropoda — Cuticle: Biochemistry.

Pp. 583-610 in: Bereiter-Hahn J, Matoltsy AG, Richards KS

(eds) Biology of the Integument. 1 Invertebrates. Springer,

Berlin / Heidelberg

©ZFMK

98 Sandra Franz-Guess & J. Matthias Starck

Hammen L van der (1966) Studies on Opilioacarida (Arach-

nida) I. Description of Opilioacarus texanus (Chamberlin &

Mulaik) and revised classification of the genera. Zoologische

Verhandelingen 86: 1-80

Hammen L van der (1967) The gnathosoma of Hermannia

convexa (C. L. Koch) (Acarida: Oribatina) and comparative

remarks on its morphology in other mites. Zoologische Ver-

handelingen 94: 3-45

Hammen L van der (1977a) A new classification of Chelicerata.

Zoologische Mededelingen 51: 307-319

Hammen L van der (1977b) The evolution of the coxa in mites

and other groups of Chelicerata. Acarologia 19: 12-19

Hammen L van der (1982) Comparative studies in Chelicerata

II. Epimerata (Palpigradi and Actinotrichida). Zoologische

Verhandelingen 196: 3—70

Hammen L van der (1986) Comparative studies in Chelicerata

IV. Apatellata, Arachnida, Scorpionida, Xiphosura. Zoolo-

gische Verhandelingen 226: 4—52

Hammen L van der (1989) An introduction to comparative

arachnology. SPB Academic Publishing, The Hague

Handlirsch A (1926) Arthropoda: Allgemeine Einleitung in die

Naturgeschichte der Gliederfiisser. Pp. 211-276 in: Krum-

bach T (ed.) Handbook of Zoology Online. De Gruyter, Ber-

lin / Boston

Hanken J, Wake DB (1993) Miniaturization of body size: or-

ganismal consequences and evolutionary significance. Annu-

al Review of Ecology and Systematics 24: 501-519

Hansen HJ (1901) On six species of Koenenia, with remarks on

the order Palpigradi. Entomologisk Tidskrift: 193-240

Hansen HJ, Sorensen W (1897) The order Palpigradi Thorell

(Koenenia mirabilis, Grassi) and its relationships to other

Arachnida. Entomologisk Tidskrift: 223—240

Hansen HJ, Sorensen WE (1905) The Tartarides: a tribe of the

order Pedipalpi. Arkiv for Zoologi 2: 1-85

Hanstrom B (1928) Vergleichende Anatomie des Nervensy-

stems der wirbellosen Tiere. Springer, Berlin / Heidelberg

Harris DJ, Mill PJ (1973) The ultrastructure of chemoreceptor

sensilla in Ciniflo (Araneida, Arachnida). Tissue and Cell 5:

679-689

Harris DJ, Mill PJ (1977) Observations on the leg receptors

of Ciniflo (Araneida: Dictynidae). Journal of Comparative

Physiology 119: 37-54

Harvey MS (2002) The neglected cousins: what do we know

about the smaller arachnid orders? Journal of Arachnology

30: 357-372

Harvey MS (2003) Catalogue of the smaller arachnid orders

of the world: Amblypygi, Uropygi, Schizomida, Palpigradi,

Ricinulei and Solifugae. CSIRO Publishing, Clayton South

Harzsch S (2003) Ontogeny of the ventral nerve cord in mala-

costracan crustaceans: a common plan for neuronal develop-

ment in Crustacea, Hexapoda and other Arthropoda? Arthro-

pod Structure & Development 32: 17—37

Harzsch S, Wildt M, Battelle B, Waloszek D (2005) Immu-

nohistochemical localization of neurotransmitters in the

nervous system of larval Limulus polyphemus (Chelicerata,

Xiphosura): evidence for a conserved protocerebral architec-

ture in Euarthropoda. Arthropod Structure & Development

34: 327-342

Haupt J (1982) Hair regeneration in a solfugid chemotactile

sensillum during moulting (Arachnida: Solifugae). Wilhelm

Roux’s Archives of Developmental Biology 191: 137—142

Hess E, Vlimant M (1982) The tarsal sensory system of Ambly-

omma variegatum Fabricius (Ixodidae, Metastriata). I. Wall

pore and terminal pore sensilla. Revue Suisse de Zoologie

89: 713-729

Bonn zoological Bulletin Suppl. 65: 1-125

Hess E, Loftus R (1984) Warm and cold receptors of two sen-

silla on the foreleg tarsi of the tropical bont tick Amblyom-

ma variegatum. Journal of Comparative Physiology A 155:

187-195

Hoffmann C (1967) Bau und Funktion der Trichobothrien von

Euscorpius carpathicus L. Zeitschrift ftir vergleichende

Physiologie 54: 290-352

Hootman SR, Conte FP (1975) Functional morphology of the

neck organ in Artemia salina nauplii. Journal of Morphology

145: 371-385

Horn ACM, Achaval M (2002) The gross anatomy of the ner-

vous system of Bothriurus bonariensis (L. C. Koch, 1842)

(Scorpiones, Bothriuridae). Brazilian Journal of Biology 62:

253-262

Ioffe ID (1963) Structure of the brain of Dermacentor pictus

Herm. (Chelicerata, Acarina). Zoologicheskiit Zhurnal 42:

1472-1484

Kaneko N (1988) Feeding habits and cheliceral size of ori-

batid mites in cool temperate forest soils in Japan. Revue

d’Ecologie et de Biologie du Sol 25: 353-363

Kastner A (1931a) Ordnung Palpigradi Thorell. Pp. 77-98 in:

Krumbach T (ed.) Handbook of Zoology Online, Vol. 2. De

Gruyter, Berlin / Boston

Kastner A (1931b) Ordnung Scorpiones (Schwertschwanze).

Pp. 117-240 in: Krumbach T (ed.) Handbook of Zoology

Online, Vol. 1. De Gruyter, Berlin / Boston

Kastner A (1931c) Ordnung Ricinulei. Pp. 99-116 in: Krum-

bach T (ed.) Handbook of Zoology Online, Vol. 2. De Gruy-

ter, Berlin / Boston

Kastner A (1931d) Ordnung Opiliones Sundevall (We-

berknechte). Pp. 300-393 in: Krumbach T (ed.) Handbook of

Zoology Online, Vol. 2. De Gruyter, Berlin / Boston

Kastner A (193le) Ordnung Solifugae (Walzenspinnen). Pp.

193-299 in: Krumbach T (ed.) Handbook of Zoology Online,

Vol. 2. De Gruyter, Berlin / Boston

K4stner A (1931f) Ordnung Pedipalpi Latreille (Geifel-Scorpi-

one). Pp. 1-76 in: Krumbach T (ed.) Handbook of Zoology

Online, Vol. 2. De Gruyter, Berlin / Boston

Kastner A (1931g) Die Hitifte und ihre Umformung zu Mund-

werkzeugen bei den Arachniden. Zeitschrift fiir Morphologie

und Okologie der Tiere 22: 721-758

Keil TA (1997) Functional morphology of insect mechanore-

ceptors. Microscopy Research and Technique 39: 506—531

Kimble M, Coursey Y, Ahmad N, Hinsch GW (2002) Behavior

of the yolk nuclei during embryogenesis, and development of

the midgut diverticulum in the horseshoe crab Limulus poly-

phemus. Invertebrate Biology 121: 365-377

Klann AE (2009) Histology and ultrastructure of solifuges.

Ernst-Moritz-Arndt-Universitat, Greifswald

Klann AE, Alberti G (2010) Histological and ultrastructural

characterization of the alimentary system of solifuges (Arach-

nida, Solifugae). Journal of Morphology 271: 225-243

Klu8mann-Fricke B-J, Wirkner CS (2016) Comparative mor-

phology of the hemolymph vascular system in Uropygi and

Amblypygi (Arachnida): complex correspondences support

Arachnopulmonata. Journal of Morphology 277: 1084-1103

KluBmann-Fricke B-J, Prendini L, Wirkner CS (2012) Evolu-

tionary morphology of the hemolymph vascular system in

scorpions: a character analysis. Arthropod Structure & De-

velopment 41: 545-560

Kovac D, Maschwitz U (2000) Protection of hydrofuge respi-

ratory structures against detrimental microbiotic growth by

terrestrial grooming in water beetles (Coleoptera: Hydrop-

bilidae, Hydraeoidae, Dryopidae, Ebnidae, Curculiooidae).

Entomologia Generalis: 277-292

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 99

Kovaé L, Mock A, Luptacik P, Palacios- Vargas JG (2002) Dis-

tribution of Eukoenenia spelaea (Peyerimhoff, 1902) (Arach-

nida, Palpigradida) in the Western Carpathians with remarks

on its biology and behaviour. Pp. 93-99 in: Tajovsky, K,

Balik, V, Pizl, V. (eds) Studies on Soil Fauna in Central Eu-

rope. Ceské Budéjovice: Institute of Soil Biology AS CR

Kovaé L, Elhottova D, Mock A, Novakova A, Kristtfek V,

Chronakova A, et al. (2014) The cave biota of Slovakia. State

Nature Conservancy of the Slovak Republic, Slovak Caves

Administration, Liptovsky Mikulas: 1-192

Kraus O (1976) Zur phylogenetischen Stellung und Evolution

der Chelicerata. Entomologica Germanica: 1-12

Lamsdell JC (2013) Revised systematics of Palaeozoic ‘horse-

shoe crabs’ and the myth of monophyletic Xiphosura:

re-evaluating the monophyly of Xiphosura. Zoological Jour-

nal of the Linnean Society 167: 1-27

Lauterbach KE (1973) Schliisselereignisse in der Evolution der

Stammeruppe der Euarthropoda. Zoologische Beitrage 19:

251-299

Legg G (1976) The external morphology of a new species of

ricinuleid (Arachnida) from Sierra Leone. Zoological Journal

of the Linnean Society 59: 1-58

Lehmacher C, Abeln B, Paululat A (2012) The ultrastructure of

Drosophila heart cells. Arthropod Structure & Development

41: 459-474

Lehmann T, HeB M, Melzer RR (2012) Wiring a periscope—

ocelli, retinula axons, visual neuropils and the ancestrality

of sea spiders. PLoS One 7: e30474. https://doi.org/10.1371/

journal.pone.0030474

Levi HW (1967) Adapations of respiratory systems of spiders.

Evolution 21: 571-583

Loesel R, Wolf H, Kenning M, Harzsch S, Sombke A (2013)

Architectural principles and evolution of the arthropod cen-

tral nervous system. Pp. 300-342 in: Minelli A, Boxshall G,

Fusco G (eds) Arthropod Biology and Evolution: Molecules,

Development, Morphology. Springer, Berlin / Heidelberg

Loftus R (1976) Temperature-dependent dry receptor on anten-

na of Periplaneta. Tonic response. Journal of Comparative

Physiology 111: 153-170

Loftus R, Corbiere-Tichané G (1981) Antennal warm and cold

receptors of the cave beetle, Speophyes lucidulus Delar, in

sensilla with a lamellated dendrite. Journal of Comparative

Physiology A 143: 443-452

Lopez A (1983) Coxal glands of the genus Metepeira (Araneae,

Araneidae). The Journal of Arachnology 11: 97-99

Lowy RJ, Conte FP (1985) Morphology of isolated crustacean

larval salt glands. American Journal of Physiology-Regulato-

ry, Integrative and Comparative Physiology 248: R709-R716

Ludwig M, Alberti G (1992) Fine structure of the midgut of

Prokoenenia wheeleri (Arachnida: Palpigradi). Zoologische

Beitrage 34: 127-134

Ludwig M, Palacios-Vargas JG, Albertif G (1994) Cellular de-

tails of the midgut of Cryptocellus boneti (Arachnida: Rici-

nulei). Journal of Morphology 220: 263-270

Mares S, Ash L, Gronenberg W (2005) Brain allometry in bum-

blebee and honey bee workers. Brain, behavior and evolution

66: 50-61

McIver SB (1975) Structure of cuticular mechanoreceptors of

arthropods. Annual Review of Entomology 20: 381-397

McNamara KJ (1986) A guide to the nomenclature of heteroch-

rony. Journal of Paleontology: 4-13

Mehnert L, Dietze Y, Hornig MK, Starck JM (2018) Body tag-

matization in pseudoscorpions. Zoologischer Anzeiger 273:

152-163

Bonn zoological Bulletin Suppl. 65: 1-125

Meijden A van der, Langer F, Boistel R, Vagovic P, Heethoff

M (2012) Functional morphology and bite performance of

raptorial chelicerae of camel spiders (Solifugae). The Journal

of Experimental Biology 215: 3411-3418

Messlinger K (1987) Fine structure of scorpion trichobothria

(Arachnida, Scorpiones). Zoomorphology 107: 49-57

Michalik P (2009) The male genital system of spiders (Arach-

nida, Araneae) with notes on the fine structure of seminal se-

cretions. Contributions to Natural History 12: 959-972

Michalik P, Reiher W, Tintelnot-Suhm M, Coyle FA, Alberti G

(2005) Female genital system of the folding-trapdoor spider

Antrodiaetus unicolor (Hentz, 1842) (Antrodiaetidae, Arane-

ae): ultrastructural study of form and function with notes on

reproductive biology of spiders. Journal of Morphology 263:

284-309

Millot J (1942) Sur Panatomie et l’histophysiologie de Koene-

nia mirabilis, Grassi (Arachnida, Palpigradi). Revue Fran-

cais d’Entomologie Paris 9: 33-51

Millot J (1943) Notes complémentaires sur l’anatomie, |’ his-

tologie et la répartition géographique en France de Koene-

nia mirabilis, Grassi (Arachnida Palpigradi). Revue Francais

d’Entomologie Paris 9: 127-135

Millot J (1949a) Ordre des Palpigrades. Pp. 520-532 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Millot, J. 1949b. Classe des Arachnides. Pp. 263-385 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Millot, J. 1949c. Ordre des Uropyges. Pp. 533-562 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Millot, J. 1949d. Ordre des Amblypyges. Pp. 563-588 in:

Grassé P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie.,

Paris

Millot, J. 1949e. Ordre des Aranéides. Pp. 589-743 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Millot, J. 1949f. Ordre des Ricinuléides. Pp. 744—760 in: Grassé

P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie., Paris

Millot, J, Vachon, M. 1949a. Ordre des Solifuges. Pp. 482-519

in: Grassé P-P (ed.) Traité de Zoologie, Vol. 6. Masson et

Cie., Paris

Millot, J, Vachon, M. 1949b. Ordre des Scorpions. Pp. 386—436

in: Grassé P-P (ed.) Traité de Zoologie, Vol. 6. Masson et

Cie., Paris

Mittmann B, Scholtz G (2003) Development of the nervous

system in the “head” of Limulus polyphemus (Chelicerata:

Xiphosura): morphological evidence for a correspondence

between the segments of the chelicerae and of the (first) an-

tennae of Mandibulata. Development Genes and Evolution

213: 9-17

Molina MR, Cripps RM (2001) Ostia, the inflow tracts of the

Drosophila heart, develop from a genetically distinct subset

of cardial cells. Mechanisms of Development 109: 51-59

Moritz M (1993) 1. Unterstamm Arachnata. Pp. 64-447 in:

Gruner H-E (ed.) Lehrbuch Der Speziellen Zoologie. Band I:

Wirbellose Tiere. 4. Teil: Arthropoda (Ohne Insecta), 4th ed.

Gustav Fischer Verlag, Jena

Neville AC (1984) Cuticle: organization. Pp. 611-625 in: Bere-

iter-Hahn J, Matoltsy AG, Richards KS (eds) Biology of the

Integument. Springer, Berlin / Heidelberg

Noble-Nesbitt J (1963) Transpiration in Podura aquatica L.

(Collembola, Isotomidae) and the wetting properties of its

cuticle. Journal of Experimental Biology 40: 681—700

Noirot C, Quennedey A (1974) Fine structure of insect epider-

mal glands. Annual Review of Entomology 19: 61—80

Obenchain FD, Oliver JH Jr (1975) The heart and arterial circu-

latory system of ticks (Acari: Ixodoidea). Journal of Arach-

nology: 57—74

©ZFMK

100 Sandra Franz-Guess & J. Matthias Starck

Ortega-Hernandez J, Janssen R, Budd GE (2017) Origin and

evolution of the panarthropod head — A palaeobiological

and developmental perspective. Arthropod Structure and De-

velopment 46: 354-379

Ozaki M, Tominaga Y (1999) Contact chemoreceptors. Pp.

143-154 in: Eguchi E, Tominaga Y (eds) Atlas of Arthropod

Sensory Receptors. Springer, Berlin / Heidelberg

Peel A (2004) The evolution of arthropod segmentation mecha-

nisms. BioEssays 26: 1108-1116

Peel AD, Chipman AD, Akam M (2005) Arthropod segmenta-

tion: beyond the Drosophila paradigm. Nature Reviews Ge-

netics 6: 905

Pepato AR, da Rocha CE, Dunlop JA (2010) Phlyogenetic po-

sition of the acariform mites: sensitivity to homology assess-

ment under total evidence. BMC Evolutionary Biology 10:

235. http://www.biomedcentral.com/1471-2148/10/235

Peters SE (1983) Postnatal development of gait behaviour and

functional allometry in the domestic cat (Felis catus). Journal

of Zoology 199: 461-486

Pinto-da-Rocha R, Machado G, Giribet G (2007) Harvestmen.

The Biology of Opiliones. Harvard University Press, Cam-

bridge / London

Pinto-da-Rocha R, Andrade R, Moreno-Gonzalez JA (2016)

Two new cave-dwelling genera of short-tailed whip-scor-

pions from Brazil (Arachnida: Schizomida: Hubbardiidae).

Zoologia (Curitiba) 33

Pitnick S, Hosken DJ, Birkhead TR (2009) Sperm morphologi-

cal diversity. Pp. 69-149 in: Sperm Biology — An Evolution-

ary Perspective. Elsevier, Oxford

Polilov AA (2008) Anatomy of the smallest Coleoptera, feath-

erwing beetles of the tribe Nanosellini (Coleoptera, Ptili-

idae), and limits of insect miniaturization. Entomological

Review 88: 26-33

Polilov AA (2015a) Consequences of miniaturization in insect

morphology. Moscow University Biological Sciences Bulle-

tin 70: 136-142

Polilov AA (2015b) Small is beautiful: features of the smallest

insects and limits to miniaturization. Annual Review of En-

tomology 60: 103-121

Polilov AA (2016) At the size limit — Effects of miniaturization

in insects. Springer International Publishing Switzerland,

Cham. https://doi.org/10.1007/978-3-319-39499-2

Polilov AA, Shmakov AS (2016) The anatomy of the thrips He-

liothrips haemorrhoidalis (Thysanoptera, Thripidae) and its

specific features caused by miniaturization. Arthropod Struc-

ture & Development 45: 496-507

Polilov AA, Makarova AA (2017) The scaling and allometry of

organ size associated with miniaturization in insects: A case

study for Coleoptera and Hymenoptera. Scientific reports 7:

43095

Polis GA (1990) The biology of scorpions. Stanford University

Press, Stanford

Polis GA, Farley RD (1979) Behavior and ecology of mating in

the cannibalistic scorpion, Paruroctonus mesaensis Stahnke

(Scorpionida: Vaejovidae). Journal of Arachnology: 33-46

Pound JM, Oliver JH Jr (1982) Synganglial and neurosecre-

tory morphology of female Ornithodoros parkeri (Cooley)

(Acari: Argasidae). Journal of Morphology 173: 159-177

Punzo F (2012) The Biology of Camel-Spiders: Arachnida, So-

lifugae. Springer Science & Business Media, Berlin / Hei-

delberg

Quesada R, Triana E, Vargas G, Douglass JK, Seid MA, Niven

JE, et al. (2011) The allometry of CNS size and consequenc-

es of miniaturization in orb-weaving and cleptoparasitic spi-

ders. Arthropod Structure & Development 40: 521-529

Bonn zoological Bulletin Suppl. 65: 1-125

Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R,

et al. (2010) Arthropod relationships revealed by phyloge-

nomic analysis of nuclear protein-coding sequences. Nature

463: 1079-1083

ReiBland A, Gorner P (1985) Trichobothria. Pp. 138-161 in:

Barth FG (ed.) Neurobiology of Arachnids, Springer, Berlin

/ Heidelberg

Reynolds ES (1963) The use of lead citrate at high pH as an

electron-opaque stain in electron microscopy. The Journal of

Cell Biology 17: 208-212

Roewer CF (1934) 4. Buch: Solifugae, Palpigradi. Pp. 1-723

in: Bronn HG (ed.) Dr. H.G. Bronns Klassen und Ordnun-

gen des Tierreichs, Vol. 5. Akademische Verlagsgesellschaft,

Leipzig

Rucker A (1901) The Texan Koenenia. The American Naturalist

35: 615-630

Rucker A (1903) A new Koenenia from Texas. Quarterly Jour-

nal of Microscopical Science: 215—231

Rtideberg C (1967) A rapid method for staining thin sections of

vestopal W-embedded tissue for light microscopy. Experien-

tia 23: 792-792

Rugendorff A, Younossi-Hartenstein A, Hartenstein V (1994)

Embryonic origin and differentiation of the Drosophila heart.

Wilhelm Roux’s Archives of Developmental Biology 203:

266-280

Scholl G (1977) Beitrage zur Embryonalentwicklung von Lim-

ulus polyphemus L. (Chelicerata, Xiphosura). Zoomorpholo-

gie 86: 99-154

Scholtz G (1998) Cleavage, germ band formation and head

segmentation: the ground pattern of the Euarthropoda. Pp.

317-332 in: Fortey RA, Thomas RH (eds) Arthropod Rela-

tionships. The Systematics Association Special Volume Se-

ries, vol 55. Springer, Dordrecht

Scholtz G, Edgecombe GD (2006) The evolution of arthropod

heads: reconciling morphological, developmental and pa-

laeontological evidence. Development Genes and Evolution

216: 395-415

Schriefer ME, Beveridge M, Sonenshine DE, Homsher PJ, Car-

son KA, Weidman CS (1987) Evidence of ecdysteroid pro-

duction by tick (Acari: Ixodidae) fat-body tissues in vitro.

Journal of Medical Entomology 24: 295-302

Seid MA, Castillo A, Wcislo WT (2011) The allometry of brain

miniaturization in ants. Brain, behavior and evolution 77:

5-13

Sharma PP (2018) Chelicerates. Current Biology 28: R774—

R778

Sharma PP, Kaluziak ST, Pérez-Porro AR, Gonzalez VL, Hor-

miga G, Wheeler WC, et al. (2014) Phylogenomic interroga-

tion of Arachnida reveals systemic conflicts in phylogenetic

signal. Molecular biology and evolution: msu235

Shear WA (1999) Introduction to Arthropoda and Cheliceri-

formes. Pp. 1-19 in: Harrison FW, Foelix RF (eds) Micro-

scopic Anatomy of Invertebrates, Vol. 8A. Wiley-Liss, New

York

Sherman RG, Bursey CR, Fourtner CR, Pax RA (1969) Cardi-

ac ganglia in spiders (Arachnida: Araneae). Experientia 25:

438—439

Shultz JW (1989) Morphology of locomotor appendages in

Arachnida: evolutionary trends and phylogenetic implica-

tions. Zoological Journal of the Linnean Society 97: 1-55

Shultz JW (1990) Evolutionary morphology and phylogeny of

Arachnida. Cladistics 6: 1-38

Shultz JW (1991) Evolution of locomotion in Arachnida: the

hydraulic pressure pump of the giant whipscorpion, Masti-

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 101

goproctus giganteus (Uropygi). Journal of Morphology 210:

13-31

Shultz JW (1993) Muscular anatomy of the giant whipscorpion

Mastigoproctus giganteus (Lucas) (Arachnida: Uropygi) and

its evolutionary significance. Zoological Journal of the Lin-

nean Society 108: 335-365

Shultz JW (1999) Muscular anatomy of a whipspider, Phrynus

longipes (Pocock) (Arachnida: Amblypygi), and its evolu-

tionary significance. Zoological Journal of the Linnean So-

ciety 126: 81-116

Shultz JW (2000) Skeletomuscular anatomy of the harvestman

Leiobunum aldrichi (Weed, 1893) (Arachnida: Opiliones:

Palpatores) and its evolutionary significance. Zoological

Journal of the Linnean Society 128: 401-438

Shultz JW (2001) Gross muscular anatomy of Limulus poly-

phemus (Xiphosura, Chelicerata) and its bearing on evolu-

tion in the Arachnida. Journal of Arachnology 29: 283-303

Shultz JW (2007a) A phylogenetic analysis of the arachnid or-

ders based on morphological characters. Zoological Journal

of the Linnean Society 150: 221—265

Shultz JW (2007b) Morphology of the prosomal endoskeleton

of Scorpiones (Arachnida) and a new hypothesis for the evo-

lution of cuticular cephalic endoskeletons in arthropods. Ar-

thropod Structure & Development 36: 77—102

Smrz J, Kovaé LV, Mikes J, LukeSova A (2013) Microwhip scor-

pions (Palpigradi) feed on heterotrophic cyanobacteria in

Slovak caves — a curiosity among Arachnida. PLOS ONE 8:

e75989. https://doi.org/10.1371/journal.pone.0075989

Smrz J, Kovaté L, Mike’ J, Sustr V, LukeSova A, Tajovsky K,

et al. (2015) Food sources of selected terrestrial cave arthro-

pods. Subterranean Biology 16: 37

Snodgrass RE (1948) The feeding organs of Arachnida, includ-

ing mites and ticks. Smithsonian Miscellaneous Collections

110: 1-93

Snodgrass RE (1965) Textbook of Arthropod Anatomy: Fac-

simile of the Edition of 1952. Publishing Company, New

York / London

Souza MFV, Ferreira RL (2010a) Eukoenenia (Palpigradi: Eu-

koenentidae) in Brazilian caves with the first troglobiotic

palpigrade from South America. Journal of Arachnology 38:

415-424

Souza M, Ferreira RL (2010b) Palpigradi: Eukoeneniidae and

its conservation status. Speleobiology Notes 4: 17—23

Souza M, Ferreira RL (2011) A new species of Eukoenenia

(Palpigradi: Eukoeneniidae) from Brazilian iron caves. Zoo-

taxa 2886: 31-38

Souza M, Ferreira RL (2011b) A new troglobitic Eukoenenia

(Palpigradi: Eukoeneniidae) from Brazil. Journal of Arach-

nology 39: 185-188

Souza M, Ferreira RL (2012) Eukoenenia virgemdalapa (Pal-

pigradi: Eukoeneniidae): a new troglobitic palpigrade from

Brazil. Zootaxa 3295: 59-64

Souza MFVR, Ferreira RL (2013) Two new species of the

enigmatic Leptokoenenia (Eukoeneniidae: Palpigradi) from

Brazil: first record of the genus outside intertidal environ-

ments. PLOS ONE 8: e77840. https://doi.org/10.1371/jour-

nal.pone.0077840

Stahnke HL (1966) Some aspects of scorpion behavior. Bulletin

of the Southern California Academy of Sciences 65: 65—80

Steinbach G (1952) Vergleichende Untersuchungen zur Che-

licerenmuskulatur einiger Araneen. Wissenschaftliche Zeit-

schrift der Humboldt-Universitat zu Berlin 2, Berlin

Steinbrecht RA (1969) Comparative morphology of olfacto-

ry receptors. Pp. 3—21 in: Pfaffmann C (ed.) Olfaction and

Taste, Vol. 3. Rockefeller University Press, New York

Bonn zoological Bulletin Suppl. 65: 1-125

Strausfeld NJ, Mok Strausfeld C, Loesel R, Rowell D, Stowe S

(2006) Arthropod phylogeny: onychophoran brain organiza-

tion suggests an archaic relationship with a chelicerate stem

lineage. Proceedings of the Royal Society B: Biological Sci-

ences 273: 1857-1866

Strubell A (1892) Zur Entwicklungsgeschichte der Pedipalpen.

Zoologischer Anzeiger 15: 87-93

Suter RB, Stratton G.E, Miller PR (2004) Taxonomic variation

among spiders in the ability to repel water: surface adhesion

and hair density. Journal of Arachnology 32: 11-21

Szalay L (1956) Der erste Fund von Palpigraden in Ungarn.

Annales historico-naturales Musei nationalis hungarici 48

(series nova 7): 439-442

Talarico G, Hernandez LG, Michalik P (2008) The male genital

system of the New World Ricinulei (Arachnida): ultrastruc-

ture of spermatozoa and spermiogenesis with special empha-

sis on its phylogenetic implications. Arthropod Structure &

Development 37: 396—409

Talarico G, Lipke E, Alberti G (2011) Gross morphology, his-

tology, and ultrastructure of the alimentary system of Ricinu-

lei (Arachnida) with emphasis on functional and phylogenet-

ic implications. Journal of Morphology 272: 89-117

Talarico G, Palacios-Vargas JG, Silva MF, Alberti G (2005)

First ultrastructural observations on the tarsal pore organ of

Pseudocellus pearsei and P. boneti (Arachnida, Ricinulei).

Journal of Arachnology 33: 604-612

Tanaka G, Hou X, Ma X, Edgecombe GD, Strausfeld NJ (2013)

Chelicerate neural ground pattern in a Cambrian great ap-

pendage arthropod. Nature 502: 364

Telford MJ, Thomas RH (1998) Expression of homeobox genes

shows chelicerate arthropods retain their deutocerebral seg-

ment. Proceedings of the National Academy of Sciences 95:

10671-10675

Teruel R, De Armas LF (2002) Un género nuevo de Hubbardii-

dae (Arachnida: Schizomida) del occidente de Cuba. Revista

Ibérica de Aracnologia 6: 91-94

Tichy H (1975) Unusual fine structure of sensory hair triad

of the millipede, Po/yxenus. Cell and Tissue Research 156:

229-238

Tichy H, Barth FG (1992) Fine structure of olfactory sensilla

in myriapods and arachnids. Microscopy Research and Tech-

nique 22: 372-391

Tichy H, Loftus R (1996) Hygroreceptors in insects and a spi-

der: humidity transduction models. Naturwissenschaften 83:

255-263

Tjonneland A, Okland S, Nylund A (1987) Evolutionary as-

pects of the arthropod heart. Zoologica Scripta 16: 167—175

Todd V (1949) The habits and ecology of the British harvestmen

(Arachnida, Opiliones), with special reference to those of the

Oxford district. The Journal of Animal Ecology: 209-229

Vachon M (1949) Ordre des Pseudoscorpions. Pp. 437-481 in:

Grassé P-P (ed.) Traité de Zoologie, Vol. 6. Masson et Cie.,

Paris

Vitzthum H (1931) Ordnung Acari (Milben). Pp. 1-160 in:

Krumbach T (ed.) Handbook of Zoology Online, Vol. 3. De

Gruyter, Berlin / Boston

Vyas AB (1974) The cheliceral muscles of the scorpion Heter-

ometrus fulvipes. Bulletin of the Southern California Acade-

my of Sciences 73: 9-14

Waldow U (1970) Elektrophysiologische Untersuchungen an

Feuchte-, Trocken-und Kalterezeptoren auf der Antenne der

Wanderheuschrecke Locusta. Journal of Comparative Phy-

siology A 69: 249-283

Waloszek D, Miller KJ (1998) Cambrian ‘Orsten’-type arthro-

pods and the phylogeny of Crustacea. Pp. 139-153 in: Fortey

©ZFMK

102 Sandra Franz-Guess & J. Matthias Starck

RA, Thomas RH (eds) Arthropod Relationships. The Sys-

tematics Association Special Volume Series, vol 55. Spring-

er, Dordrecht

Waloszek D, Chen J, Maas A, Wang X (2005) Early Cambrian

arthropods — new insights into arthropod head and structural

evolution. Arthropod Structure & Development 34: 189-205

Wang B, Dunlop JA, Selden PA, Garwood RJ, Shear WA,

Miller P, et al. (2018) Cretaceous arachnid Chimerarachne

yingi gen. et sp. nov. illuminates spider origins. Nature Ecol-

ogy & Evolution 2: 614-622

Wegerhoff R, Breidbach O (1995) Comparative aspects of the

chelicerate nervous systems. Pp. 159-179 in: Breidbach O,

Kutsch W (eds) The Nervous Systems of Invertebrates:

An Evolutionary and Comparative Approach. Experientia

Supplementum, vol 72. Birkhauser, Basel

Weygoldt P (1969) The Biology of Pseudoscorpions. Harvard

University Press, Cambridge

Weygoldt P (1985) Ontogeny of the arachnid central nervous

system. Pp. 20-37 in: Barth FG (ed.) Neurobiology of Arach-

nids. Springer, Berlin / Heidelberg

Weygoldt P (1996) Evolutionary morphology of whip spiders:

towards a phylogenetic system (Chelicerata: Arachnida: Am-

blypygi). Journal of Zoological Systematics and Evolutiona-

ry Research 34: 185—202

Weygoldt P, Paulus HF (1979a) Untersuchungen zur Morpho-

logie, Taxonomie und Phylogenie der Chelicerata II. Clado-

gramme und die Entfaltung der Chelicerata. Journal of Zoo-

logical Systematics and Evolutionary Research 17: 177—200

Weygoldt P, Paulus HF (1979b) Untersuchungen zur Morpho-

logie, Taxonomie und Phylogenie der Chelicerata I. Morpho-

logische Untersuchungen. Journal of Zoological Systematics

and Evolutionary Research 17: 85-116

Bonn zoological Bulletin Suppl. 65: 1-125

Weygoldt P, Rahmadi C, Huber S (2010) Notes on the reproduc-

tive biology of Phrynus exsul Harvey, 2002 (Arachnida: Am-

blypygi: Phrynidae). Zoologischer Anzeiger 249: 113-119

Wheeler WM (1900) A singular arachnid Koenenia mirabilis

(Grassi) occurring in Texas. The American Naturalist 34:

837-850

Whitehead WF, Rempel JG (1959) A study of the musculature

of the black widow spider, Latrodectus mactans (Fabr.). Ca-

nadian Journal of Zoology 37: 831-870

Wiens JJ, Donoghue MJ (2004) Historical biogeography, ecol-

ogy and species richness. Trends in Ecology & Evolution 19:

639-644

Wirkner CS, Huckstorf K (2013) The circulatory system of spi-

ders. Pp. 15—27 in: Nentwig W (ed.) Spider Ecophysiology.

Springer, Berlin / Heidelberg

Wirkner CS, Prendini L (2007) Comparative morphology of the

hemolymph vascular system in scorpions — A survey using

corrosion casting, MicroCT, and 3D-reconstruction. Journal

of Morphology 268: 401-413

Wirkner CS, Togel M, Pass G (2013) The arthropod circulato-

ry system. Pp. 343-391 in: Minelli A, Boxshall G, Fusco G

(eds) Arthropod Biology and Evolution: Molecules, Devel-

opment, Morphology. Springer, Berlin / Heidelberg

Wolf H (2016) Scorpiones. Pp. 443-452 in: Schmidt-Rhaesa

A, Harzsch S, Purschke G (eds) Structure and Evolution of

Invertebrate Nervous Systems. Oxford University Press, Ox-

ford

Yokohari F (1999) Hygro- and Thermoreceptors. Pp. 191—210

in: Eguchi E, Tominaga Y (eds) Atlas of Arthropod Sensory

Receptors. Springer, Berlin / Heidelberg

Zwicky KT, Hodgson SM (1965) Occurrence of myogenic

hearts in arthropods. Nature 207: 778

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 103

Appendix I.

Table 5. Eukoenenia spelaea, list of musculature. Each muscle or muscle branch is listed with its points of origin and insertion.

When two or more points of origin are listed, two or more branches originate and fuse into one strand. When two or more points of

insertion are listed, the muscle branches. Within extremities, the term “proximal” is used to describe the location of origin/insertion

of a muscle oriented toward the body, “distal” is used for the location away from the body. This terminology is used for the entire

extremities as well as their separate articles. Muscles associated with the box-truss axial muscle system (Shultz 2001, 2007b) are

marked grey. Abbreviations: C1—13 = cheliceral muscle; D1—2 = dorsal muscle; DV1—5 = dorsoventral muscle; E1—20 = endoster-

nite muscle; F1—3 = flagellar muscle; Gf = genital muscle female; Gm1—4 = genital muscle male; JI1—4 = intersegmental muscle 1;

JI[1—6 = intersegmental muscle 2; JII[1—6 = intersegmental muscle 3; JIV1—6 = intersegmental muscle 4; JV1—6 = intersegmental

muscle 5; JVI1—5 = intersegmental muscle 6; JVII1—4 = intersegmental muscle 7; JVII1—3 = intersegmental muscle 8; JIX1—2 =

intersegmental muscle 9; LI1—14t = leg 1 muscle/tendon; LII1—9 = leg 2 muscle; LHI1—10 = leg 3 muscle; LIV1—14 = leg 4 mus-

cle; P1—-13 = prosomal muscle; PP1—10t = pedipalps muscle/tendon; TI1—2 = transversal muscle 1; TII[1—2 = transversal muscle 2;

TIII1—2 = transversal muscle 3; TIV1—2 = transversal muscle 4; TV1—2 = transversal muscle 5; TVI1 = transversal muscle 6; V =

ventral muscle.

Insertion

Muscle Origin Fig.

Extremities:

Cheliceral muscles

propeltidium, dorsolateral above the ;

GI prosternum, lateral to C4, extrinsic mus- Nise at the P a of the cence 19B, 20A

ae where chelicera articulates with prosoma

Anteroventral in chelicera, between in- osterolateral in cheliceral basal article

C2 sertion of C1 and C3, where chelicera Be SHE aS > 194, 20A

articulates with prosoma MeEP,

propeltidium, anterodorsal and medial, ventrolateral and proximal, where cheli-

C3 splits in two strands that extend to each PSE ERT Si URLS 19B, 20A

chelicera, extrinsic muscle M aD

43 dorsomedial and proximal, where cheli-

C4 SA te ok tae aL POSE OT cera articulates with prosoma, adjacent 19B,20A

: to C5

cheliceral basal article, dorsomedial and __ ventrolateral and immediately proximal

C5 proximal, where chelicera articulates to the joint connecting cheliceral base 19A, 20A

with prosoma with chela

lateral and proximal in cheliceral basal ventrolateral and proximal, where

C6 article, where chelicera articulates with cheliceral base articulates with chela, 19A, 20A

prosoma opposite C5

ventrolateral and proximal in cheliceral

C7 basal article, where chelicera articulates same as C5 19A, 20A

with prosoma

dorsal and distal in cheliceral basal ar-

C8 ticle, starts as two strands which join same as C5 19A, 20A

ventral

ventrolateral from cheliceral base, imme- as tendon, in the center of the dorsoven-

€9 diately proximal to the joint connecting tral axis, immediately distal to the joint 19A

cheliceral base and chela connecting fixed digit and movable digit

ventrolateral and immediately proximal

C10 to the joint connecting cheliceral base same as C9 19A, 20A

and chela

dorsolateral and immediately proximal to

Cll the joint connecting cheliceral base and same as C9 19A

chela

dorsolateral and immediately proximal to

C12 the joint connecting cheliceral base and same as C9 19A, 20A

chela

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

104 Sandra Franz-Guess & J. Matthias Starck

Muscle Origin Insertion Fig.

C3 dorsal, immediately proximal to the joint iano 19A, 20A

connecting cheliceral base and chela

Muscles of the pedipalp

PP propeltidiim: posieiedorsal 16.3, ,pos- dorsomedial and proximal on coxa 19B, 20A

terolateral to C4, extrinsic muscle

medioproximal, immediately distal to dorsomedial, immediately distal to the

PRZ ae : ; 19A, 20C

where coxa articulates with prosoma joint connecting coxa and article 2

posterior, immediately distal to the joint

posteroventral, immediately distal to the connecting article 2 and 3 I9A, 20C

PPS joint connecting coxa and article 2, splits eer

in two branches just prior to end anterior, immediately distal to the joint 19A. 20C

connecting article 2 and 3 :

posterodorsal, immediately distal to the 19A. 20C

joint connecting article 3 and 4 °

anterodorsal, immediately distal to the 19A, 20C

joint connecting article 3 and 5

dorsal, in the center of the anterior-pos-

PPA terior axis, and distal in article 2, splits in aS tendon ventral and in the center of

four branches in the center of the anteri- _ the anterior-posterior axis, immediately

; mre a : 19A, 20C

or-posterior axis in article 3 distal to the joint connecting article 3

and 4

ventral and in the center of the anteri-

or-posterior axis, immediately distal to 19A, 20C

the joint connecting article 4 and 5

dorsal, in the center of the anterior-poste- ventral and in the center of the anteri-

PPS or-posterior axis, immediately proximal 19A,20C

rior axis, and distal in article 4 Me ;

to the joint connecting article 6 and 7

as tendon ventral, immediately distal to venta) ane ie iter Onis ane

PP6t at Z y Or-posterior axis, immediately distal to 19A, 20C

the joint connecting article 4 and 5 ie

the joint connecting article 5 and 6

as tendon ventral, immediately distal to senirakand aniahe Center Olt eante nts

PP7t we - y or-posterior axis, immediately distal to 19A, 20C

the joint connecting article 5 and 6 oy ;

the joint connecting article 6 and 7

as tendon ventral and in the center of the _ ventral and in the center of the anteri-

PP8t anterior-posterior axis, immediately distal or-posterior axis, immediately distal to 19A, 20C

to the joint connecting article 6 and 7 the joint connecting article 7 and 8

as tendon ventral and in the center of the ventral and in the center of the anteri-

PPS anterior-posterior axis, immediately distal or-posterior axis, immediately distal to 19A, 20C

to the joint connecting article 7 and 8 the joint connecting article 8 and 9

as tendon ventral and in the center of the ; ;

; ee ventral and in the center of the anteri-

PP10t anterior-posterior axis, immediately distal 19A, 20C

A : or-posterior axis at tarsal claw

to the joint connecting article 8 and 9

Leg |

Ll propel, eee ane eet dorsomedial and proximal on coxa 19B, 20A

Cl and PP1, extrinsic muscle

lateral on anterior transverse bridge of

LI2 endosternite in segment 5, extrinsic mus- inahe center Oeite proatinal: disiahaxs 19A, 20B

ie and anterior in coxa

LI3 ie LOGS a nadia ty isla lOyunele posteroventral and proximal inarticle2 19A,20C

coxa articulates with prosoma

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 105

Muscle Origin Insertion Fig.

dorsal and in the center of the anteri- posterior, immediately distal to the joint 19A.20C

or-posterior axis, immediately distal to connecting article 2 and 3 ’

LI4 the joint connecting coxa and article 2, en ‘i

splits in two branches in the center of the anterior, immediately distal to the joint 194 49¢

anterior-posterior axis in article 2 connecting article 3 and 4 :

LIS posterior, immediately distal to the joint —_ posterior, immediately distal to the joint 19A. 20C

connecting article 3 and 4 connecting article 4 and 5 ;

dorsal and in the center of the anteri-

or-posterior axis, immediately distal to '

the joint connecting article 2 and 3 as tendon anteroventral, immediately

LI6 distal to the joint connecting article 5 19A, 20C

posterodorsal and proximal in article 3 and 6

anterodorsal and proximal in article 3

posteroventral, immediately distal to the 19A. 20C

joint connecting article 5 and 6 ;

LI7 posterolateral, immediately distal to the as tendon ventral and in the center of

joint connecting article 4 and 5 the anterior-posterior axis, immediately 94 99¢

distal to the joint connecting article 5 ;

and 6

dorsal and in the center of the anteri- Ventana sae Pent mous aut.

LI8 me or-posterior axis, immediately proximal 19A,20C

or-posterior axis in article 5 ae

to the joint connecting article 6 and 7

as tendon ventral and in the center of as tendon ventral and in the center of

LI9t the anterior-posterior axis, immediately the anterior-posterior axis, immediately 19A. 20C

proximal to the joint connecting article distal to the joint connecting article 6 ;

5 and 6 and 7

SSC eur anda i De Ce UetgOr: as tendon anteroventral, immediately

LH0t Pcsanler OF posterionans, mmitedialely. distal to the joint connecting article 7 19A, 20C

proximal to the joint connecting article ;

and 8

6 and 7

; tendon anteroventral, immediately

as tendon anteroventral, immediately dis- aa ae as

LI11t tal to the joint connecting article 7 and 8 ore to the joint connecting article 8 19A, 20C

as tendon ventral and in the center of

Lut as tendon anteroventral, immediately dis- the anterior-posterior axis, immediately 19A.20C

tal to the joint connecting article 8and9 _ distal to the joint connecting article 9 ;

and 10

as tendon ventral and in the center of the See ee tar

LATS anterior-posterior axis, immediately distal |. Pe if ; y 19A, 20C

to the joint connecting article 9 and 10 iat x the jomt connecting article 10

as tendon ventral and in the center of the iS aes eritst OF themantenompustenior

LI14t anterior-posterior axis, immediately distal aie Se aa ke P 19A, 20C

to the joint connecting article 10 and 11

Leg 2

wt dorsal and in the center of the anteri- 19B. 20B

propeltidium, dorsolateral, posterior to or-posterior axis on coxa, thicker }

LIT P6, splits in two branches medial, extrin-

sic muscle lateral at pleural membrane between leg

; 19B, 20B

2 and leg 3, thinner :

medial on anterior transverse bridge and Posterior, immediately proximal to the

Lin central bridge of endosternite in the re. JInt connecting coxa and article 2, 19A, 20B

gion of leg 2 and 3, splits in two branches thinner

medial, extrinsic muscle posterior in coxa, thicker 19A, 20B

106

Sandra Franz-Guess & J. Matthias Starck

Muscle Origin Insertion Fig.

in the center of the anterior-posterior

axis, immediately distal to where coxa dorsal and in the center of the anteri-

LIB articulates with prosoma or-posterior axis, immediately distalto 19A, 20C

in the center of the proximal-distal axis the Joint connecting coxa and article 2

and anterior in coxa

LII4 ante Ocoee at, oe torthe anterodorsal and proximal in article 2 19A, 20C

joint connecting coxa and article 2 ?

ventral and in the center of the anteri- posterior, immediately distal to the joint 19A. 20C

or-posterior axis, immediately distal to connecting article 3 and 4 ;

115 the joint connecting coxa and article 2,

splits in two branches in the center of the posterodorsal and proximal in article5 19A, 20C

anterior-posterior axis in article 3

dorsal and in the center of the anteri-

LI6 or-posterior axis, immediately distal to posterodorsal and proximal in article 4 19A, 20C

the joint connecting article 2 and 3

dorsal, in the center of the anterior-poste- ventral and in the center of the anteri-

1 Ue 2 CO bs ; P or-posterior axis, immediately proximal 19A,20C

rior axis and distal in article 3 ae ;

to the joint connecting article 5 and 6

: ventral and in the center of the anteri-

LII8 posterodorsal, immediately distal to the or-posterior axis, immediately distal to 19A, 20C

joint connecting article 4 and 5 oath

the joint connecting article 5 and 6

dorsal, in the center of the anterior-poste- in the center of the anterior-posterior

LH9 é ; Ce ; 19A, 20C

rior axis and distal in article 5 axis at tarsal claw

Leg 3

Prope G iar sgorspmedidls Above able ts osterodorsal and in center of the proxi-

LI or bridge of endosternite in the region of P Abs P 19A, 20B

nthe mal-distal axis in coxa

leg 3, extrinsic muscle

at central bridge, medial at posterior posterior, immediately proximal to the

bridge, and at upturned U section of joint connecting coxa and article 2, 19A, 20B

LII2 endosternite in the region of leg 3 and 4, __ thinner

splits in two branches medial, extrinsic =

muscle posterior in coxa, thicker 19A, 20B

in the center of the anterior-posterior

an Te ca distal to where coxa dorsal and in the center of the anteri-

LIN3 atueulates Wil prosoma or-posterior axis, immediately distalto 19A, 20C

in the center of the proximal-distal axis the Joint connecting coxa and article 2

and anterior in coxa

LIII4 posterodorsal, immediately distal He posterodorsal and proximal in article 3 19A, 20C

joint connecting coxa and article 2

ventral and in the center of the anteri- ventral and in the center of the anteri-

LIWS or-posterior axis, immediately distal to or-posterior axis, immediately distal to 19A, 20C

the joint connecting coxa and article 2 the joint connecting article 3 and 4

LIII6 anterodorsal and distal in article 2 posienedaeral mime diately, pale 19A, 20C

the joint connecting article 3 and 4

LIW7 anteroventral and distal in article 2 aDLETONeuaL lanniediate pro zuaal{o 19A, 20C

the joint connecting article 3 and 4

ventral and in the center of the anteri-

dorsal, in the center of the anterior-poste- =

LIV8 é : ; , or-posterior axis, immediately proximal 19A,20C

rior axis, and distal in article 3 erat

to the joint connecting article 5 and 6

LIII9 anterodorsal and distal in article 4 anterior; immediately proximal to the 19A, 20C

joint connecting article 5 and 6

LIMO dorsal, in the center of the anterior-poste- in the center of the anterior-posterior 19A, 20C

rior axis, and distal in article 5

Bonn zoological Bulletin Suppl. 65: 1-125

axis at tarsal claw

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

Muscle Origin Insertion Fig.

Leg 4

ventrolateral at posterior section of end- _lateral at pleural membrane between first 19A. 20B

LIVI osternite in the region of leg 4, splits in _ articles of leg 3 and 4 ;

two branches just after origin, extrinsic rE

muscle posterior in coxa, adjacent to LIV3 19A, 20B

ventrolateral and posterior to LIV1 at in the center of the anterior-posterior

LIVZ endosternite in the region of leg 4, extrin- axis between LIV4 and LIVS, fuses with 19A,20B

sic muscle LIV4 and LIVS

LIV3 era: ae ee an s posterior in coxa, adjacent to LIV1 19A, 20B

dorsal and in the center of the anteri-

LIV4 eae eee eae to where or-posterior axis, immediately distal to 19A, 20C

P the joint connecting coxa and article 2

ventral and in the center of the anteri- RATE: cone trot he ane

LIV5 or-posterior axis, immediately distal to Neues Se a a aLOA DUG

where coxa articulates with prosoma Heitgr, orl ako inaaels JNanele

pope Nabe in The ce nine e ante anterodorsal, immediately proximal to

LIV6 or-posterior axis, immediately distal to i) TN ea TEA EE Fe ada 19A, 20C

the joint connecting coxa and article 2 J 8

LIV7 posteroventral and distal in article 2 posterior in article 3 19A, 20C

LIV8 anteroventral and distal in article 2 ante Odessa un ed lalele roma 19A, 20C

the joint connecting article 3 and 4 ;

Hee posteroventral and distal in article 3 posterior, immediately proximal to the mace

posterior and proximal in article 4 joint connecting article 4 and 5

LIVI1O ventral, in the center of the anterior-pos- —_ anterior, immediately proximal to the 19A. 20C

terior axis, and distal in article 3 joint connecting article 4 and 5 ;

LIVI posteroventral, immediately distal to the — posterior, immediately distal to the joint 19A.20C

joint connecting article 4 and 5 connecting article 5 and 6 ;

: dorsal and in the center of the anteri-

LIV12 et ae pie ea ee or-posterior axis, immediately proximal 19A,20C

‘ to the joint connecting article 5 and 6

anterior, immediately distal to the joint anterior, immediately distal to the joint

pie connecting article 4 and 5 connecting article 5 and 6 ERIN ee

LIV14 anteroventral and distal in article 5 ventral and in the center of the anteri- = 194 99¢

or-posterior axis at tarsal claw

Prosomal body wall:

Pl unpaired anteromedial in upper lip of rostrosoma cpmome hal in upper Wpohrosiasoma, 19B, 20A

just anterior to mouth opening

P2 paired anterolateral in upper lip of rostrosoma Sorsdlateralan upper Mp Orrost soma: 19B, 20A

just anterior to mouth opening

P3 unpaired dorsal on pharynx dorsomedial at intercheliceral sclerite 19B, 20A

P4 paired lateral on pharynx ventrolateral in rostrosoma 19B, 20A

propeltidium, ventrolateral and anterior, — propeltidium, anterolateral, in the region

Be paired in the region of the chelicerae of the chelicerae eo

propeltidium, dorsolateral, inthe region _lateral at pleural membrane between

Ee pated of leg 1, between C1 and LII1 coxa of leg 1 and 2, dorsal to E7 BE Oe

a3 lateral at pleural membrane and in the

P7 paired propellidiums dorsolateral siheweglon center of the anterior-posterior axis in 19B, 20A

of leg 2, between LIT] and P8

Bonn zoological Bulletin Suppl. 65: 1-125

the region of leg 3

©ZFMK

108 Sandra Franz-Guess & J. Matthias Starck

Muscle Origin Insertion Fig.

; propeltidium, dorsolateral, in the region fle rapa seu alien ane ana nals

P8 paired Pleo NASD COntO Ey center of the anterior-posterior axis in 19B, 20A

BP the region of leg 3, anteroventral to P7

lateral dorsal plate, dorsolateral and pos-

Pg paired terior in the region of leg 3, between both ictal dorsal plate, Adral an the-tepon 19B, 20A

of leg 2 A

branches of P11

propeltidium, dorsolateral and posterior — anterolateral at pleural membrane in the

ES paired in the region of leg 2, outside of P11 region of leg 4 PB 20

metapeltidium, dorsal and in the center

r a of the anterior-posterior axis in the re- 19C, 20A

FEMALE: propeltidium, dorsomedial in gion of leg 3, thicker

paired the region of leg 1, splits in two branches ;

posterior posterolateral at pleural membrane in

the region of leg 3, posterodorsal to 19C, 20A

LI, thinner

eae.

metapeltidium, dorsal and in the center

o, ss of the anterior-posterior axis in the re- 19B, 20B

MALE: propeltidium, dorsomedial in the gion of leg 3, thicker

paired region of leg 2, splits in two branches

medial posterolateral at pleural membrane in

the region of leg 3, posterodorsal to 19B, 20B

LIT, thinner

FEMALE: metapeltidium, medial andin metapeltidium, medial and in the center

unpaired the center of the anterior-posterior axis in of the anterior-posterior axis in the re- 19C, 20A

Bis the region of leg 3 gion of leg 4

MALE: metapeltidium, dorsomedial and dorsolateral at tergite and in the center

paired in the center of the anterior-posterior axis _ of the anterior-posterior axis insegment 19B, 20B

in the region of leg 3 8

icra paltadiamacndstetolatondinahesre: metapeltidium, lateral and in the center

PAS paired ree >P of the anterior-posterior axis in the re- 19B, 20A

gion of leg 3

gion of leg 4

Endosternite:

ventromedial at intersegmental mem-

brane and in the center of the anteri- 1

ae Or-posterior axis in the region of the

ventral at branch of endosternite in the pedipalp, lateral to ventral plate

El paired region of pedipalp and leg 1, splits in two

branches just after origin prosternum, ventromedial and in the

center of the anterior-posterior axis in 1

the region of leg 1, posterior to first

branch

pO nee anterior at branch of endosternite in the ventrolateral at pleural membrane adja- 1

P region of pedipalp and leg 1 cent to coxa of pedipalp

propeltidium, dorsolateral in the region 41

of leg 1

anterodorsal at branch of endosternite in nyc ave Pe a thchers ON ATES AERA HORTA

E3 paired the region of leg 1, just posterior to E1, Mind vy ye ts eae oN 21

me .°. Of leg 1, posterior to first branch

splits into three branches just after origin

propeltidium, dorsolateral in the region >|

of leg 1, posterior to second branch

EA pane anterodorsal at branch of endosternite in _ propeltidium, dorsolateral and posterior 41

P the region of leg 1, just posterior to E3 in the region of leg 2

ES pha anterodorsal at branch of endosternite in _— propeltidium, dorsolateral and anterior 41

iy the region of leg 1, just posterior to E4 in the region of leg 2

E6 paired anterodorsal at branch of endosternite in —_ anterodorsal at pleural membrane, where 41

the region of leg 1, just posterior to ES

Bonn zoological Bulletin Suppl. 65: 1-125

chelicera articulates with prosoma

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 109

Muscle Origin Insertion Fig.

E7 act ventral at branch of endosternite in the ventrolateral at pleural membrane be- 1

Me region of leg 2 tween coxa of leg 1 and 2, ventral to P6

E8 unpaired eae oe obendasvernite: ii posteroventral and medial on pharynx 21

Ane ventrolateral at pleural membrane, just

E9 paired eee ne ee Ps ease tS et posterior where leg 1 articulates with 21

dorsolateral and in the center of the an- Pe cenle eralarinltaraitmrem traci

E10 paired terior-posterior axis on anterior bridge of Ae ake eras Z eA |

endosternite in the region of leg 2 S 8

dorsolateral and posterior on anterior Faneltidatmedarniateratardcdostene:

Ell paired bridge of endosternite in the region of P aie F leo 3 P on

ee in the region of leg

SOESG Foie) Qn (PSE on Sn EMRE as metapeltidium, dorsolateral in the region

El2 paired bridge of endosternite in the region of leg rl P sh E14 and E16 e at

3, outside of E11 peer SE AY

ie ventral at tergite, at the border of seg-

E13 paired leben rane Ol Stoo eunre ae ments 8 and 9, immediately anteriorto 21

region of leg 3, posterior to E12 ay

origin of V

dorsolateral and in the center of the ante- _ propeltidium, dorsolateral and posterior

E14 paired rior-posterior axis on posterior bridge of in the region of leg 3, just anteromedial 21

endosternite in the region of leg 3 of Ell

dorsolateral and posterior on posterior oe

EIS paired bridge of endosternite in the region of leg ee roe eat Be ee 21

Aisin ori gion of leg 4, adjacent to an

E16 haa posterolateral on upturned U section of anterolateral at pleural membrane in the 41

E endosternite in the region of leg 4 region of leg 4

dorsolateral at posterior section of endos- PF iA pele iedorce lore enaienes

E17 paired ternite in the region of leg 4, two branch- oes ee : 21

a gion of leg 4, adjacent to E12 and E14

es merge just after origin

lateral at posterior section of endosternite Lateral at pleural membrane, where leg

ame poned in the region of leg 4, ventral to E16 4 articulates with prosoma a

ventrolateral and posterior at posterior anterolateral at pleural membrane,

E20E19 paired section of endosternite in the region of where leg 4 articulates with prosoma, 21

leg 4 between E15 and E17

posterolateral at posterior section of lateral at pleural membrane and in the

E20 paired endosternite in the region of leg 4, just center of the anterior-posterior axis in 21

posterior to E19 segment 8

Opisthosoma:

: metapeltidium, dorsolateral and anterior _— dorsal at tergite of segment 9, just me-

2 Denes in the region of leg 4 dial of D2 Hae

: dorsal at tergite of segment 17, immedi-

D2 paired ae as EIA Uy SES. ately posterior to the border of segments 19C, 20B

. 16 and 17

FEMALE: anterodorsal at tergite of seg- anteroventral at sternite of segment 9, 19A. 200A

ment 9, lateral to D1 just medial of V :

DVI paired MALE: anterodorsal at tergite of segment

9, outside of D1 anteroventral at sternite of segment 9, 19B. 20B

MALE: posterodorsal to the segmental Just medial of V

overhang of segment 9

110 Sandra Franz-Guess & J. Matthias Starck

Muscle Origin Insertion Fig.

anteroventral at sternite of segment 10,

Seas aE ae ae wee just posterior to genital lobe and medial 19A,20A

? of V

DV2 paired dorsolateral at genital atrium 19B, 20B

MALE: anterodorsal at tergite of segment

10, between D2 and JII1, splits in two anteroventral at sternite of segment 10,

branches ventral just posterior to genital lobe 3 and me- 19B, 20B

dial of V

BMPS AO Sie eget ane Til anteroventral at sternite of segments 11

DV3-4 paired ~—_and 12, between D2 and JIIII and JIV1, ae ar cialis 19A, B, 20B

respectively Heo daa te

anteroventral at sternite of segment 13, 19A. B.20B

anterodorsal at tergite of segment 13, be- Just medial of V, thicker im

DV5 paired tween D2 and JV1, splits in two branches entral and in the center of the anteri-

at half point or-posterior axis at sternite of segment 19A,B, 20B

13, just medial of V, thinner

anterior at tergite of segment 9, outside of Nee ieee aye oa eas. f

ju-4 paired DI, strands 1-4 evenly distributed from 5" candy 1-4 evenly distributed f 19C, 20A,B

dorsal to ventrolateral altands SVD ye sin uler or

dorsal to ventrolateral

in the center of the anterior-posterior axis

: at tergite of segment 9 cate toJIcand. +7 MeHOr at tere ite of segment 10, outside

JI1-6 paired J ida De. cians 16 evenly distrib- of D2, strands 1—6 evenly distributed 19C, 20A,B

uted from dorsal to ventrolateral noun dorss Lio veut Olate tal

osterior at tergite of segment 10, pos- : ’

ae: to JII oy outside of D2 ee ani) loner iere ie Or seem nn me ier

JUI1-6 paired 16 avenie-distributed font aca fo of D2, strands 1—6 evenly distributed 19C, 20A,B

eee ee from dorsal to ventrolateral

osterior at tergite of segment 11, pos- ;

tater to JI ie outside of D2 ets anterior at tergite of segment 12, outside

JIV1-6 paired {= even ie iateBiuted tran Areal a of D2, strands 1—6 evenly distributed 19C, 20A,B

deat from dorsal to ventrolateral

osterior at tergite of segment 12, pos- é

tetior to JIV aa oubsidSo D2 sane SDI Sor AL leteMlAOr SgemeMh Ss ouisae 19C

JV1-6 paired were ° of D2, strands 1—6 evenly distributed ;

1-6 evenly distributed from dorsal to fei ea | | 20B20A,B

sanicalataeal rom dorsal to ventrolatera

posterior at tergite of segment 13, pos- ;

; anterior at tergite of segment 14, outside

JVI1-5 paired pian Sars a is weet oo of D2, strands 1—5 evenly distributed 19C, 20A,B

Sea ie at from dorsal to ventrolateral

posterior at tergite of segment 14, pos- in the center of the anterior-posterior

terior to JVI and outside of D2, strands axis at sclerite of segment 15, outside of

A ae 1-4 evenly distributed from lateral to D2, strands 1-4 evenly distributed from Page

ventrolateral lateral to ventrolateral

osterior at sclerite of segment 15, pos- ;

terior to JVII and outside of D2 strands posterior at sclerite of segment 16, out- 19C

JVI-3 paired hearer lee Hat one IRCA side of D2, strands 1-3 evenly distribut- .0B20A B

a ed from lateral to ventrolateral :

posterolateral at sclerite of segment 16,

JIX1-2 paired posterior to JVIII, strands 1—2 evenly Saat oe vee See ts 19C, 20A,B

scetbuted lateral strands 1—2 evenly distributed latera

FEMALE: ventrolateral and in the center E eniiGlatstaldind Sue rlotakeccesse

TH paired of the anterior-posterior axis at pleural "y 19A, 20A

membrane of segment 9

Bonn zoological Bulletin Suppl. 65: 1-125

gland posterior in segment 9

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 111

Muscle Origin Insertion Fig.

ventrolateral and in the center of the ante- ventromedial and in the center of the

TI2 paired rior-posterior axis at pleural membrane of anterior-posterior axis at sternite of 19A,B 20A,B

segment 9, in females ventral to TI1 segment 9

ventrolateral and in the center of the an- — ventromedial and in the center of the

THI paired terior-posterior axis at pleural membrane _ anterior-posterior axis at sternite of 19A, 20A,B

of segment 10 segment 10

ventrolateral and in the center of the an- — ventromedial and in the center of the

TH2 paired terior-posterior axis at pleural membrane —_anterior-posterior axis at sternite of 19A,B20A B

of segment 10, ventral and inward of TII1 segment 10, ventral to TIII

ventrolateral at tergite of segments 11— ventromedial at sternite of segments

TIH- ait 13, TIT] posterior, TIV1 inthe center of 11-13, TIL] posterior, TIV1 in the cen- 194. B20A

TV1 P the anterior-posterior axis, TV1 anterior ter of the anterior-posterior axis, TV1 ;

in the respective segment anterior in the respective segment

FEMALE: ventrolateral at pleural mem- —_ ventromedial at sternite of segments

brane of segments 11—13, ventral to 11-13, ventral to TIW1-TV1, TIII2

THI1-TV1, TI2 posterior, TIV2 inthe — posterior, TIV2 in the center of the an- 19A, 20A

center of the anterior-posterior axis, TV2 _ terior-posterior axis, TV2 anterior in the

ae anterior in the respective segment respective segment

> paired hs

TV2 MALE: ventrolateral at pleural mem ventromedial at sternite of segments

brane of segments 11—13, ventral and 1-13

—13, ventral and outward of TIII1—

inward of TIHI1—TV1, THI2 posterior,

; TV1, TIII2 posterior, TIV2 inthe center 19B, 20B

TIV2 in the center of the anterior-poste-

; a ale of the anterior-posterior axis, TV2 ante-

rior axis, TV2 anterior in the respective ei

segment rior in the respective segment

ventrolateral, posterior and outward at ventrolateral and posterior at pleural

Evi paired tergite of segment 14 membrane of segment 14, outside of V IDB. 208

anteroventral to its origin in the genital

dorsomedial in the genital operculum in operculum in segment 9 I9A, 20A

Gf paired segment 9, splits in two branches just . ee

after origin posteroventral to its origin in the genital 194. 20A

operculum in segment 9 j

Gmi Bee ventromedial at the genital atrium in anteroventral and medial in genital lobe 19B. 20B

P segment 9 1 in segment 9 ;

ventromedial at the genital atrium in ventromedial in genital lobe 2, just out-

Se paued segment 9, just posteromedial of Gm1 side of Gm1 in segment 9 1B 2206

: ventromedial at the genital atrium in ventromedial in genital lobe 2, just pos-

oe palipd segment 9, just posteromedial of Gm2 terior and outside of Gm2 in segment 9 BRA 08

posteroventral and medial at genital atri- anileroyehiial ayaletnite, Ol seement 0;

Gm4 paired just posterior to genital lobe 3 and me- 19B, 20B

um in segment 9

dial of V

ventral at sclerite at the border of seg- 19A. B20A_B

ments 17 and 19 3 7

ventral at tergite, at the border of seg- ventrolateral at pleural membrane poste-

V acd ments 8 and 9, immediately posterior to Fe ree ie Mg 19A, B20A,B

: the insertion of E13, gives off two side 8

strands anterior in segments 9 and 11 ventrolateral at pleural membrane just

anterior to the border of segments 9 and 19A,B20A,B

lateral at sclerite at the anterior end of Be a eee pists toe ie

F1-3 paired segment 19, strands 1-3 distributed from y 19 and fi + b . 3 19C, 20B

dorsal to ventrolateral ae ad es ea a Et Sa

distributed from dorsal to ventrolateral

112

Sandra Franz-Guess & J. Matthias Starck

Table 6. Changes applied to the character states in the character matrix of Shultz’s (2007a) phylogeny based on morphological

characters and additional character coding changes applied to the original data matrix of Shultz (2007a). Character 61 was deleted,

and characters 12a, 128a, 135a, 143a, 148a, 178a, 179a and 203 are introduced. Characters with their original/new description and

original/new coding are marked grey, taxa with their corresponding coding are white.

Taxa

Eukoenenia

Prokoenenia

E. spelaea

Eukoenenia

Prokoenenia

E. spelaea

Schizomida

Solifugae

E. spelaea

Eukoenenia

Prokoenenia

Eukoenenia

Prokoenenia

E. spelaea

Character

12a

Description by Shultz (2007a)

Anterior end of dorsal prosoma

with median marginal or sub-

marginal pointed process

carapace with distinct pro-,

meso- or metapeltidial sclerites

rostrosoma: long, narrow, sub-

cylindrical epistome projecting

anteriorly with base fixed to

dorsal surface of palpal coxae,

bordered laterally by lobes

projecting from palpal coxae;

ventral wall of preoral chamber

formed by anterior element of

prosoma (sternapophysis)

apotele (claw), position

Bonn zoological Bulletin Suppl. 65: 1-125

New description

prosternum con-

sisting of the

fused sclerites of

segments 2-4

rostrosoma: long,

narrow, subcylin-

drical epistome

projecting ante-

riorly, bordered

laterally by lobes

Original coding New coding

O = absent,

1 = present

0 = absent

O = absent,

1 = present

n/a

O = absent,

| = present

O = absent

O = terminal,

1 = subterminal,

— = inapplica-

ble, coded only

for taxa with a

distinct apotele

1 = present

O = absent,

1 = two peltidia,

2 = three peltidia

1 =two peltidia

2 = three peltidia

O = absent,

1 = present

? = unknown

O = absent,

1 = fixed to pedipal-

pal coxae,

2 = no association

with pedipalp

2 = no association

with pedipalp

©ZFMK

Taxa

Palaeo-

charinus

Eukoenenia

Prokoenenia

E. spelaea

Mastigo-

proctus

E. spelaea

Character

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

Description by Shultz (2007a) Newdescription Original coding

? = unknown

appendage III (= arachnid leg 1)

extremely elongate, antenniform

O = absent,

1 = present

? = unknown

deleted, because

it was a supposed

autapomorphy for

Palpigradi

trochanter-femur joint with dor-

sal hinge or pivot operated by

flexor muscles only

superior trochanter-femur mus-

cle (or homologue) originating r 0 = absent,

broadly in femur, inserting on 1 = present

distal margin of trochanter

O = absent

patella of appendage of postoral

somite III (= arachnid leg 1) Le

proportionally much longer than — : 2 ensene

those of more posterior append- P

ages

0 = absent

O = monocondy-

lar, several

axes of move-

ment and

multifunctional

muscles,

1 = bicondylar

hinge, one

axis of move-

ment and

antagonistic

muscles,

2 = hinge, one

axis of

movement,

flexor muscles

without mus-

cular

antagonists

femur-patella joint —

2 = hinge, one

axis of move-

ment, flexor

muscles without

muscular antag-

onists

Bonn zoological Bulletin Suppl. 65: 1-125

New coding

0 = terminal

1 = present

? = unknown

113

0 = monocondylar,

several axes of

movement and mul-

tifunctional muscles

©ZFMK

114

Taxa

Mastigo-

proctus

E. spelaea

Eukoenenia

Prokoenenia

E. spelaea

Character

73,74

106

116

128

128a

Sandra Franz-Guess & J. Matthias Starck

Description by Shultz (2007a)

patella-tibia joint

dependent on

character 72

anterior femur-—tibia or femo-

ropatella—tibia (transpatellar)

muscle

circumtarsal ring

megoperculum

number of metasomal somites

anterior oblique muscles of

BTAMS posterior to postoral

somite VI

Bonn zoological Bulletin Suppl. 65: 1-125

New description

anterior oblique

muscles of

BTAMS anterior

to postoral somite

VI (state 1 only in

E. spelaea)

Original coding

2 = hinge, one

axis of move-

ment, flexor

muscles without

muscular antag-

onists

O = absent,

1 = present

0 = monocondy-

lar with or with-

out CZY (73)

O = absent,

1 = present

0 = absent

0 = absent,

1 = present

? = unknown

O = absent,

1 = present

0 = absent

O = absent,

| = present

1 = present

O = zero,

1 = two,

2 = three,

3 = five,

4 =nine

2 = three

O = absent,

1 = present

? = unknown

New coding

1 = bicondylar

hinge, one axis of

movement and an-

tagonistic muscles

2 =hinge, one axis

of movement, mus-

cles without muscu-

lar antagonists

— = inapplicable

1 = present

? = unknown

0 = absent

O = zero,

1 =two,

2 = three,

3 = four,

4 = five,

5 = nine

3 = four

0 = absent

O = absent,

ik= present

1 = present

©ZFMK

Taxa

Eukoenenia

Prokoenenia

E. spelaea

Acari

E. spelaea

Eukoenenia

Prokoenenia

E. spelaea

Eukoenenia

Prokoenenia

Eukoenenia

Prokoenenia

E. spelaea

Character

130

131

132

135a

143a

148

148a

162

163

164

165

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

Description by Shultz (2007a)

endosternite fenestrate

suboral suspensor: a tendon that

arises from the BTAMS and

inserts on the ventral surface of

the oral cavity via muscle

perineural vascular membrane

in adult

intercheliceral median organ

nucleus (of sperm) with manch-

ette of microtubules

axoneme (of sperm)

coiled axoneme (of sperm)

microtubule arrangement in

axoneme (of sperm)

Bonn zoological Bulletin Suppl. 65: 1-125

New description

suboral suspen-

sor: a tendon that

arises from the

BTAMS and in-

serts on the ven-

tral surface of the

foregut via muscle

arcuate body in

protocerebrum

trichobothria with

dendrites reaching

into the hair shaft

supracheliceral

median organ

lateral organ

Original coding

n/a

O = absent,

1 = present

O = absent

O = absent,

1 = present

(ventral on oral

cavity)

O = absent,

1 = present

n/a

O = absent,

1 = present

n/a

O = absent,

1 = present

O = absent

O = absent,

1 = present

O = absent

O = absent,

1 = present

— = inapplicable

O = absent,

1 = present

New coding

? = unknown

1 = present

O = absent,

115

1 = ventral on oral

cavity,

2 = ventral and

posterior on pharynx

2 = ventral and

posterior on pharynx

O = absent

O = absent,

1 = present

O = absent

? = unknown

O = absent,

1 = present

? = unknown

O = absent,

1 = present

? = unknown

©ZFMK

116

Taxa

E. spelaea

Acari

E. spelaea

Eukoenenia

Prokoenenia

E. spelaea

Eukoenenia

Prokoenenia

E. spelaea

Prokoenenia

E. spelaea

Eukoenenia

Prokoenenia

Character

166

167

178a

179a

194

198

Loy

203

Sandra Franz-Guess & J. Matthias Starck

Description by Shultz (2007a) = New description

helical or corkscrew shaped

nucleus (of sperm)

Vacuolated-type sperm -

coxal organ with

tubule differenti-

ated into proximal

and distal section

- ventral plate

dorsal dilator muscle of precere-

bral pharynx attaching to dorsal

surface of prosoma or intercheli-

ceral sclerite

dilator muscle of

precerebral phar-

ynx and/or preoral

cavity attaching to

dilator muscle of precerebral

pharynx and/or preoral cavity

attaching to ventral surface of

prosoma prosoma or rostro-

soma

postcerebral pharynx -

hindgut with cu-

ticular lining

Bonn zoological Bulletin Suppl. 65: 1-125

Original coding

— = inapplicable

O = absent,

1 = present

0 = absent

O = absent,

1 = present

0 = absent

n/a

O = absent,

1 = present

(dorsal on pro-

soma)

O = absent,

1 = present

(ventral on

prosoma)

O = absent,

1 = present

n/a

New coding

? = unknown

1 = present

O = absent,

1 = present

? = unknown

O = absent,

1 = present

? = unknown

O = absent,

1 = dorsal on pro-

soma,

2 = dorsal on inter-

cheliceral sclerite

2 = dorsal on inter-

cheliceral sclerite

O = absent,

1 = ventral on pro-

soma,

2 = ventrolateral on

rostrosoma

2 = ventrolateral on

rostrosoma

0 = absent

O = absent,

= present

O = absent

? = unknown

©ZFMK

Appendix II.

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

117

Table A1. Character matrix based on the analysis of Shultz (2007a). Updated character codes are marked red, added character codes

are marked blue

Taxon Character coding

— 0000227000 00001100720 0702-00001 10000-0700

000--00000 0002000001 0111-0020 02--011011

Trombidiformes 000--00011 00102?000-0 000000-001 fe 0-0:02-— 4

Trombidiidae 00000427229 29929000-013 011001000000 1001001000

000--00000 2701001101101 0011100000 0-0000000-—

Allothrombium Law

— 0000021000 00001100220 0200-00001 10100-0700

000--00000 0002000001 000120020 62-3020 1

Trombidiformes 000--00011 00101000-0 000000-001 ASO i

Mievdidas 00000072229 2292000-013 2711001000000 12701002929

097999 99:99 29990117 1901 0010100000 0-67 99900"

Alycus 509

— 0000121000 04001100720 0200-00001 10100-0700

000--00000 0002000001 000000020 O00. bl

Oribatida 000--00011 00102000-0 000000-001 O01 24

ikfvobchunoniilée 00000029929 2299000-00- ~11001000000 10011000

000--00000 001001101001 0010100000 0-0000000-—

Archegozetes 01

ar 0000220000 00001100720 0207-00001 10000-0200

000--00000 0002000001 0111-0020 OF TO

Trombidiformes 000--00011 00102000-0 000000-001 fers =0.0°0= =)

Caeculidae 00000472929 299990017013 011001000000 1001001270

02:0 2-0:0-7:00 2701001201101 0012100000 0-0000000-

Microcaeculus ee

ee 0000121000 04001100220 0200-00001 10100-0700

000--00000 0002000001 0111-0020 OF4=0 1 POL

Sarcoptiformes 000--00011 00102000-0 000000-001 foO200 oO =41

Pulabacaridas 00000027229 2292001202? 2711001000000 1201011000

O20 FORO 2701001101001 0010100000 0-0270000-

Palaeacarus Li

00000-0000 00011000110 000001000 ~0000-0000

Araneae

0110000000 0000000110 0000010022 0010011011

Mygalomorphae 0100-00001 00115010-0 0000011010 1110000--1

Theedphosidhe 11000010200 00031111012 011000000011 1001000000

0111310000 100000010001 1001101000 (Ort 10:6 taal

Aphonopelma 101

00000-0000 00011000110 0000001000 ~0000-0000

Araneae

0110000000 0000000110 000010022 0010011011

Araneomorphae 0100-00001 00115010-0 0000011010 P1060 02 —41

Prpoohitides 11000010200 CONSO D1 T0423 011000000011 1001000000

0111310000 100000010001 2001101000 1110100111

Hypochilus

101

Bonn zoological Bulletin Suppl. 65: 1-125

©ZFMK

118 Sandra Franz-Guess & J. Matthias Starck

Taxon Character coding

banaee 00000-0000 00011000110 0000001000 —0000-0000

0110000000 0000000110 000010022 0010011011

Mesothelae 0100-00001 00115010-0 0000010010 1101000--1

Liphistiidae 11000010700 00030111012 011000000011 1001000000

0111310000 100000010001 1001101000 1110100111

Liphistius 101

00100-0000 00011000110 0000100000 —-000100?01

Amblypygi 0010010000 000?001011 000010023 1000101011

Berns 0101100000 01105000-0 0000010000 10-—-020--1

11000010700 10150110012 0110000000?? 1011001100

Charinus 0111310000 111000010001 1001100000 1110001011

101

00100-0000 00011000110 0000100000 —000100101

Amblypygi 0000-10000 0001001011 000010023 1000101011

0101100000 00105000-0 0000010000 00--020--1

Phrynidae

11000010700 10150110012 0110000000?? 1011001100

Phrynus 0111310000 111000010001 00-1100000 1110001011

101

Meant 00000-0711 LG Bs a aie ay dil Ia a a 279292997-2292999 29299-29999

ELITR EEE ELS Ed Mca a A nt ae as dae a de oY a PPR PLE ET LT?

¥ Chasmataspidida 2222729209 12226201-0 O72 29929 99 Y=) TALEO

fi Chasmataenicidae 72007-22299 POEVIVIVIIAA PLQVPIOVIEVD? 2227022972

+ Chasmataspis

Merostomata

+ Chasmataspidida

+ Diploaspididae

+ Diploaspis

Merostomata

+ Chasmataspidida

+ Diploaspididae

+ Octoberaspis*

+ Eurypterida

+ Eurypteridae

+ Baltoeurypterus*

00000-0711

0010000010

2100-00001

Bonn zoological Bulletin Suppl. 65: 1-125

0290772271272?

PLP F ET FAN

LOO le aie al Ur ae e

PLP F ET FAN

0000??00000

011700070?

00??5000-0

ae eer ee ee)

2011-0020

0??72?0-070

2207 —=00-070:0

WO OPO

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 119

Taxon Character coding

00000-0711 00002700000 PIVV 99 E99 ~0200-27770

+ Eurypterida 001000000? 012200070? 29011-0070 0770029 979

2120-20001 002?2?5000-0 02292?0-02? 20 —?03 10-0

+ Stylonuridae

id ia Ol er a a cas PPI9IA TPO LO FODP IED FOF? 2 PER FED

Stylonurus* [PER IAI e RETIN PF PO Pg ay a iV A dl ae Bs EPRES PEON

22?

00110-0200 0002999997999 FIP? F996 Ps ai at kOe ay a

Wanchith 02299292000 0002909922 200092909? 079292299999

219210200?? 0?2?29A?00-0 02220120270 20--007001

+ Haptopoda

2 PINT D Pee ge TERS RII SD PEEP UR LO PEF 2 a Rav nO We Be,

+ Plesiosiro* G22 F999 FEVED. FID ISG PEPE IETS EP PET LI OD

ae 00001-0000 00011000020 0000-00001 10100-0200

0010010000 0002000701 0011791120 OF 097 O79

Parasitiformes 0101000011 011274000-0 000000-000 00-0001

Opilioacarida 0000052729222 SUPT G TO 13 0100--000010 POO.T Meats

000--01000 POPOL T 1:00.01 00-0100000 0-0000000-

Neocarus hs

poe 00001-0000 00011000020 0000-00001 Pe. 0-0-0.7 0-0

0010010000 000?000270? 20112112? 09-02 2098

Parasitiformes 27101070011 012274000-0 000000-000 002200024

@pilisacarida 00000522992? 29990 1060-012 211000000010 1201112270

0702-91902 2299017910001 290100000 0-079999999

Siamacarus Ld

0000100100 00000-00000 1020-00000 —~0000-2000

Arachnida

0010000001 1000000001 000000010 0.1 — = OO I

Opiliones 0101010001 00112000-0 000000-000 00-=060:13—1

Caddidae 00000170200 0101011200- —~100--000000 10-1110001

0229200000 202200000000 10-0100110 10?001000-

Caddo Lf

0000100100 01000-00000 1010-00000 —~0000-1000

Arachnida

0010000001 1000000001 000000020 01 -—041-0-—111

Opiliones 0100-00001 00112000-0 000000-000 0:05 0hO: PsA

Pettalidae 00000170200 01010129290?? 2100--000000 1001110001

072222900270? 202200000000 10-?100110 0-0001000-

Chileogovea es

0000100100 01000-00000 1020-00000 —~0000-1000

Arachnida

0010000001 1000000001 000000020 O1=—=010-11

Opiliones 0100-00001 00112000-0 000000-000 0:05 = 00= bss

Sionidas 00000170200 0101019790?? 2100--000000 1001110001

001100010? 202200000000 1. "10:01. 0 0-0001000-

Cyphophthamus hal

120 Sandra Franz-Guess & J. Matthias Starck

Taxon Character coding

0000000100 00000-00000 1010-00000 —~0001-?000

Arachnida

0010000001 1000000001 000000010 01 -eree 2011

Opiliones 0101010001 00112000-0 000000-000 00--0013-1

Gonyleptidae 00000170?00 0100011C00- 1-0 = 0:00 00 1001110001

010--00000 10000000000? 10-0100110 10?001000-

Gonyleptes Bak

0000100100 00000-00000 1010-00000 —~0000-0000

Arachnida

0010000001 1000000001 000000010 O1=S01-307 4

Opiliones 0101010001 00112000-0 000000-000 00-——0013—1

Bi erotuaunnieas 00000170200 010B011C00- —~100--000000 1001110001

000--00000 100000000000 10-0100110 101001000-

Leiobunum ome

. 0000000100 00000-00000 1020-00000 —~0001-?000

Arachnida

0010000001 1000000001 000000010 O1--012011

Opiliones 0101010001 00112000-0 000000-000 00-00-13 1

tl ernbuditise 00000170200 0100011C00- —~100--000000 1001110001

0?0--00000 100000000000 10-0100110 10?001000-

Sclerobunus iG

0010110000 00010-00000 0000000000 —~2000-0000

Arachnida

0010010000 0000000001 000000700 03200. 1014

Palpigradi 0121000001 00104000-0 000000-010 00--031201

Batocnentdes 00000010101 2003100-00- —~01100001100 10010027270

Eukoenenia spelaea

Arachnida

Palpigradi

Eukoenentidae

Eukoenenia

Arachnida

Palpigradi

Prokoenentidae

Prokoenenia

Acari

Parasitiformes

Allothyridae

Allothyrus

0010110000

0010010000

0101000001

00000012200

000--0002?

0010110000

0010010000

0101000001

0000001220?

000--000?2

00?

01000-0100

0011000000

0210-00011

OPO IDO

Bonn zoological Bulletin Suppl. 65: 1-125

00010-00000

0000000001

00104000-0

1103100-00-

00010-00000

0000000701

001?4000-0

?103100-00-

00011000020

000?000?0?

OLE? 20 0:0. 0

2?2227000-00-

EEE OP Pe O07

0001100000

0000000000

000000020

000001-010

—01?00001100

0001100000

0000?00000

000000020

000001-010

—01?700001100

0001100000

0000-00001

2?000?1020

000000-100

—-100--000010

00-1100000

101200020-

—2000-0000

00000?1011

00--031201

100100???70

101100010-

—2000-?700

00000110??

OS FOr <2 Oa

10:01 CO? 20

1011000101

10100-0700

02—-=0 7707?

EN =0'.0 0. =Al

Ne 0 Md Ie 0 Ra ta 8

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) 121

Taxon Character coding

Pe! 00000-0000 00000-00020 0000-00001 10000-0700

000--00000 0002000001 000001020 (2-20-10 14

Parasitiformes 02710-00011 01100000-0 000000-000 G02 -B4F0— 1

edits 00000327200 271722000-014 0100--000010 1201100000

O07 109 000001011001 00-1100000 0-0270000-

Amblyomma a

ace 00000-0000 00000-00020 0000-00001 10000-02700

000--00000 0002000001 000001020 02S 16-1

Parasitiformes 0710-00011 00100000-0 000000-000 602-00

Aeostdine 00000327200 27122000-014 0100--000010 1201100000

0270--21702 000001011001 00-1100000 O—0:72-0:0:0:0-—

Argas Le

asa 01000-0100 00001000020 0000-00001 10100-0700

0011000000 000200070? 200071020 OD 099 079

Parasitiformes 0710-00011 01222000-0 000000-100 C0010 02-1

Allothenidae 000003297929 2999000-014 27100--000010 1001102220

000--01000 9299-02 11004 00-1100000 0-00029299?

Australothyrus es

ee 01000-0000 00011000020 0020-00001 10100-0200

0011000000 0002000001 000001020 02--011011

Parasitiformes 0210-00011 01102000-0 000000-000 00--000--1

ooh nate 00000327200 7979. 000 O-0= ~100--000000 1001100000

000--01000 2700001011001 00-1100000 0-0000000-

Glyptholaspis vai

0000100000 03010-00121 0000-10100 ~1000-0010

Arachnida

100--00100 0000000001 000010010 01-2047 100

Pseudoscorpiones 000--00001 01115000-0 000000-000 00 = 0:00. 41]

Cheliferidae 00000210200 0127010-014 011000000000 1001001000

0011200011 110000000000 10-1100000 0-0000000-—

Chelifer Pie

0000100000 03010-00120 0000-10100 ~1000-0000

Arachnida

100--00100 0000000001 000010010 01--012100

Pseudoscorpiones 0E00-00001 01115000-0 000000-000 00--000--1

Chtoniidae 00000210200 0127010-013 011000000000 1001001000

0011200011 110000000000 10-1100000 0-0000000-—

Chthonius he

0000100000 03010-00120 0000-10100 ~1000-0000

Arachnida

100--00100 0000000001 000010010 Galt Oni Dal O°0

Pseudoscorpiones 000--00001 01115000-0 000000-000 00--000--1

Feaelliidae 00000210200 01770104013 011000000000 1001001000

0011200011 110000000000 10-1100000 0-0000000-

Feaella 7.24

122 Sandra Franz-Guess & J. Matthias Starck

Taxon Character coding

Taine 0000100000 03010-00121 0000-10100 ~1000-0010

100--00100 0000000001 000010010 O 1 ere? 1.0:0

Pseudoscorpiones 0100-00001 01115000-0 000000-000 O:052-O1GG sal

Neobisiidae 00000210200 0127010-013 011000000000 1001001000

0011200011 110000000000 10-1100000 0-0000000-—

Neobisium sg

ontae 00000-0000 12011000100 0220-00001 20000-0700

0010001000 0002000001 001100020 02--000-11

Ricinulei 0101000001 00105120-0 100000-000 en) earn ae pe]

Ricinoididae 00000329229 271795010-014 27100-0000011 1001000270

0111200070 202201010001 00-1100000 0-0000000-—

Cryptocellus ae

00000-0200 120299270999 EP? AEF ya a a VE ao

Arachnida

02222702000 0002202999 20112702? DIRVIVETID

Ricinulei 1 agen ame a OE 02995120-0 ($2990 799 De (0

Sepalinchebalee POPP IF 9-899 2925290-213 ya ke en Caw wae) 95) 299-9 95999

0:2092-9 999 PRP IND OD POPE IH 9 PHVA TPIS 9

+ Poliochera 299

00000-0000 12011000100 02270-00001 20000-0700

Arachnida

0010001000 0002000001 001100020 09220001 1

Ricinulei 0101000001 00105120-0 100000-000 Ob 1205-4

Ricinoididae 0000032992? 271275010-014 27100-0000011 1001000270

0111200070 207201010001 00-1100000 0-0000000-—

Ricinoides Ong

es: 00000-0200 170799707999 PEEFA99999 FFP FHA 9799

racnnida

02222702000 0002202992 20112702? 9 P99 999 19

Ricinulei

+ Palaeoricinulei

+ Terpsicroton*

Arachnidae

Schizomida

Protoschizomidae

Protoschizomus

Arachnidae

Schizomida

Hubbardiidae

Stenochrus

0010120000

0010010000

0101100000

10000010°?0?

0111310000

001

0010120000

0010010000

0101100000

10000010°01

0111310000

001

Bonn zoological Bulletin Suppl. 65: 1-125

ORES AO 0

opel Oa Er LS

PUPIL LS POS

02011000110

000?00??11

001?5000-0

70-757 10-0 0—

11?000010001

02011000110

0000001711

00115000-0

2014110-014

11?000010001

IPP OO Pee?

0000?00001

000070123

0100010000

-11010000000

00-1100000

0000?00001

000010123

0100010000

211010000000

00-1100000

4 ee 0 Sol

LOPS DY)

0.0 =02 Tee dl

11?1001110

1??00010?0

0000110100

0000001011

0.0 S=021 211

Tole. WOO ITT.

11100010°?0

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902) | Wass!

Taxon Character coding

. 0000100000 01000-10000 0111-00000 —0000-1000

Arachnida

100--0000I1 1000000001 000010010 01--012110

Scorpiones 0100-10001 00116000—-0 001000-000 00--0311-I1

Buthidae 00110010°?00 OO0OO051110011 111000010011 1101101000

0010110100 000010010000 10-1100111 O-10010011

Centruroides 001

. 0000100000 01000-10000 0111-00000 —0000-1000

Arachnida

100-—-—-00001 1000000001 000010010 01--012110

Scorpiones 0100-10001 00116000-0 001000-000 00--0311-I1

@araboctonidae 00110010?00 OO0OO0S51 110011 111000010011 1101101000

0010110111 000010010000 10-1100111 O-10010011

Hadrurus 001

’ 0000100000 01000-10000 0111-00000 —0000-1000

Arachnida

100--O00001 1000000001 000010010 01--012110

Scorpiones 0100-10001 00116000-0 001000-—000 00--0311-1

Scorpionidae 00110010?00 OO0OO0S51110011 111000010011 1101101000

00102?0111 000010010000 10-1100111 O-10010011

Heterometrus

001

. 0000?00?700 ODOM? 7077? 2779797-222970 -—-0?00-277?

Arachnida

100--0000? Ce PaO? Oe? 2000?00?? 0 a Aa aa i co a le

Scorpiones 2?1?0-?0001 02???6000-0 0?2??00-0?0 ??--0311-?

+ Palaeoscorpionidae 7 it a i! al a a A i a ie i ar ak 2 Us a ot it oes ek P2LPEOTIIF?

OPP RIR I? 72 Sa a a Die alco aa ake ol ae al se ie al a dae al a eA oe a a ea ane

+ Palaeoscorpius ea.

0000100200 010777706979 27997-2299 70 —-0?00-272929?

Arachnida LOO 0:0: F721 COOL? PATI? daa ik i a i 2 PREP ELE?

yA ga NL AL ae a 22972929000-0 0972?70-77? ?7?--0311-?

Scorpiones

el Be ilo Se al Sat A Ao fe dal es a Oe at oe Want at ae a ad 2 Ye a aa es ae A a

+ Prearcturus Get eT PIE Ee te a oo Sar ee J ke a PP TERY PE Ss ai ae oo a a

age

; 0000700700 000??7?7?000? 29797-29279 90 —-0?00-?777?

Arachnida

1-050 = 0:0'0:0-2 ol Rica des OE ats dg 2?000?00?? QE PP aE 2

Scorpiones 2?1?0-?0001 0?9??6000-0 0???00-070 ??--0311-?

+ Proscorpiidae Ait fae Ol i SOs ak A EPEEIP LITE 1O PEE? F Gale? Ve? PEP IS | Pee

1 aie Se a Ee at ie aid 2 ee ie as Aa TRAY SUE OED a a Bad a wa

+ Proscorpius sae

. 0000200200 00022270002 2297-2997] 1200-2229

Arachnida

100--0000? CO RP Pe eee? 2000?700?? L PPR oP VPP?

Scorpiones ?922?1-?0001 1???6000-0 0???00-1?71 ??--0311-?

+ Proscorpiidae Wd is a EO a i Rene? eee LO ee is da a a ON Me a EP PEDER TE?

EM Ss A ga A Sa As ae weep pe pp eee Paha Se ENP Papen) ate apne

+ Stoermeroscorpio' ae

124 Sandra Franz-Guess & J. Matthias Starck

Taxon Character coding

: 0000120000 03010-00121 0000-00010 —~1000-0000

Arachnida

0010170100 0000000001 0011-0000 02--010-10

Solifugae 1101010001 01114000-0 000000-000 0:0-—-—000——41

Eeemdbatiine 00000270270? pale ee Cae el I 0100--100000 1001000000

000--00100 101000010000 01-1100001 0-00000110

Eremocosta

001

0000120000 03010-00121 0000-00010 —~1000-0000

Arachnida

0010120100 0000000001 0011-0000 O82 2010=1 0

Solifugae 1101010001 01114000-0 000000-000 00--000--1

Galeodidae 00000270270? Pee Sel EP Beles 0100--100000 1001000000

000--00100 101000010000 01291100001 0-00000110

Galeodes 001

. 00110-0000 02011000110 0000100001 0000100100

Arachnida

000--10000 0000100111 100010113 0000001011

Uropygi 0101100000 00115000-0 0100010000 00-=03:13041

Thalyphanidas 110000102701 1015111001B 011010000000 1111001110

0111310000 111000010001 00-1100000 1110001010

Mastigoproctus 001

00100-0700 036799 00.193 i ad eB oat a | 0.07 PhP2-ENG

Reena 0029210000 000270297299 EPCS FO VERE PG

a oat eal abr 08 Ba 022?2?5000-0 02229010070 1 O2-020.1271

Uropygi

+ Proschizomus

Arachnida

+ Trigonotarbida

+ Palaeocharinidae

+ Gilboarachne

Arachnida

+ Trigonotarbida

+ Palaeocharinidae

+ Palaeocharinus

Euchelicerata

Xiphosurida

+ Euproopidae

+ Euproops

00000-0000

00???0?000

71020’ 7-0°G-081

00000-0000

0010007000

2?100-?0001

00010-0711

9999999 999

Bonn zoological Bulletin Suppl. 65: 1-125

eT ae een

000???00110

000?000?0?

0???4100-1

0001??00110

000?000?0?

0??2?4100-1

Oana 9 Meg ara bles

Diana ay ak ay ana

0??72010070

PLETE EOOO 7 2?

9299 POY 999

20002002?

0???7010070

2122722000???

Pepe PAR? Pe

i oa al OO

DERE PEED HS

©ZFMK

Microscopic anatomy of Eukoenenia spelaea (Peyerimhoff, 1902)

125

Taxon

Euchelicerata

Xiphosurida

+ Planaterga

+ Limuloides

Euchelicerata

Xiphosurida

Limulidae

Limulus

Euchelicerata

Xiphosurida

Limulidae

Tachypleus

Chelicerata

+ Prosomapoda

+ Weinbergina

00010-0?11

yee aia a ee Wa)

or ax

10010-0011

0010000000

0:0:0—-—01 101

ODT 10-5 Del PDO.

0010200000

011

10010-0011

0010000000

000--01101

OL 1-06—0 17-10

0010000000

O11

00010-0711

2?1?0-?0001

fi Ress al Me Be

Character coding

OPO ae? Pol nek?

PRED LA DET

2727300000

SOROS ROOT

00000-01000

O11?110001

1000200010

O66 [Ll2 eo

000100000000

00000-01000

0117110001

1000200010

OGG [liz eo

000100000000

OPO Ta REO at

Mai wae wae

0??2?300000

ee hia ie ay ae

0000-00000

100010021

000110-000

1000-—-000000

10-0010000

0000-00000

100010021

000110—-000

1000--000000

10-0010000

Pee S12 =O

POR PO epee

—0010-0000

O00 OLLI

00-?0010-0

0000000000

0-O0000I111

-0010-0000

OO0OO01011011

00-?0010-0

0000000000

0-O0000I111

27-7 1220-9

DR Pye

Arachnida

Pantetrapulmonata

+ Chimerarachne yingi

00?00-?770

0?0--0000?

Oe a |

000?2?7701?7?

LEIDER IS FID

2101031201

DOT O RT

' Stoermeroscorpio Kjellesvig-Waering, 1966 is a synonym of Proscorpius osborni Scudder, 1885 — this was obviously not consid-

ered in the original character matrix. We maintained this.

Summary

Palpigradi Is an enigmatic group of arachnids that have been

neglected by zoological research for almost 100 years. This

monograph reports about the microscopic anatomy of Eukoenenia

spelaea (Peyerimhoff, 1902), a European species of Palpigradi. It is

a treasure of morphological data on an almost unknown group of

chelicerates. The detailed morphological study documents numerous

so far unknown structures and presents new views on existing

interpretations of body tagmatization. Despite their small body size,

Palpigradi are most probably not miniaturized but derived trom

already small ancestors. The overall simplified and hyperplesiomorphic

morphology of Eukoenenia spelaea is interpreted as resulting

from paedomorphosis, developmental truncation and “reverse

recapitulation”. A phylogenetic analysis suggests that Palpigradi are

the sistergroup to the Acaromorpha.

Bonn zoological Bulletin —Supplementum Vol. 65 (2020)

Managing Editor: Thomas Wesener

foologisches Forschungsmuseum Alexander Koenig —

Leibniz-Institut Tr Biodiversitat der Tiere (ZFMK)

Adenauerallee 160, D-53113 Bonn, Germany

ISSN: 0302-6/1%

Cover illustration:

Fukoenenia speleea (Peyernmbhott, 1902). Light microscopic micrograph of a histological cross-section

through the prosoma of a female at the level of the rostrosoma, including cheliceral and pedipalpal

articulation. Please see caption of Figure 3/ in this publication tor details.

» | Bundesministerium,,... - a ee

Oke fiir Bildung Ministerium fur Innovation,

8 5 sb | | |

und Forschung Wissenschaft und Forschung

des Landes Nordrhein-Westtfalen

Leibniz-Gemeinschaft

winjuswayddns — uljayng jesbojooz uuog

(OZ07) $9 Sswnyoy,