(ISSN 0161-8202)

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“ Journal of
ARACHNOLOGY

PUBLISHED BY THE AMERICAN ARACHNOLOGICAL SOCIETY

VOLUME 44

2016

NUMBER 2

JOURNAL OF ARACHNOLOGY

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Publication date: 15 July 2016

©This paper meets the requirements of ANSI/NISO Z39.48-1992 (Permanence of Paper).

2016. Journal of Arachnology 44:105-141

Revision of the Nearctic Eratigena and Tegenaria species (Araneae: Agelenidae)

Angelo Bolzern and Ambros Hanggi: Biosciences, Natural History Museum Basel, Augustinergasse 2, CH-4001 Basel,
Switzerland: E-mail: angelo.bolzern@arachnodet.com

Abstract. Based on specimens from several museum collections and recently sampled spiders during a field excursion to
Mexico in 2014, the 11 species of Tegenaria s. 1. endemic to the United States of America and Mexico are revised.
Morphological characters and mitochondrial DNA sequences (COl, NADHl, 16S) serve as the basis for proposed new
combinations and new species. Tegenaria chiricalmae Roth, 1968 remains the only endemic Tegenaria species in the
Western Hemisphere. All other specific names {T. blanda Gertsch, 1971, T. caverna Gertsch, 1971, T. decora Gertsch, 1971,
T.flexuosa F.O. Pickard-Cambridge, 1902, T.ftorea Brignoli, 1974, T. gertschi Roth, 1968, T. mexicana Roth, 1968, T.
rothi Gertsch, 1971, T. selva Roth, 1968, and T. tlaxcala Roth, 1968) are transferred to the genus Eratigena Bolzern,
Burckhardt & Hanggi, 2013. Six new species are described: E. edimindoi, E. fernandoi, E. giiauato, E. queretaro, E. xilitia,
and E. yarini. In addition, females of E.flexnosa, and E. gertschi, and the male of E.ftorea are described for the first time.

A phylogeny based on maximum likelihood analysis of combined mtDNA sequences, an identification key and images of
all diagnosed species are provided.

Keywords: Eratigena, mtDNA, new combination, new species, morphology

http://zoobank.org/?lsid=urn:lsid:zoobank.org:pub:4F518AA0-7745-403A-9EDF-A84622E9BEB7

Among the 114 known spider families, the Agelenidae
comprises more than 1,160 described species and ranks as the
11*’’ most diverse group (World Spider Catalog 2015). Due to
the obvious funnel-webs produced by many species, some of
them are well known: for example, the large, long-legged
European house spider {Eratigena atrica (C.L. Koch, 1843),
formerly Tegenaria atrica), or the American grass spiders
{Agelenopsis Giebel, 1869; 13 species). However, new species
are still being discovered frequently (Bolzern & Herve 2010;
Bolzern et al. 2009, 2013a, b; Bosmans 201 1; Maya-Morales &
Jimenez 2013). Our knowledge of the taxonomy and
phylogeny of this spider group has fundamentally improved
in recent years (Bolzern et al. 2010, 2013a; Miller et al. 2010),
and currently the family can be divided into two subfamilies.
Ageleninae generally shows a Holarctic distribution, but
includes five genera found only in the Afrotropical (two
genera) or the Neotropical (three genera) region. One of these
genera was first described in 2013 with six new species and is
only known from the Baja California peninsula in Mexico
(Maya-Morales & Jimenez 2013). The second subfamily,
Coelotinae, forms a Holarctic lineage most diverse in Asia,
with only two genera exclusively in North America.

The subfamily Ageleninae includes the genus Tegenaria s. 1.,
composed of species endemic to the Palearctic or Nearctic
regions. The European representatives of this genus were
recently revised and grouped in four monophyletic genera
(Bolzern et al. 2010, 2013a): Aterigena Bolzern, Hanggi &
Burckhardt, 2010, Eratigena Bolzern, Burckhardt & Hanggi,
2013, Malthonica Simon, 1898, and Tegenaria Latreille, 1804.
The generic affiliation of the 16 Nearctic species is resolved
only for the presumably introduced European species, but not
for the 11 endemic species. Roth (1952) published the first
revision of Tegenaria in North America. His original
hypothesis, that all Tegenaria s. 1. species were introduced
into the Western Hemisphere from Europe (Roth 1956), was
refuted after the discovery of endemic species from Mexico
and Arizona (Roth 1968). After that, additional endemic

species were described by Gertsch (1971) and Brignoli (1974).
In his work, Brignoli noted that this species-complex was
extremely problematic due to a lack of images, the very close
relationships of the involved species and a lack of diagnostic
features. Furthermore, until now, four species were described
by one sex only. In view of these complexities and the high
proportion of introduced species, a taxonomic clarification is
essential for further research or nature conservation ap¬
proaches.

Therefore, the aims of this paper are threefold: firstly, the
taxonomical clarification of the endemic Nearctic and
Neotropical Tegenaria s. 1. species and the publication of
images of all endemic taxa; secondly, the description of newly
discovered forms or species; and finally, the provision of
mitochondrial gene sequences for certain species.

METHODS

Sampling and material examined. — Type specimens (includ¬
ing all type material) and additional material mentioned below
were examined from the following institutions: American
Museum of Natural History, New York, United States
(AMNH: Norman Platnick, Lorenzo Prendini, Luis Sorkin);
Biologfa Comparada, Taxonomia y Sistematica de Araneo-
morphae, Universidad Naciona! Autonoma de Mexico,
Mexico (FC-UNAM: Fernando Alvarez Padilla); Coleccion
Nacional de Aracnidos, Instituto de Biologia, Universidad
Nacional Autonoma de Mexico, Mexico (CNAN: Oscar
Francke, Diego A. Barrales); Museo Civico di Storia
Naturale, Verona, Italy (MCSNV: Roberta Salmaso);
Museum National d’Histoire naturelle, Paris, France
(MNHN: Christine Rollard); Naturhistorisches Museum
Basel, Switzerland (NMB); and The Natural History Museum,
London, Great Britain (NHM: Janet Beccaloni). To all
specimens examined (excluding existing type material), an
identifier was added to the vial (e.g., AB1234). Newly collected
specimens are shared between FC-UNAM and the NMB. For

105

106

JOURNAL OF ARACHNOLOGY

the collection at the NMB, official collection numbers are
provided (e.g., NMB-ARAN-12345). Barcoding sequences
(COl and NADHI) are referenced to the adequate specimens
by providing the GenBank accession-number following the
identifier.

In addition to the museum collections, specimens were
sampled during a field excursion to Mexico in October 2014
(A. Bolzern and E. Gonzalez Santillan). All specimens were
collected by hand and transferred directly into pure ethanol.

Distribution maps or single references of all georeferenced
specimens are available on the scratchpads platform, online at
http://agelenidsoftheworld.myspecies.info/specimen_
observation. In addition, downloadable high resolution
images of representatives of the included species are available
on the same website (Bolzern 2014).

Molecular methods and analyses. — For DNA extractions,
two legs were removed from a freshly sampled and alcohol-
fixed (pure ethanol) specimen. The ethanol was removed by
incubating the cut tissue at 56°C for 10 min. Then the leg was
processed according to the protocol for the purification of
total DNA from animal tissues (Spin-Column protocol) using
the DNeasy Blood & Tissue Kit (Qiagen). The DNA
concentration of the resulting solution was measured using
the fluorescent dye Picogreen and a Spectrofluorometer.
Polymerase chain reaction (PCR) amplification of two loci
was undertaken by using the following primer pairs: LCO1490
(Folmer et al. 1994) and Cl-N-2191 (Simon et al. 1994) for the
mitochondrial cytochrome c oxidase subunit 1 gene (COl, 678
bp), and TL-l-N-12718 (Hedin & Maddison 2001; numbered
following Simon et al. 1994) and M510 (Murphy et al. 2006)
for the mitochondrial nicotinamide adenine dinucleotide
dehydrogenase subunit 1 (NADHI, 591 bp). For PCR, the
Qiagen Hotstar polymerase reagents (Qiagen, Germany) were
used. The following thermo cycling conditions were applied:
initial denaturation step of 95°C for 15 min, followed by 15
touchdown cycles of: 94°C for 35 s, an annealing temperature
of 60°C to 45°C for 90 s, and an extension temperature of 72°C
for 90 s. After the touchdown cycling 30 additional cycles were
added at 94°C for 35 s, 50°C for 90 s, and 72°C for 90 s.
Finally, the cycling was followed by an additional extension of
72°C for 5 min. To eliminate incorporated nucleosides and
primers, the PCR products were treated with ExoSAP-IT (GE
Healthcare). The fragments were then sequenced in both
directions using an ABI PRISM BigDye Terminator Cycle
Sequencing Ready Reaction Kit (Applied Biosystems). Se¬
quences were then analyzed using an ABI Prism 3730 Genetic
Analyzer.

Each sequence was proof-read by checking the chromato¬
grams by eye using the software FinchTV v. 1.4 (Geospiza
Inc.). The complementary sequences (5' and 3' directions) of
each specimen were aligned using ClustalW 2 (Larkin et al.
2007) on the EBI website (Li et al. 2015) to test the sequence
quality. Each sequence was checked for contamination by
using the ‘Basic Local Alignment Search Tool’ (BLAST) on
the NCBI website. The alignments of the mitochondrial gene
regions were carried out manually, using the translation to
amino acids as a guide and checking for any inappropriately
placed stop codons and insertions or deletions. All sequences
were then cut to a length of 678 bp (COl) or 591 bp

(NADHI). Within these two alignments no indels or stop
codons occurred.

In addition to the protein coding markers, GenBank was
searched for 16S sequences of already included taxa. In favor
of repeatability and objectivity, the 39 adequate sequences
were aligned by using a fixed automatic alignment instead of
manually edited alignments or alignments based on secondary
structures. Therefore, multiple sequence alignments were
carried out using the software package Opal (Wheeler &
Kececioglu 2007) implemented in Mesquite (Maddison &
Maddison 2015), applying the default parameters (A<->G:
56; C<->T: 53; transversions: 100; gap costs: open: 260;
terminal open: 100; extension: 69; terminal extension: 66).

The three alignments were concatenated using Mesquite,
resulting in an alignment comprising 103 taxa and 1697 bp,
and with an overall coverage of 68% (COl: 101 seq.; NADH:
70 seq.; 16S: 39 seq.). Details are available as Supplemental SI,
a list of included taxonomic units for the molecular analysis
with GenBank accession-numbers (online at http://dx.doi.org/
10.1636/R15-81.S1), and Supplemental S2, a PHYLIP file
showing the alignment of the mitochondrial COl, NADHI
and 16S sequences (online at http://dx.doi.org/10.1636/
R15-81.s2). Both files are also available online at http://
agelenidsoftheworld.myspecies. info/content/downloads.

Maximum likelihood analysis and bootstrap runs were
performed using GARLI 2.01 (Zwickl 2006) at the CIPRES
Science Gateway (M.A. Miller et al. 2010). For the two
mitochondrial partitions, the codon model was applied, for the
1 6S partition a GTR-l-G-l-I model was used as suggested by the
model search function in MEGA 6.0 (Tamura et al. 2013).
Bootstrap values were subsequently drawn on the best ML
tree using the program SumTrees within DendroPy (Suku-
maran & Holder 2010, 2015). Parsimony analysis was
performed in TNT Version 1.1 (Goloboff et al. 2008) applying
a heuristic tree search with TBR, implied weighting (K=10),
and 1000 random additions of taxa while holding 100 trees per
iteration. Branch support was estimated by applying a jack¬
knife resampling method (1000 replicates) with default
removal probability of characters (0.36). For both phyloge¬
netic analyses, Amaurobius ferox (Walckenaer, 1830) (Amaur-
obiidae) was used as the outgroup, and resulting trees were
rooted at the Amaurobius branch/clade.

Morphological methods and abbreviations. — Preserved spec¬
imens were examined under a Leica MZ12 and a Leica Ml 65
C microscope. Images were taken using a Leica MCI 70 HD
camera attached to the Leica Ml 65 C, and processed with the
stacking program CombineZP (Alan Hadley) and Adobe
Photoshop. To remove soft tissue, dissected female genitalia
were first transferred to distilled water for several hours.
Subsequently, they were put in an enzymatic lens cleaner
solution overnight, washed, and transferred back to ethanol.
The morphological terminology follows Bolzern et al. (2013a).
The following abbreviations are used (see also Figs. 8, 9, 11,
14-16, 18-20, 22, 27, 28): AER, anterior eye row; ALE,
anterior lateral eyes; ALS, anterior lateral spinnerets; AME,
anterior median eyes; bulbL, distance of the cymbium base to
the most distal tip of the male bulb (including conductor); C,
conductor; CB, cymbium breadth; CD, copulatory duct; CL,
carapace length; CLYl, clypeus height under AME; CLY2,
clypeus height under ALE; CO, copulatory opening at female

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

107

epigyne; CW, carapace width; DB, dorsal branch of RTA; DP,
distal portion of conductor; DS, distal sclerite at MA; E,
embolus; FD, fertilization duct; MA, median apophysis of
male bulb; OL, opisthosoma length; OW, opisthosoma width;
PER, posterior eye row; PEE, posterior lateral eyes; PLS,
posterior lateral spinnerets; PM, posterior membrane (internal
posterior limit of female genital area); PME, posterior median
eyes; PMS, posterior median spinnerets; PS, epigynal posterior
sclerite; PT, epigynal ‘pseudo teeth’; R, retroventral ridge of
palpal tibia; RC, receptaculum; RTA, retrolateral tibial
apophysis (used here as the sum of all structures on the
retrolateral aspect of the tibia of the male pedipalp); STL,
sternum length; STW, sternum width; T, tegulum; TR,
transversal ridge at conductor; VB, ventral branch of RTA.

Taxonomical nomenclature follows the World Spider
Catalog (2015).

RESULTS

Molecular data. — Maximum likelihood and parsimony
(MP) analyses resulted in essentially identical best trees (Fig.
1, MP tree not shown). The higher level classification
proposed by Bolzern et al. (2013a) is supported by the
molecular data presented here and summarized as follows (see
also Fig. 1): (1), Agelenidae is split into the two monophyletic
subfamilies Ageleninae and Coelotinae; (2), the genera
Tegenaria and Eratigena are separate monophyletic clades.
However, the relationships between genera are unclear due to
low supporting values. Based on mitochondrial DNA, the
Mexican-clade represents a monophyletic sister-group to all
other included Eratigena species (Fig. 1). Within this Mexican-
clade, E. yarini sp. nov. and E. ednnmdoi sp. nov. represent a
closely related, well supported monophyletic subgroup, the
flexuosa-gxou^. Within the remaining species of the Mexican-
clade, the species E. mexicana (Roth, 1968) and E. tiaxcala
(Roth, 1968) are very closely related. A inexicana-group, as
suggested by morphological data, is not supported by mtDNA
data.

Morphological data. — The finding that the Mexican species
previously described in Tegenaria s. 1. are closely related to
Palearctic Eratigena species is supported by the following
morphological characters, which match the genus definition of
Eratigena Bolzern et al. (2013a): (1), dentition of the
retromargin of the chelicerae (six or more teeth, decreasing
in size proximally); (2), the PMS bearing one conspicuously
prominent spigot; (3), colulus developed as a trapezoidal plate
with distal margin w-shaped; (4), absence of a retroventral
ridge on male palpal tibia; (5), presence of a transverse hyaline
(lamelliform) ridge on the conductor; and (6), presence of
appendages at the genital ducts in females {mexicana-gxoup).
Therefore, all Mexican Tegenaria species are here transferred
from Tegenaria to Eratigena.

Based on genital morphology (for details see Taxonomy
section), the Mexican Eratigena species can be divided into a
southern (the /?e.YMOja-group, also supported by mtDNA) and
a northern species group (the mexicana-gxoup) .

Tegenaria chiricahuae Roth, 1968, the only species exclu¬
sively known from Northern America (USA), differs mor¬
phologically from the Mexican species in all characters
mentioned above and matches the definition of Tegenaria.

Therefore, it remains the single endemic representative of the
genus Tegenaria in the Western Hemisphere.

SYSTEMATICS

Family Agelenidae C.L. Koch, 1837
Genus Tegenaria Latreille, 1844

Tegenaria Latreille, 1844: 134. Full synonymy: see World
Spider Catalog (2015).

Type species. — Araneus doniesticus Clerck, 1757, by subse¬
quent designation of Kluge (2007).

Diagnosis. — A detailed diagnosis for the genus Tegenaria
was provided by Bolzern et al. (2013a: 774-775).

Distribution. — The currently 104 described species (World
Spider Catalog 2015; this publication) are primarily distribut¬
ed in the Mediterranean Region, spreading towards Asia.
Tegeneria domestica (Clerck, 1757), T. pagana C. L. Koch,
1840 and T. parietina (Fourcroy, 1785) expanded their
distribution areas extensively, most likely due to introductions
by humans. In the Western Hemisphere, T. chiricahuae
represents the only known endemic species.

Tegenaria chiricahuae Roth, 1968
Figs. 8-16

Tegenaria chiricahuae Roth, 1968: 7, figs. 9-11.

Type material. — Holotype male. UNITED STATES: Arizo¬
na: Cochise Co., Chiricahua Mountains, Cave Creek Canyon,
4.83 km W. of Portal, 28 November 1963, V. Roth (AMNH).

Paratypes. UNITED STATES: Arizona: 1 9 allotype, same
data as holotype except 30 June 1963 (AMNH); 1 9, same
data except 30 November 1962 (MNHN).

Other material examined. — UNITED STATES: Arizona: 1
9, Cochise Co., Chiricahua Mountains, Cave Creek Canyon,
small cave 3.22 km W. of Portal, 2 June 1972, G. Dingerkus
(AMNH: AB1155); 1 c?, 1 9, Huachuca Mountains, Carr
Canyon, 1829 m, in cave with some litter, 23 March 1964, L.
La Pre, M. Eells (AMNH: AB1180). New Mexico: 1 d, “new
cave”, 18 December 1976, P. Strinati (MSCNV); 1 <5, 4 9,
Eddy Co., Carlsbad Caverns National Park, Midnight
Canyon, Ringtail Cave (Flea Cave), 26 May 1973, Wm.
Elliott, W.C. Welbourn (AMNH: AB1150); 1 (?, 1 9, same
data except Arch Cave, 27 November 1975, W.C. Welbourn
(AMNH: AB1121); 1 S, same data except Dome Cave, 15
February 1975 (AMNH: AB1149); 2 9, same data except
Helen’s Cave, 31 August 1974 (AMNH: AB1152); 1 6, same
data except cave, goat trap, 19 February 1976 (AMNH).
Texas: 2 9, Culberson Co., Guadalupe Mountains National
Park, cave, upper sloth, 17 April 1976, W.C. Welbourn
(AMNH).

Diagnosis. — Male Tegenaria chiricahuae can be separated
from all other Tegenaria species by the simple RTA (Figs. 13,
16), the large median apophysis with a pocket-like distal
sclerite and the distally keel-shaped conductor tip (Figs. 14,
15). Females can be distinguished from other species by the
distinct conformation of the epigyne and vulva (Figs. 8-12).

Description. — Essential information was provided by Roth
(1968).

108

JOURNAL OF ARACHNOLOGY

■ Amaurobius fenestralis

' Amaurobius ferox
100 Callobius sp.
- Callobius ko
99

:oreanus

Draconarius coreanus
Draoonarius kayasanensis

Pireneitega spinivulva
Tegecoelotes secundus

Histopona torpida
loot Textrix cf. caudata
' Textrix caudata
Textrix denticulata
■1^1 Maimuna cretica

Lycosoides coarctata
Agelena canadensis
)e gideoni

Novalena sp. mex13
Novalena sp. Huix
Novalena sp. mex36
Rualena goleta
- Rualena cruzana

Novalena intermedia
Calilena califomica
Calilena restricta

Calilena stylophora
mnr Hololena curta
‘ • Hololena sp. 1
Hololena nedra
Hololena sp. 2
Hololena adnexa
Melpomene sp. mex25
“ Melpomene sp. mex36
Tortolena giaucopis mex34
Tortolena giaucopis mex10
Agelena limbata
Agelena labyrinthica
— — Allagelena koreana
Allagelena gracilens
Allagelena difficilis
Barronopsis texana
Barronopsis barrowsi
Agelenopsis utahana
Agelenopsis oregonensis
' Agelenopsis spatula
Agelenopsis afeenae
Agelenopsis aperta
— — Agelenopsis iongistyla
Agelenopsis Oklahoma
Agelenopsis naevia
Agelenopsis emertoni
Agelenopsis pennsylvanica
Agelenopsis potten

Aterigena aculeata
na ligurica
Malthontca oceanica

Tegenaria domestica
'■“lenaria ariadnae
egenaria ariadnae
Tegenaria vankeerorum
Tegenaria haspeti
- Tegenaria dalmatica
Tegenaria dalmatica
Tegenaria campestris
99 1 Tegenaria ferruginea

Mj— — Tegenaria parietina
■ Tegenaria parietina
Tegenaria tndentina
Tegenaria eleonorae
enaria parmenidis

Iwogumoa songminjae

Tegenaria

r— - legenana parmenidis
1 lOOp Tegenaria parmenidis
Tegenaria parmenidis
Eratigena aft. herculea
Eratigena sardoa

Eratigena

ciauyeiia leiiisnea

—— Eratigena incognita
1,00 [ Eratigena agrestis

Eratigena atrica (saeva)

Eratigena atrica

• Eratigena.atri,ca,^, . .

• Eratigena queretaro n. sp ' '

Erateria queretaro n. sp. ^ .

Eraticifriayarini n. sp. ' • ;

.ratigena edmui idol n. sp.' :■

Eratigena edmundoi n. sp.'
iratigena decora . . .
Erangeria cavema: ' '

Eraflgena guanato n, sp. . :

' Eratigena mexicana- 0
Eratigena Haxcala :
Eratigena tlaxcala" .
Eratigena mmandoi n. so,'
Eratigena xilitla n. sp.

Eratigena xilitia n. sp.'

■ Eratigena rothi .. -
Eratigena rathl.- _ . .:.

Mexican -clade^

. ' 's. , ,'1

O

CB

3

3

03

(D

3

OJ

C»

>

CQ

2.

(B

g

CL

03

03

Figure 1. — Best maximum likelihood tree of mitochondrial genes (COl, NADH, 16S). Bootstrap values are given left and above nodes. Clades
with jackknife support higher than 50 % (+) or 85 % (+-I-) from maximum parsimony analysis of the same alignment are indicated left and below
nodes.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

109

Distribution. — Reported from several caves in Arizona and
New Mexico (United States).

Tegeitaria domestica (Clerck, 1757)

Araneus domesticus Clerck, 1757: 76-79, pi. 2, tab. 9, figs. 1-4
(in part). Full synonymy: see Roth (1968) and Bolzern et
al. (2013a).

Diagnosis. — Male Tegenaria domestica can be separated
from all other Tegenaria species by the distinct RTA (Roth
1968: figs. 14, 15), the truncated terminal end of the embolus,
and the terminally bifid conductor (Bolzern et al. 2013a: fig. 16
W, X). Females differ in having a strongly sclerotized
posterior sclerite with the anterior margin concave in
combination with a simple, subglobular vulva (Bolzern et al.
2013a: figs. 2 f, 18 b, c).

Distribution. — Introduced from Europe. Cosmopolitan
(synanthropic species).

Tegenaria pagana C.L. Koch, 1840

Tegenaria pagana C.L. Koch, 1840: 31, pi. 262, figs. 612, 613.
Full synonymy: see Roth (1968) and Bolzern et al.
(2013a).

Diagnosis. — Male Tegenaria pagana can be separated from
all other Tegenaria species by the transversally arranged
conductor, the elongated finger-shaped distal sclerite of the
MA, and the distinct RTA (Roth 1968: figs. 30, 31; Bolzern et
al. 2013a: fig. 28 K-L). Females show a high variability in
epigynal morphology but differ from others in having a
protruding suboval median plate with distinct anterolateral

pockets, and a distinct triple twisted vulva (Bolzern et al.
2013a: figs. 28 P-W).

Distribution. — Europe to Central Asia, introduced to
North- and South America and New Zealand.

Tegenaria parietina (Fourcroy, 1785)

Aranea parietina Fourcroy, 1785: 533. Full synonymy: see
Bolzern et al. (2013a).

Diagnosis. — Tegenaria parietina specimens are most similar
to specimens of Tegenaria ferruginea (Panzer, 1 804) but differ
from all other species in having a unique RTA and a very
strongly U-shaped anterior margin of the posterior sclerite in
females (Bolzern et al. 2013a: fig. 21 N-R). Males can be
separated from T. ferruginea by the much shorter conductor,
females by the much less convoluted vulva.

Distribution. — Introduced from Europe. In the Western
Hemisphere, reported from the West Indies to Argentina.

Genus Eratigena Bolzern, Burckhardt & Hanggi, 2013
Eratigena Bolzern, Burckhardt & Hanggi, 2013: 738

Type Species. — Tegenaria atrica C. L. Koch, 1843, by
original designation.

Diagnosis. — A detailed diagnosis for the genus Eratigena
was provided by Bolzern et al. (2013a; 738-741).

Distribution. — The currently 34 described species (World
Spider Catalog 2015; this publication) are primarily distribut¬
ed in the West Mediterranean Region, but with representatives
reaching as far east as Laos and 15 endemic species in Mexico.
Eratigena atrica (C. L. Koch, 1843) and E. agrestis (Walck-
enaer, 1802) were introduced into Northern America.

KEY TO THE NEARCTIC SPECIES OF ERATIGENA

1 male palpal tibia with short dorsal spike, median apophysis pocket-like; female vulva with strongly convoluted and

enclosed duct . 2

male palpal tibia without short dorsal spike, median apophysis protruding; female vulva with duct not enclosed . 3

2 male with massive and broad conductor with strongly sclerotized, pointed terminal appendages; female epigyne with

protruding posterior sclerite and deep anterior cavity . agrestis

male with strong conductor, terminally with elongated tip; female epigyne with large median area without protuberance

or cavity . atrica

3 male conductor distally distinctly elongated (Figs. 27, 34, 52, 64); female with flattened or coiled copulatory duct without

appendages (Figs. 19, 41, 45, 61) . flexiiosa-group, 4

distal portion of male conductor not distinctly elongated (Figs. 79, 82, 94, 112, 136, 142, 160, 166, 175, 187, 196, 208);
female with short and straight copulatory duct with appendages (Figs. 73, 87, 101, 124, 133, 147, 149, 158, 185, 192, 206,

218) . . . mexicana-group, 1

4 carapace length smaller than 3 mm; subtegular sperm duct of male bulb c-shaped (Fig. 64); copulatory duct only

flattened, not coiled (Fig. 61) . yarini

carapace longer than 3 mm; subtegular sperm duct of male bulb moderately s-shaped (Figs. 27, 34, 52); copulatory duct
coiled (Figs. 19, 41, 43) . 5

5 tegular sperm duct strongly undulated (arrow in Fig. 34); epigynal atrium bordered by pronounced broad ridge (Fig. 38)

. . . . fiexuosa

tegular sperm duct almost straight (Figs. 27, 52); epigynal atrium not bordered by protruding ridge (Figs. 18, 42) . 6

6 median apophysis long, with long triangular distal sclerite (Figs. 26, 28); copulatory duct with only one coil (Fig. 19)

. ednnmdoi

median apophysis very short, with reduced distal sclerite (arrows in Figs. 51-53); copulatory duct with two coils (Figs.
43-56) . . florea

1 carapace longer than 5 mm; legs exceptionally long (patella-tibia length leg I > 9 mm); indistinct abdominal pattern,

grayish . . selva

carapace shorter, if equally long, legs shorter and with distinct abdominal pattern . 8

110

JOURNAL OF ARACHNOLOGY

8 eyes at least moderately reduced (Figs. 66, 84) . . . . . . . . . . . . . 9

eyes well developed . . . . . . . 10

9 eyes strongly reduced (Fig. 84) . . . . . caverna

eyes moderately reduced (Fig. 66) . . . blanda

10 AME larger than PME (Figs. 163, 164, 212) . . . . 11

AME equally sized or smaller than PME . . . . . . . 12

1 1 CL shorter than 4 mm; male with distal sclerite of MA spoon-shaped, rounded (Figs. 208, 209); female with epigynal

posterior sclerite dumbbell-shaped (Fig. 215) . . . xililla

CL longer than 4.5 mm; male with distal sclerite of MA long, subtriangular and pointed (Figs. 165, 167); female with
epigynal posterior sclerite as in Fig. 170 . . . rothi

12 male pedipalp with elongated femur and tibia (Fig. 140); female with posterior membrane (internal posterior limit of

genital area) distinctly protruding anteriad (Figs. 148, 149) . . . mexicana

male palpal femur and tibia not elongated; female with posterior membrane only moderately protruding, sometimes with
median notch . . . . . . . . . . . . . . . .... 13

13 RTA with dorsal branch distally hook-shaped (Fig. 136); female with dumbbell-shaped posterior sclerite (posterior and

anterior margins concave, Fig. 131) . . . . . . . . guanato

RTA with dorsal branch distally pointed or truncated; posterior epigynal sclerite with anterior margin concave only. . 14

14 MA with distal sclerite distally pointed (Figs. 95, 161); female with prominent or elongated appendages at genital duct

(Figs. 99-101, 158) . 15

MA with distal sclerite spoon-shaped, distally rounded; female with subcircular, less prominent appendages at genital
duct . . . 16

15 MA with distal sclerite long triangular, sharply pointed (Figs. 159, 161); female vulva with arc-shaped anterior part (Fig.

158) . queretaro

MA with distal sclerite subtriangular (Figs. 93, 95); female with distinctly elongated appendages at genital duct (Figs. 99-
101) . . . . . . . . . . . . . decora

16 sternum with distinct pale patch only at the anterior half (Fig. 103); PLS with distal segment as long as basal segment
(Fig. 104); sperm duct at tegulum without distinct curve (Fig. Ill); female with semicircular posterior sclerite (Fig. 105)

. fernandoi

sternum with different pattern; PLS with distal segment longer than basal segment (Fig. 118); sperm duct at tegulum with
distinct curve (Fig. 195); female posterior sclerite with anterior margin concave, posterior margin straight . 17

17 sternum with pale median line; MA with distal sclerite narrow, connection between tegulum and conductor narrow (Figs.

195-197); female as in Figs. 202-206 . . . . . . . tlaxcala

sternum without pattern; MA with distal sclerite broad, connection between tegulum and conductor broad (Figs. 115,
116); female as in Figs. 122-125 . . gerlschi

Emtigena agrestis (Walckenaer, 1802)

Aranea agrestis Walckenaer, 1802: 216. Full synonymy: see
Bolzern et al. (2013a).

Diagnosis. — Male Eratigena agrestis are similar to speci¬
mens of E. atrica in having a pocket-like (Roth 1968: “shell¬
like”) median apophysis but differ from all other Eratigena
species in having a massive and broad conductor with strongly
sclerotized, pointed terminal appendages (Bolzern et al. 2013a:
figs. 8 C-D, 9 A-B). Females differ in having a distinct
epigyne with protruding posterior sclerite and deep anterior
cavity (Bolzern et al. 2013a: fig. 9 D).

Distribution. — Introduced from Europe. In the Western
Hemisphere, reported from several north-western states and
New York (USA), and south-western parts of Canada.

Eratigena atrica (C. L. Koch, 1843)

Tegenaria atrica C. L. Koch, 1843: 105, fig. 825. Full
synonymy: see Bolzern et al. (2013a).

Diagnosis. — Eratigena atrica specimens are similar to
specimens of E. agrestis in having a pocket-like median
apophysis, but differ from all other species in having the
strongly sclerotized, finger-shaped and pointed dorsal branch

of the RTA originating on a protuberance, and a strong
conductor (Bolzern et al. 2013a: fig. 9 J-O). Females differ in
having the large epigynal area as long as wide with distinct
‘pseudo teeth’, and having a uniquely shaped vulva (Bolzern et
al. 2013a: fig. 10 A-F).

Distribution. — Introduced from Europe. In the Western
Hemisphere, reported from the north-western United States
and southern Canada (from east to west).

THE FLEXUOSA-GROVP

The flexiiosa-group comprises four species: E. edmundoi sp.
nov., E.flexuosa (F. O. Pickard-Cambridge, 1902) comb, nov.,
E. florea (Brignoli, 1974) comb, nov., and E. yarini sp. nov.
The elongated distal part of the conductor (Figs. 27, 34, 52,
64) and the strongly elongated and coiled copulatory duct
(anterior part, Figs. 19, 41, 45, 61) separate them from species
of the mexicana-gmup.

Eratigena edmundoi sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.
org:act:67CC827B-EF04-4C8F-AB60-6D7C7340C29A
Figs. 2, 3, 17-29

Type material. — Holotype female. MEXICO: Veracruz: Pico
de Orizaba Volcano, Atotonilco de Calcahualco, plot 1, 2300

BOLZERN & HANGGI^NEARCTIC TEGENARIA AND ERATIGENA

111

Figures 2-7. — Photographs of habitats and webs. 2 & 3, Eraligena edmundoi sp. nov. from Ajalpan, street between Pala and Nicolas Bravo,
2619 m (Mexico); 4 & 5, Eratigena decora Gertsch, 1971 from Xilitla, Ahuacatlan, ESE. of Potrerillos, Cueva de Potrerillos, 1181 m (Mexico); 6,
Eratigenci queretaro sp. nov. from Pinal de Amoles, at road 120 between Jalpan de Serra and Pinal de Amoles, close to La Curva del Chuveje, 20
km W. of Jalpan, 1288 m (Mexico); 7, Eratigena caverna Gertsch, 1971 from Penamiller, Cueva Puerto del Leon, 6.5 km SE. of Rio Blanco, 2484
m (Mexico).

m, 14 February 2012, Lab. Aracnologia, F. Alvarez Padilla
(FC-UNAM; AB1340).

Paratypes. MEXICO: Veracruz: 1 S , same data as holotype
(FC-UNAM; ABl 340); 1 5 , same data (FC-UNAM: ABl 343:
accession-nr. LN887151, LN887174); 1 S, same data (FC-
UNAM; AB1338).

Other material examined. — MEXICO: Puebla: 19,3 juv.,
Ajalpan, street between Pala and Nicolas Bravo, 2619 m, oak-
pine forest, at terrain break at steep slope near path in the
forest, 7 October 2014, A. Bolzern, E. Gonzalez Santillan
(NMB-ARAN-27500: AB1276); 1 d, 4 9, 2 juv., same data

except 2545 m, pine forest, at terrain break (NMB-ARAN-
27501 to 27502: AB1275, AB1255: accession-nr. LN887150,
LN887173); 2 9, Zoquitlan, 2nd river cave, 31 December
1977, P. Strickland (AMNH: ABl 148). Veracruz: 1 9, Volcan
San Martin, near San Andres, 1524 m, 14 July 1953, C.J.
Goodnight (AMNH: ABl 168).

Etymology. — The specific name is a patronym in honor of
Edmundo Gonzalez Santillan, a Mexican arachnologist,
expedition guide and friend.

Diagnosis. — Male E. edmundoi sp. nov. specimens differ
from others of the group by the long triangular distal sclerite

112

JOURNAL OF ARACHNOLOGY

at the median apophysis (Figs. 27, 28; rather than spoon
shaped in E. yarini sp. nov. and E. fiexiiosa, moderately
reduced in E. florea). They differ from E. yarini sp. nov. by the
larger size (CL longer than 3.5 mm; in E. yarini sp. nov.
shorter than 3 mm), and by the s-shaped subtegular sperm
duct (Fig. 27; rather than c-shaped), and from E. flexuosa by
the only moderately undulated tegular sperm duct (Fig. 27;
rather than strongly undulated). Female specimens differ from
all others of the group by the single coiled copulatory duct
(Fig. 19; rather than only flattened in E. yarini sp. nov., double
coiled in E. flexuosa and E. florea). In addition, specimens of
E. edmimdoi sp. nov. differ in having the distal segment of the
PLS only proximally darkened (Fig. 17).

Description. — Measurements: Male (paratype): CL 4.0, CW
3.17, STL 1.97, STW 1.93, OL 4.27, OW 2.6. Leg I (6.8, 1.73,

6.4, 6.8, 3.3), II (5.87, 1.67, 3.67, 5.8, 2.93), III (5.27, 1.33, 4.07,

5.4, 2.4), IV (6.47, 1.6, 5.53, 7.4, 3.33), Pedipalp (1.83, 0.67,
1.0, 1.67), bulbL 1.0. Female (holotype): CL 4.01, CW 3.0,
STL 1.8, STW 1.8, OL 5.33, OW 3.87. Leg I (5.2, 1.53, 4.66,
4.93, 2.6), II (4.27, 1.4, 3.53, 4.27, 2.0), III (3.93, 1.2, 3.2, 3.93,

1.87) , IV (5.2, 1.4, 4.2, 5.4, 2.27). Pedipalp (1.77, 0.73, 1.23,

1.87) . EPL 0.33, EPW 0.63. Eyes; eye rows moderately
procurved (Fig. 23). PME 0.21, PLE 0.26, AME 0.24, ALE
0.23. Eye distances: PME-PME 1 x PME, PME-AME 0.5-
0.75 X PME, PME-PLE 0.5 x PME, PME-ALE 0.75-1 x
PME, AME-AME 0.5 x AME, AME-ALE 0.25 x AME.
CLYl 1.5 X AME, CLY2 0.5 x ALE. Male pedipalp: RTA
with two branches, lateral branch simple, lobe-like, moder¬
ately protruding, dorsal branch strongly sclerotized, narrow
finger shaped, sharply pointed. Short dorsal spike on palpal
tibia absent. Embolus length about 3.5 x CB, originating at
7-8 o’clock position, distal tip at 3-4 o’clock position.
Conductor lamelliform, distal portion (DP) distinctly elongat¬
ed, lateral margin folded (Figs. 27, 28). Terminal end of
conductor strongly elongated, pointed. Transversal ridge (TR)
of conductor expressed as hyaline ridge (Fig. 27). Conductor
membranously connected to tegulum. MA originating at 4-5
o’clock position, protruding, longer than wide, distal sclerite
(DS) translucent, long triangularly shaped (Figs. 27, 28). MA
membranously connected to tegulum. Epigyne and vulva:
Epigyne medially with a pale, hyaline area (Fig. 18). Posterior
sclerite protruding anteroventral as moderately sclerotized bar
with anterior margin concave (semicircular. Fig. 18), posterior
membrane (PM, internal posterior limitation of genital area)
notched (Fig. 19). CO laterally of posterior sclerite. Epigynal
‘pseudo teeth’ present, small (Fig. 20). Vulva consists of
combined narrowly convoluted duct, CD less sclerotized, with
one coil, without appendages, RC stronger sclerotized (Fig.
19). FD only represented by small, leaf-shaped appendages
(Fig. 22). Other important characters'. Cheliceral promargin
with 4-5, retromargin with 7-8 teeth, more proximally, the
teeth become smaller. Colulus developed as trapezoidal plate
with distal margin w-shaped. PMS bearing one conspicuously
prominent spigot. PLS with distal segment nearly as long as
basal segment (Fig. 25). Trichobothria on cymbium and palpal
tarsus absent. Tarsal trichobothria at leg I 6-7. Small teeth on
paired claws of leg I 10-1 1. Leg spination: male pedipalp (2-0-
0 or 2-1-0, 2-0-0, 1-1+lp-O), female pedipalp (3-0-0, 2-0-0, 2-2-
0), leg femora (1 -3-2-0, 1 -2-2-0 or 1 -3-2-0, 1 -2-2-0, 1-0- 1-0 or 1-
0-2-0 or 1-1-2-0), patellae (all 2-0-0), tibiae (0-0-0-4p or 0-1-0-

l+3p, 0-0-0- lp+2+lp or O-l-O-lp-l-2+lp, l-2-2-2+2p or 2-2-2-
2+2p, 2-2-2-3+lp), metatarsi (0-0-0-4p+l, 0-l-0-4p+l, 0-3-2-
4p+l or 0-3-3-4p+l, 0-3-3-3p-t-l-l-lp+l), tarsi (0, 0, 0-2-3-0, 0-2-
3-0). Coloration'. Carapace with two longitudinal symmetrical
dark bands, irregularly expressed, margins narrowly darkened,
head region dorsolaterally with two distinct, longitudinally
curved dark bands (Fig. 17). Chelicerae frontally with distinct
dark patches (Fig. 23). Sternum darkened anteriorly, some¬
times also posteriorly (less distinct) with pale median band
(Fig. 24). Opisthosoma dark, black, with reddish pale median
band, anteriorly bordered by black bands or almost com¬
pletely black, posteriorly with yellowish, reddish chevrons,
indistinct (Fig. 17). Legs irregularly annulated. Colulus
laterally darkened. ALS moderately, basal segment of PLS
distinctly darkened, distal segment of PLS only proximal half
darkened.

Distribution. — Reported from the two states Puebla and
Veracruz (Mexico).

Eratigena flexuosa (F.O. Pickard-Cambridge, 1902), comb.

nov.

Figs. 30-41

Tegenaria flexuosa F.O. Pickard-Cambridge, 1902: 334, pi. 31,
fig. 34; Roth, 1968: 14, figs. 19, 20.

Type material. — Holotype male. MEXICO: Guerrero'. Omil-
temi (“Omilteme” on label), 28 April 1900, F.D. Godman
(NHML).

Other material examined. — MEXICO: Guerrero: 1 $ ,
Chilpancingo, Cueva del Borrego, 3.5 km E of Omiltemi,
2623 m, pine-oak forest, 23 July 2009, A. Valdez, O. Francke,

H. Montano, C. Santibanez, T. Palafox, C. Trajano (CNAN:
ABl 199); 3 2 , same data except Cueva del Borrego, 2 km E of
Omiltemi, 1835 m, vegetation outside the cave, 20 June 2007,
O. Francke, H. Montano, L. Escalante, A. Ballesteros
(CNAN: ABl 202).

Diagnosis. — Male E. flexuosa specimens differ from others
of the group by the strongly undulated tegular sperm duct
(Fig. 34; rather than moderately undulated). They differ from
E. edmimdoi sp. nov. and E. florea by the spoon-like distal
sclerite of the MA (Figs. 33, 34; rather than long triangular or
moderately reduced). They differ from E. yarini sp. nov. by the
larger size (CL longer than 3.5 mm; in E. yarini sp. nov.
shorter than 3 mm), and by the s-shaped subtegular sperm
duct (Fig. 34; rather than c-shaped). Female specimens differ
from E. edmimdoi sp. nov. and E. yarini sp. nov. by the double
coiled copulatory duct (Fig. 41; rather than single coiled or
only flattened), and from E. florea in having the epigynal
median area bordered by a pronounced broad ridge (Fig. 38).

Description. — Essential information for the male was
provided by F.O. Pickard-Cambridge (1902). Measurements:
Female (ABl 199); CL 3.75, CW 2.67, STL 2.0, STW 1.63, OL
3.67, OW 2.33. Leg I (6.13, 1.60, 5.67, 6.00, 2.8), II (5.00, 1.47,
4.47, 5.13, 2.33), III (4.40, 1.33, 3.60, 4.87, 2.20), IV (5.27,

I. 47, 5.0, 6.87, 2.40). Pedipalp (1.97, 0.67, 1.17, 2.0). EPL 0.56,
EPW 0.88. Eyes: anterior eye row straight, posterior eye row
procurved (Fig. 37). PME 0.17, PLE 0.19 , AME 0.15, ALE
0.22. Eye distances; PME-PME 0.75 x PME, PME-AME 1 x
PME, PME-PLE 1 X PME, PME-ALE 1.5 x PME, AME-
AME 0.5 X AME, AME-ALE 0.5 x AME. CLYl 1.5 x AME,

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

113

CLY2 1.25 X ALE. Epigyne and vulva: Epigyne with hyaline
area subrectangular, bordered by pronounced broad ridge
(Fig. 38). Posterior membrane without notch (Fig. 41). CO
anterolateral to posterior sclerite. Epigynal ‘pseudo teeth’
indistinct (Fig. 39). Vulva consists of combined narrowly
convoluted duct, CD less sclerotized, almost double coiled,
without appendages, RC more strongly sclerotized (Fig 41).
FD only represented by small, leaf-shaped appendages. Other
important characters: Cheliceral promargin with 4, retromar-
gin with 6 teeth, more proximally, the teeth become smaller
(Fig. 36). Colulus developed as trapezoidal plate with distal
margin moderately w-shaped. PMS bearing one conspicuously
prominent spigot. PLS with distal segment moderately longer
than basal segment (Fig. 32). Trichobothria on cymbium and
palpal tarsus absent. Tarsal trichobothria of leg I 7. Small
teeth on paired claws of leg I 10-11. Leg spination: female
pedipalp (1-0-0 or 2-0-0, 2-0-0, 2-1-2), leg femora (1-3-1-0 or 1-
3-2-0, 1 -2-2-0 or 1 -3-2-0, 1 -0-2-0 or 1- 1-2-0, 1-0- 1-0), patellae
(all 2-0-0), tibiae (O-O-O-l or O-l-O-l, 0-1-0-lp or 1-1-0-lp, 2-1-
l-2p, 2-1 -1-1), metatarsi (0-0-0- Ip+l+lp+l, 0-0-0- lp+2+lp-|-l,
0-1-1 -4p4-l or 0-l-2-4p-|-l, 0-2-l-4p-|-l or 0-2-2-4p-|-l ), tarsi (0,
0, 0-0- 1-0, 0-0-2-0). Coloration: Carapace with two longitudi¬
nal symmetrical dark bands, irregularly and indistinctly
expressed, margins narrowly darkened, head region dorsolat¬
eral with two indistinct, longitudinally curved dark bands
(Fig. 30). Chelicerae frontally without dark patches (Fig. 37).
Sternum darkened, with indistinct pale median band (Fig. 31).
Opisthosoma dark brownish to black, with yellowish pale
median band, anteriorly with two longitudinally dark bands,
posteriorly with yellowish chevrons (Fig. 30). Legs irregularly
annulated. Colulus pale or laterally moderately darkened.
ALS and both segments of PLS moderately darkened.

Distribution. — Reported only from the state of Guerrero
(Mexico).

Comments. — Based on the drawing provided by F.O.
Pickard-Cambridge (1902), Roth stated that the median
apophysis is lacking (Roth 1968: 14), but this was a
misinterpretation (see Figs. 33-35). Males and females have
never been collected together (the only known male is the
holotype). However, based on their morphological similarity
and their collection close to the type locality, these females are
tentatively placed with this species.

Eratigena ftorea (Brignoli, 1974), comb. nov.

Figs.42~53

Tegenaria florea Brignoli, 1974: 228, fig. lOA, C.

Tegenaria florea: Brignoli, 1974: 231, fig. lOB.

Type material. — Holotype female. MEXICO: Chiapas:
Comitan, Cueva de las Florecillas, 2265 m, 18 March 1971,
A. Zollini (MCSNV).

Other material examined. — MEXICO: Chiapas: 1 9 , Ama-
tenango, Cueva I de Tulanca, 2200 m, 4 March 1971, V.
Sbordoni (MCSNV); 1 $ , Comitan, Cueva de la Cruz Belen,
2210 m, 19 March 1971, R. Argano (MCSNV, sub prope
florea)', 1 ?, 5 miles West of San Cristobal, 24 August 1966,
W. & J. Ivie (AMNH: AB1112); 1 $, same data except 2438
m, pine-oak forest, 16 August to 3 September 1969, S. & J.
Peck (AMNH: AB1166); 1 d, Laguna Belgica Educational
Park, 16 km NW. of Ocozocoautla de Espinosa, 14 June 1990,

H. Howden (AMNH: AB1141). Oaxaca: 1 9, Santa Marfa
Tlahuitoltepec, Distrito Mixes, 2032 m, pine forest, disturbed,
14 September 2009, A. Valdez, C. Santibanez, R. Paredes
(CNAN: AB1193); 1 9, Yautepec, Santo Tomas Teipan, 2346
m, cloud forest, 20 March 2002, S. Reynaud (CNAN:
AB1200).

Diagnosis. — Male E. florea specimens differ from others of
the group by the moderately reduced distal sclerite of the
median apophysis (Figs. 51, 52; rather than long and
triangular in E. edmundoi sp. nov., spoon shaped in E. yarini
sp. nov. and E. flexuosa). Female specimens differ from
E. edmundoi sp. nov. and E. yarini sp. nov. by the double
coiled copulatory duct (Fig. 43; rather than single coiled or
only flattened), and from E. flexuosa in having the epigynal
median area not bordered by a pronounced broad ridge (Fig.
42).

Description. — Essential information for the female was
provided by Brignoli (1974). Measurements: Male (AB1141):
CL 2.9, CW 2.17, STL 1.42, STW 1.34, OL 3.17, OW 2.1. Leg
I (5.25, 1.15, 5.15, 5.8, 3.05), II (4.6, 1.0, 3.98, 4.5, 2.1), III (4.1,

I. 0, 3.65, 4.5, 2.25), IV (5.35, 1.0, 4.75, 6.4, 2.8), Pedipalp (1.3,
0.44, 0.56, 1.18), bulbL 0.84. Eyes: eye rows moderately
procurved (Fig. 48). PME 0.17, PLE 0.18 , AME 0.16, ALE
0.22. Eye distances: PME-PME 0.5 x PME, PME-AME 0.5 x
PME, PME-PLE 0.4 x PME, PME-ALE 0.7 x PME, AME
AME 0.5 X AME, AME-ALE 0.25 x AME. CLYl 1.5-2 x
AME, CLY2 0.75-1 x ALE. Male pedipalp: RTA with two
branches, lateral branch simple, lobe-like, moderately pro¬
truding, dorsal branch strongly sclerotized, narrow finger
shaped, sharply pointed. Short dorsal spike on palpal tibia
absent. Embolus length about 3.5 x CB, originating at 7-8
o’clock position, distal tip at 4 o’clock position. Conductor
lamelliform, distal portion distinctly elongated, lateral margin
folded (Figs. 51-53). Terminal end of conductor strongly
elongated, pointed. Transversal ridge of conductor expressed
as hyaline ridge. Conductor membranously connected to
tegulum. MA originating at 4-5 o’clock position, as long as
wide, distal sclerite translucent, plate like, moderately reduced
(Figs. 51-53). Other important characters: Cheliceral promar¬
gin with 4-5, retromargin with 7-8 teeth, more proximally, the
teeth become smaller. Colulus developed as rectangular to
trapezoidal plate with distal margin w-shaped. PLS with distal
segment slightly shorter than basal segment (Fig. 50).
Trichobothria on cymbium and palpal tarsus absent. Tarsal
trichobothria at leg I 6-7. Small teeth on paired claws of leg I
8. Leg spination: male pedipalp (2-0-0, 2-0-0, 1-1-0), leg
femora (1 -2-0-0, 1 -0-0-0 or 1-1 -0-0, 1 -0-0-0, 1 -0-0-0), patellae
(all 2-0-0), tibiae (O-O-O-l, O-O-O-l or 1 -0-0-1, 2-1-0-1, 2-1-1-1),
metatarsi (0-0-0- lp-fl-l-lp-l-1, 0-0-0- Ip-fl-t-lp-t-l or 0-0-0-
lp+2-|-lp-|-l, 0-2-1 -lp-|-l-|-2p-|-l, 0-2-l-Ip-|-2-|-lp-|-l), tarsi (0, 0,
0, O-O-l-O). Coloration: Carapace with two longitudinal
symmetrical dark bands, irregularly expressed, margins
narrowly darkened (Fig. 47). Chelicerae frontally with
indistinct dark patches (Fig. 48). Sternum indistinctly dark¬
ened, anteriorly with pale median band (Fig. 49). Opisthoso¬
ma dark, grayish brown, without reddish pigments, pale
median band, posteriorly with pale, yellowish chevrons,
indistinct (Fig. 47). Legs irregularly annulated, indistinct.
Colulus moderately darkened. ALS and both segments of PLS
darkened.

114

JOURNAL OF ARACHNOLOGY

Distribution. — Reported from several localities in the states
of Chiapas and Oaxaca (Mexico).

Comments. — Males and females have never been collected
together. However, based on their morphological similarity
and the collection site being within the distribution range of
the females, the male from Laguna Belgica Educational Park,
16 km NW. of Ocozocoautla de Espinosa, is tentatively placed
in this species.

Eratigem yarini sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.
org:act:FAFC2F99-20AC-439C-B177-E767115CA13B
Figs. 54-65

Type material. — Holotype female. MEXICO: Veracruz: Pico
de Orizaba Volcano, Atotonilco de Calcahualco, plot 1, 2300
m, 24 February 2013, Lab. Aracnologia, F. Alvarez Padilla
(FC-UNAM: AB1342).

Paratypes. MEXICO: Veracruz: 1 6 , same data as holotype
(FC-UNAM: AB1341); 1 d, 1 9, same data (NMB-ARAN-
27503 to 27504: AB1332, AB1334: accession-nr. LN887162,
LN887181).

Other material examined. — MEXICO: Oaxaca: 1 9, “El
Cumbre” on ridge E. of Cerro San Felipe, 2438 m, 28
September 1961, C.M. & M.R. Bogert (AMNH: AB1124).

Etymology. — The specific name is a patronym dedicated to
Yarin Bolzern, the second born child of the first author.

Diagnosis. — E. yarini sp. nov. specimens differ from others
of the group by the smaller size (CL shorter than 3.0 mm; all
others longer than 3.5 mm). Males differ by the c-shaped
subtegular sperm duct (arrow in Fig. 64; rather than s-shaped),
females by the only flattened copulatory duct (Figs. 59-61;
rather than single or double coiled).

Description. — Measurements: Male (paratype): CL 1.9, CW
1.5, STL 1.97, STW 1.93, OL 2.04, OW 1.26. Leg I (2.5, 1.0,
2.23, 2.02, 1.42), II (2.04, 0.68, 1.64, 1.78, 1.26), III (1.9, 0.62,
1.34, 1.8, 1.1), IV (2.67, 0.733, 2.2, 2.67, 1.4), Pedipalp (0.78,
0.32, 0.44, 0.68), bulbL 0.4. Female (holotype): CL 2.53, CW
1.83, STL 1.12, STW 1.14, OL 3.0, OW 2.12. Leg I (2.67, 0.87,
2.4, 2.4, 1.67), II (2.4, 0.7, 1.68, 1.88, 1.1), III (2.1, 0.68, 1.54,
1.9, 1.2), IV (2.87, 0.8, 2.28, 3.03, 1.3). Pedipalp (0.96, 0.42,
0.64, 1.08). EPL 0.38, EPW 0.58. Eyes: eye rows moderately
procLirved (Fig. 55). PME 0.1, PLE 0.12 , AME 0.08, ALE 0.1.
Eye distances: PME-PME 0.5-1 x PME, PME-AME 0.5-1 x
PME, PME PLE 0.5 1 x PME, PME-ALE 1 x PME, AME-
AME <0.5 X AME, AME-ALE <0.5 x AME. CLYl 2 x
AME, CLY2 0.5-1 x ALE. Male pedipalp: RTA with two
branches, lateral branch triangular, protruding, dorsal branch
strongly sclerotized, narrow finger shaped, sharply pointed
(Fig. 65). Short dorsal spike on palpal tibia absent. Embolus
length about 2 x CB, originating at 8 o’clock position, distal
tip at 4 o’clock position. Conductor lamelliform, distal portion
distinctly elongated, lateral margin folded (Figs. 63-65).
Terminal end of conductor strongly elongated, pointed.
Transversal ridge of conductor expressed as hyaline ridge.
Conductor membranously connected to tegulum. MA origi¬
nating at 6 o’clock position, protruding, longer than wide,
distal sclerite translucent, spoon-like (Figs. 63, 64). MA
membranously connected to tegulum. Epigyne and vulva:
Epigyne medially with a pale, hyaline area (Fig. 58). Posterior
sclerite with anterior margin strongly concave, posterior

membrane broadly notched (Fig. 61). CO anterolateral to
posterior sclerite. Epigynal ‘pseudo teeth’ present, small (Fig.
62). Vulva consists of combined narrowly convoluted duct,
CD flattened, medially elongated, without appendages, RC
stronger sclerotized (Figs. 59-61). FD only represented by
small, leaf-shaped appendages. Other important characters:
Cheliceral promargin with 4, retromargin with 5 (male) or 6
(female) teeth, most proximal tooth smaller. Colulus devel¬
oped as trapezoidal plate with distal margin w-shaped. PMS
bearing one conspicuously prominent spigot. PLS with distal
segment nearly as long as basal segment (Fig. 57). Tricho-
bothria on cymbium and palpal tarsus absent. Tarsal
trichobothria at leg I 5-6. Small teeth on paired claws of leg
I 7. Leg spination: male pedipalp (2-0-0, 2-0-0, 1-2-0), female
pedipalp (2-0-0, 2-0-0, 2-2-0), leg femora (2-2-0-0, 2-0-0-0, 2-0-

1- 0, l-O-l-O), patellae (all 2-0-0), tibiae (2-0-0- 1+1 p,2-l-0-2p, 2-

2- 1-3, 2-2-2-3), metatarsi (0-0-0-3p+l, 0-l-0-3p+l, 0-3-2-
lp+l+2p+l, 0-3-2- lp+l+2p+l), tarsi (0, 0, 0-1-2-0, 0-2-3-0).
Coloration: Carapace and head region with two broad
longitudinal symmetrical dark bands, with triangular darker
spots (Fig. 54). Chelicerae frontally with indistinct dark
patches (Fig. 55). Sternum darkened, distinct pale median
band, laterally with indistinct paler spots (Fig. 56). Opistho-
soma dark, brownish, sprinkled with small pale spots,
indistinct pale median band, posteriorly with yellowish
chevrons, indistinct (Fig. 54). Leg femora broad but moder¬
ately annulated, other segments moderately annulated, tarsi
pale. Colulus, ALS and PLS distinctly darkened (Fig. 57).

Distribution. — Reported only from the state of Veracruz
(the type locality) and one locality in the state of Oaxaca
(Mexico).

THE MEXICANA-GKOCV

The mexicana-gxow'p comprises 12 species: E. blanda
(Gertsch, 1971) comb, nov., E. caverna (Gertsch, 1971) comb,
nov., E. decora (Gertsch, 1971) comb, nov., E. fernandoi sp.
nov., E. gertschi (Roth, 1968) comb, nov., E. guanato sp. nov.,
E. mexicana (Roth, 1968) comb, nov., E. queretaro sp. nov.,
E. rothi (Gertsch, 1971) comb., nov., E. selva (Roth, 1968)
comb, nov., E. tlaxcala (Roth, 1968) comb, nov., and E. xilitla
sp. nov. The only moderately elongated distal portion of the
conductor (Figs. 79, 82, 94, 112, 136, 142, 160, 166, 175, 187,
196, 208) and the short, straight copulatory duct with mostly
distinct appendages (Figs. 73, 87, 101, 124, 133, 147, 149, 158,
185, 192, 206, 218) separate them from specimens of the
flexuosa-gvou'p.

Emtigena blanda (Gertsch, 1971), comb. nov.

Figs. 66-71

Tegenaria blanda Gtxisch, 1971: 105.

Type material. — Holotype female. MEXICO: Tamaulipas:
El Porvenir, Cueva de la Capilla, 13.5 km NW Gomez Farias,
28 January 1969, J. Reddell, R. Mitchell, F. Rose, J. George
(AMNH).

Other material examined. — MEXICO: Tamaulipas: 1 9,
Gomez Farias, Cueva de la Perra, 15 mi. NW Gomez Fan'as,
2164 m, 28 January 1968, J. Reddell, R. Mitchell, F. Rose, J.
George (AMNH: AB1153).

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

115

Diagnosis. — Females of E. blanda differ from all other
Nearctic Eratigena species in having moderately reduced eyes
(Fig. 66; rather than strongly reduced eyes in E. caverna, eyes
normally developed in all other species).

Description. — Essential information was provided by
Gertsch (1971).

Distribution. — Reported from two caves in the state of
Tamaulipas (Mexico).

Eratigena caverna (Gertsch, 1971), comb. nov.

Figs. 7, 81-89

Tegenaria caverna Gertsch, 1971; 106, figs. 158-160.

Type material. — Hoiotype male. MEXICO: Queretaro:
Penamiller, Cueva Puerto del Leon, 6.5 km SE Rio Blanco,
9 July 1967, J. Reddell, J. Fish, P. Russell (AMNH: AB1156).

Paratypes. MEXICO: Queretaro: 2 9 allotypes, same data
as hoiotype (AMNH: AB1156).

Other material examined. — MEXICO: Queretaro: 5 juv.,
same data as hoiotype except deep inside cave, 2484 m, 14
October 2014, A. Bolzern, E. Gonzalez Santillan (NMB-
ARAN-27505 to 27507: AB1244, AB1305, AB1284: accession-
nr. LN887148, LN887187).

Diagnosis. — Male and female E. caverna specimens differ
from all other West Nearctic Eratigena species by having
strongly reduced eyes (Fig. 84; rather than moderately reduced
in E. blanda, eyes normally developed in all other species).

Description. — Essential information was provided by
Gertsch (1971).

Distribution. — Reported only from a cave near Rio Blanco
in the state of Queretaro (Mexico).

Comments. — Specimens could not be detected at the
entrance of the cave but only very deep inside the cave. There,
they were quite abundant, building their webs (Fig. 7) between
large rocks which covered the lower part of the cave.
Interestingly, there was no tube- or funnel-shaped retreat
detectable in their webs. The spiders were just sitting in the
middle of their web on a circular, more densely woven patch.

Eratigena decora (Gertsch, 1971), comb. nov.

Figs. 4, 5, 90-101

Tegenaria decora Gertsch, 1971: 104, figs. 164, 165.

Type materia!. — Hoiotype male. MEXICO: San Luis Potosr.
Cueva de Potrerillos, 1.5 km W. of Ahuacatlan, 12 July 1967,
J. Reddell, J. Fish, P. Russell (AMNH).

Paratypes. MEXICO: San Luis Potosr. 7 9, same data as
hoiotype (AMNH: AB1167).

Other material examined. — MEXICO: San Luis Potosr. 1 S ,
1 9, Xilitla, Ahuacatlan, ESE. of Potrerillos, Cueva de
Potrerillos, 1181 m, cave entrance with tropical forest in the
middle of pasture land, at rock faces and woody vegetation, 12
October 2014, A. Bolzern, E. Gonzalez Santillan (NMB-
ARAN-27508: AB1237: accession-nr. LN887149); 2 (?, 5 9, 3
juv., same data (FC-UNAM: AB1240, AB1280).

Diagnosis. — Female E. decora specimen differ from all
others of the group in having elongated appendages at the
genital duct (Figs. 99-101). Males are similar to E. guanato sp.
nov., E. queretaro sp. nov., E. rothi and E. selva in having at
least a moderately pointed distal sclerite of the MA (Figs. 93,

95). They differ from E. guanato sp. nov., E. rothi and E. selva
in having a relatively slim, finger-shaped and simply pointed
dorsal branch of the RTA (rather than distally hook-shaped in
E. quanato sp., nov. strong and basally bent in E. rothi and E.
selva), and from E. queretaro sp. nov. in having a subtrian-
gular, moderately pointed distal sclerite of the MA (rather
than triangular and sharply pointed).

Description. — Essential information was provided by
Gertsch (1971).

Distribution. — Reported only from one cave in the state of
San Luis Potosi (Mexico).

Comments. — In 2014, specimens of E. decora were collected
at the entrance of Cueva de Potrerillos (Figs. 4, 5), the type
locality. Interestingly, this cave entrance, a large woody hole,
is located in the middle of pasture land, surrounded by cattle
and secured by a fence. It is remarkable that the webs of this
species were also attached to the woody vegetation, and not
exclusively to rocks (Fig. 5).

Eratigena fernandoi sp. nov.
http://zoobank.org/?lsid=urn;lsid;zoobank.
org:act:19066CFl-0625-4D40-8FB7-35E0CF74AD8B
Figs. 102-113

Type material. —Hoiotype male. MEXICO: Veracruz: Ato-
tonilco de Calcahualco, Pico de Orizaba Volcano, plot 2, 24
February 2013, Lab. Aracnologia FC-UNAM (FC-UNAM:
AB1335: accession-nr. LN887152, LN887175).

Paratypes. MEXICO; Veracruz: I 9, same data as hoiotype
except 14 February 2012 (FC-UNAM: AB1333).

Other material examined. — MEXICO: Veracruz: 3 6,
Huatusco, 7 km E Huatusco, cloud forest, 22 Jun 1983, S. &
J. Peck (AMNH: AB1137).

Etymology. — The specific name is a patronym in honor of
Fernando Alvarez Padilla, a Mexican arachnologist and active
and strong supporter of the current work.

Diagnosis. — Male and female of E. fernandoi sp. nov. differ
from related species in having a distinct pale patch only at the
anterior half of the sternum (Fig. 103). Males are most similar
to E. gertschi in having the posterior sclerite of the MA
broadly spoon-shaped and distally rounded (rather than
distally pointed in E. decora, E. guanato sp. nov., E. rothi
and E. selva), but differ from that species in having the distal
segment of the PLS as long as the basal segment (Fig. 104;
rather than distal segment longer than basal segment), and the
spermatic duct of the tegulum without a distinct curve (arrow
in Fig. Ill; rather than distinctly curved). Females differ by
the semicircular posterior sclerite (Fig. 105), and the anteriorly
differently convoluted genital ducts (Fig. 106).

Description. — Measurements: Male (hoiotype): CL 2.26,
CW 1.7, STL 1.06, STW 1.14, OL 3.0, OW 1.74. Leg I (4.0,
0.93, 3.8, 3.8, 2.33), II (3.57, 0.9, 2.97, 3.6, 2.03), III (3.13, 0.8,
2.63, 3.43, 1.7), IV (3.93, 0.93, 3.43, 4.35, 2.1), Pedipalp (1.2,
0.42, 0.68, 1.03), bulbL 0.44. Female (paratype): CL 3.2, CW
2.4, STL 1 .8, STW 1 .8, OL 3.3, OW 2.47. Leg I (4.25, 1.1, 3.75,
3.95, 2.3), II (3.87, 1.1, 3.23, 3.6, 1.76), III (3.3, 1.0, 2.73, 3.47,
1.6), IV (4.25, 1.0, 3.7, 4.65, 1.92). Pedipalp (1.36, 0.56, 0.94,
1.11). EPL 0.33, EPW 0.54. Eyes: anterior eye row straight,
posterior moderately procurved (Fig. 102). PME 0.13, PLE
0.14 , AME 0.12, ALE 0.17. Eye distances: PME-PME 1 x
PME, PME-AME 0.5-0.75 x PME, PME-PLE 1 x PME,

116

JOURNAL OF ARACHNOLOGY

PME-ALE 1.25 x PME, AME-AME 0.5-0.75 x AME,
AME-ALE 0.5 x AME. CLYl 2 x AME, CLY2 1.5 x ALE.
Male pedipalp: RTA with two branches, lateral branch simple,
lobe-like, moderately protruding, dorsal branch strongly
sclerotized, narrow finger shaped, sharply pointed (broken
off in Figs. 112, 113). Short dorsal spike on palpal tibia absent.
Embolus length about 1.5 x CB, originating at 9-10 o’clock
position, distal tip at 4 o’clock position. Conductor lamelli-
form, distal portion only moderately elongated, lateral margin
folded (Figs. 112, 113). Terminal end of conductor strongly
elongated, pointed. Transversal ridge of conductor expressed
as hyaline ridge (Figs. Ill, 112). Conductor membranously
connected to tegulum. MA originating at 5 o’clock position,
protruding, longer than wide, distal sclerite translucent, broad
spoon shaped. MA membranously connected to tegulum.
Epigyne and vulva: Epigyne medially with a pale, hyaline area,
oval (Fig. 105). Posterior sclerite protruding anteroventral as
moderately sclerotized bar with anterior margin concave
(semicircular, Fig. 105), posterior membrane notched (Fig.
106). CO anterolateral to posterior sclerite. Epigynal ‘pseudo
teeth’ present, small. Vulva consists of combined narrowly
convoluted duct, CD less sclerotized, with indistinct append¬
ages, RC stronger sclerotized (Figs. 106, 107). ED only
represented by small, leaf-shaped appendages. Other important
characters: Cheliceral promargin with 4, retromargin with 6
small teeth in two separated groups of 3, most proximal tooth
smaller. Colulus developed as trapezoidal plate with distal
margin moderately w-shaped (Fig. 104). PMS bearing one
conspicuously prominent spigot. PLS with distal segment
nearly as long as basal segment (Fig. 104). Trichobothria on
cymbium and palpal tarsus absent. Tarsal trichobothria at leg
I 6. Small teeth on paired claws of leg I 7-10. Leg spination:
male pedipalp (2-0-0, 2-0-0, 1-2-0), female pedipalp (3-0-0, 2-0-
0, 2-2-0), leg femora (1 -2-0-0 or 1-3- 1-0, 1 -2-2-0 or 1-1 -2-0, 1-0-
2-0 or 1 -2-2-0, 1 -0-2-0), patellae (all 2-0-0), tibiae (0-1 -0-1 or 1-
1-0-1, 0-1-0- 1 or 1-1-0- 1, 2-1 -1-1 or 2-2- 1-2, 2-2-2-3), metatarsi
(0-0-0-4|^l, 0-0-0-4|^l or 0-l-0-4p+l, 0-2-2-4p+l or 0-3-3-
4i^l, 0-3-3-4i^l), tarsi (0, 0, O-O-l-O, 0-1-3-0 or 0-2-2-0).
Coloration: Carapace with two longitudinal symmetrical dark
bands, irregularly expressed, margins narrowly darkened,
head region dorsolaterally with two distinct, longitudinally
curved dark bands (Fig. 109). Chelicerae frontally with
distinct dark patches (Fig. 102). Sternum darkened, anterior
half with pale median band (Fig. 103). Opisthosoma greenish
to dark gray, without reddish pigments, indistinct yellowish
median band, anteriorly bordered by dark bands, posteriorly
with yellowish chevrons, indistinct (Fig. 109). Legs irregularly
but distinctly annulated. Colulus completely darkened. ALS
moderately, basal segment of PLS distinctly darkened, distal
segment of PLS only proximal half darkened.

Distribution.- Reported only from two localities in the state
of Veracruz (Mexico).

Eratigena gertschi {Roth, 1968), comb. nov.

Figs. 1 14-125, cf. gertschi Figs. 72-80

Tegenaria mexicana gertschi Roth, 1968: 22, fig. 27.

Tegenaria gertschi Roth: Brignoli, 1974: 230.

Type material.- male. MEXICO: Nuevo Leon:

Resumidero (cave) de Pablillo at Hacienda Pablillo, 30 km

south of Galeana, 4 June 1966, J. Reddell, D. McKenzie
(AMNH).

Other material examined (of E. gertschi s. s.). — MEXICO:
Nuevo Leon: 1 9, Monterrey, Chipinque Mesa, small caves,
1645 m, 24 June 1969, S. & J. Peck, R. Norten (AMNH:
AB1106). Tamaulipas: 2 9, Gomez Farias, 6 miles NW. of
Gomez Farias, mine cave, March 1969, J. Reddell, C. Tucker
(AMNH: AB1161); 1 9, Rancho del Cielo, mine cave, 3 June
1967, R. Mitchell (AMNH: AB1135); 1 9, same data except
10 January 1971, J. Reddell (AMNH: AB1163).

Other material examined (of E. cf. gertschi). — MEXICO:
Tamaulipas: 1 9, Ejido Conrado Castillo, Cerro Zapatero,
Cueva del Coral, 19 March 1979, D. Pate, J. Atkinson, M.
Shumate (AMNH, AB1102); 1 6, San Juan, La Cueva sin
Nombra, 4 June 1967, R. Mitchell (AMNH: AB1165); 1 S,
Sotano de Monumento, 26 km WNW of Ocampo, near
Allende, 5 September 1979, W.R. Elliott, D.C. Rudolph
(AMNH: AB1142).

Diagnosis. — Male and female of E. gertschi are similar to
E. mexicana in having the distal segment of the PLS twice as
long as the basal segment (Fig. 118; E. xilitla sp. nov. with
distal to basal segment 3/2). Males differ from E. mexicana by
the normal proportions of the palpal femur and tibia (Fig.
117; rather than both strongly elongated), females in having
an only moderately elevated and medially notched posterior
membrane (Fig. 125; rather than strongly elevated). Males of
E. gertschi are similar to E. fernandoi sp. nov. in having the
posterior sclerite of the MA broadly spoon-shaped and
distally rounded (Fig. 116; rather than distally pointed in
E. decora, E. guanato sp. nov., E. rothi and E. selva), but differ
from this species in having the spermatic duct of the tegulum
distinctly curved (arrow in Fig. 114; rather than without
distinct curve). Females are similar to specimens of E. guanato
sp. nov., E. rothi, E. tlaxcala, and E. xilitla sp. nov. but differ
from E. guanato sp. nov. in having the posterior sclerite not
dumbbell-shaped (Fig. 122), from E. rothi and E. xilitla sp.
nov. in having the AME only as large as the PME, and from
E. tlaxcala by the terminal part of the vulva (Figs. 123, 124).

Description, — Essential information for the male was
provided by Roth (1968: 22-23). Measurements: Female
(AB1106): CL 4.1, CW 3.1, STL 1.9, STW 1.72, OL 5.25,
OW 3.6. Leg I (6.45, 1.7, 6.1, 6.2, 2.65), II (5.6, 1.45, 4.55, 5.2,
2.25), III (5.05, 1.25, 3.95, 5.2, 2.05), IV (6.5, 1.57, 5.55, 7.25,
2.5). Pedipalp (2.17, 0.8, 1.33, 1.97). EPL 0.43, EPW 0.81.
Eyes: anterior eye row straight, posterior eye row moderately
procurved. PME 0.18, PLE 0.23, AME 0.14, ALE 0.21. Eye
distances: PME-PME 1.5 x PME, PME-AME 1.25 x PME,
PME-PLE 1.25 X PME, PME-ALE 1.5 x PME, AME-AME
0.75-1 X AME, AME-ALE 0.8 x AME. CLYl 2.5 x AME,
CLY2 1 .3 X ALE. Epigyne and vulva: Epigyne medially with a
pale, hyaline area (Fig. 122). Posterior sclerite protruding
anteroventral as moderately sclerotized bar with anterior
margin concave (Figs. 122, 125), posterior membrane notched
(Fig. 125). CO laterally of posterior sclerite. Epigynal ‘pseudo
teeth’ present, small (Fig. 122). Vulva consists of combined
narrowly convoluted duct, CD less sclerotized, with append¬
ages (Figs. 124, 125), RC stronger sclerotized, terminally
strongly convoluted (Figs. 123, 124). FD only represented by
small, leaf-shaped appendages. Other important characters:
Cheliceral promargin with 4, retromargin with 6 teeth, more

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

117

proximally, the teeth become smaller. Colulus developed as
trapezoidal plate with distal margin w-shaped. PMS bearing
one conspicuously prominent spigot. PLS with distal segment
twice as long as basal segment (Fig. 118). Trichobothria on
cymbium and palpal tarsus absent. Tarsal trichobothria at leg
I 7. Small teeth on paired claws of leg I 10-11. Leg spination:
female pedipalp (0-0-0 or 1-0-0, 2-0-0, 2-1+lp-O), leg femora
(1 -2-0-0, 1 -1-1-0, 1 -0-0-0, 1 -0-0-0), patellae (all 2-0-0), tibiae
(O-O-O-O, O-O-O-O, O-O-O-O or 2-0-0-0, 2-0-0- 1), metatarsi (0-0-0-
4p+l, 0-l-0-4i>fl, 0-2-3-4i>fl, 0-2-3-4i>f1), tarsi (0, 0, 0, 0-0-1-
0). Coloration: Carapace with two longitudinal symmetrical
dark bands, irregularly expressed, margins narrowly darkened,
head region darker, laterally with indistinct dark patches (Fig.
121). Chelicerae frontally without dark patches. Sternum
darkened, medially moderately paler (Fig. 119). Opisthosoma
dark grayish, without red pigments, anteriorly with dark
patch, bordered by yellowish bands, posteriorly with yellowish
chevrons (Fig. 120). Legs very indistinctly annulated. Colulus
pale. ALS pale, basal segment of PLS darkened, distal
segment of PLS only proximally darkened (2/3).

Distribution. — Reported from localities in the states of
Nuevo Leon and Tamaulipas (Mexico).

Comments. — One female and two males from different
localities in Tamaulipas differ from E. gertschi s. s. by the
different vulva with an apparently fused duct (Figs. 73, 74),
and the narrow and pointed distal sclerite of the MA (Figs.
78-80). Due to the fact that: (i) the species group in focus
comprises a complex of very closely related species, (ii) the
males and female were not collected at the same locality and
(iii) only one female is available, these morphs are not
described as a new species and treated here as E. cf. gertschi.

Eratigena guanato sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.
org:act:9AC79754-8337-4AC5-97A2-387C757A78C2
Figs. 126-137

Type material. — Holotype female. MEXICO: Guanajuato:
Guanajuato, at road 110 from Dolores Hidalgo to Guana¬
juato, 1 km N of Santa Rosa de Lima, 2510 m, oak forest, 16
October 2014, A. Bolzern, E. Gonzalez Santillan (FC-UNAM:
AB1260).

Paratypes. MEXICO: Guanajuato: 5 juv., same data as
holotype (FC-UNAM: AB1260); 3 $, 3 juv., same data
(NMB-ARAN-27509: AB1243: accession-nr. LN887176).

Other material examined (of E. guanato s. s.). — MEXICO:
Unknown: 1 ?, 280 Cueva las Calevas, 6 April 1941, Mich
(AMNH: AB1130). Colima: 1 ?, Nevado de Colima, 20
January 1943, F. Bonet (AMNH: AB1126). Jalico: 1 $,
Mascota, km 21at street from Mascota to Puerto Vallarta,
1662 m, pine forest, night catch, 1 April 2012, L. Olguin, J.
Mendoza, G. Contreraz, C. Santibahez, D. Ortiz (CNAN).
Michoacdn: 1 6 , Basencheve National Park, 7 May 1963, W.J.
Gertsch, W. Ivie (AMNH: AB1103); 1 9, Garnica Pass, 2834
m, 8 May 1963, W.J. Gertsch, W. Ivie (AMNH: ABl 127); 1 9 ,
Cd. Hidalgo, Gruta de Tziranda, 1855 m, 29 April 2011, A.
Valdez, O. Francke, J.A. Cruz, R. Monjaraz, E. Miranda
(CNAN); 1 9 , Zitacuaro, ca. 30 km SE of Zitacuaro, Butterfly
forest, 2896 m, 16 December 2011, S. Huber (NMB-ARAN-
27510: AB1089).

Other material examined (of E. cf. guanato), — MEXICO:
Hidalgo: 2 9,5 miles SW. of Jacala, 21 April 1963, W.J.
Gertsch, W. Ivie (AMNH: 1176). Michoacdn: 1 9, Coalco-
man, Cueva de Cascada Chica, 10 km NE Coalcoman de
Matamoros, 1356 m, 1 May 1984, L. Elliott, D. McKenzie
(AMNH: ABl 100); 1 9, 15 km NE. of Coalcoman de
Matamoros, Cueva de Torrecillas, 2 May 1984, D. McKenzie,
L. Elliot (AMNH: ABl 108).

Etymology. — The specific name is a noun in apposition
shortened from the type locality.

Diagnosis. — Males of E. guanato sp. nov. can be separated
from all other related species by the distally hook-shaped
dorsal branch of the RTA (Fig. 136), and the lateral margin
folded only towards the terminal end of the distal portion of
the conductor (arrow in Fig. 136). Females are similar to
specimens of E. gertschi, E. rothi, E. tlaxcala, and E. xilitla sp.
nov. but differ in having the posterior sclerite distinctly
dumbbell-shaped (Figs. 130, 131; rather than posterior margin
almost straight), and differ from E. rothi and E. xilitla sp. nov.
in having the AME only as large as the PME, and differ from
E. gertschi in having the distal segment of the PMS not twice
as long as the basal segment (Fig. 129).

Description. — Measurements: Male (ABl 127): CL 2.73, CW
2.2, STL 1.2, STW 1.22, OL 2.93, OW 1.83. Leg I (3.73, 0.92,
3.4, 3.7, 2.23), II (3.33, 0.87, 2.9, 3.47, 2.13), III (3.2, 0.8, 2.44,
3.37, 2.1), IV (3.7, 0.84, 3.56, 4.5, 2.4), Pedipalp (1.22, 0.42,
0.72, 1.1), bulbL 0.52. Female (holotype): CL 3.53, CW 2.6,
STL 1.6, STW 1.58, OL 4.95, OW 3.75. Leg I (4.45, 1.35, 3.85,
3.9, 2.0), II (4.0, 1.2, 3.5, 3.73, 1.87), III (3.6, 1.15, 2.8, 3.6,
1.73), IV (4.7, 1.2, 4.05, 5.15, 2.0). Pedipalp (1.6, 0.733, 1.03,
1.47). EPL 0.33, EPW 0.58. Eyes: anterior eye row straight,
posterior moderately procurved (Fig. 127). PME 0.19, PLE
0.21, AME 0.17, ALE 0.19. Eye distances: PME-PME 1 x
PME, PME-AME 0.8-1 x PME, PME-PLE 1 x PME, PME-
ALE 1.2 X PME, AME-AME 1 x AME, AME-ALE 0.5-0.75
X AME. CLYl 2-2.5 x AME, CLY2 1.2-1. 5 x ALE. Male
pedipalp: RTA with two branches, lateral branch simple,
broadly lobe-like, dorsal branch distally hook-shaped (Fig.
136). Short dorsal spike on palpal tibia absent. Embolus
length about 1.5 x CB, originating at 10 o’clock position,
distal tip at 4 o’clock position. Conductor lamelliform, distal
portion only moderately elongated, lateral margin only
moderately folded towards terminal end (arrow in Fig. 136).
Terminal end of conductor strongly elongated, pointed.
Transversal ridge of conductor expressed as hyaline ridge,
indistinct. Conductor membranously connected to tegulum.
MA originating at 6-7 o’clock position, protruding, longer
than wide, distal sclerite translucent, subtriangular, moder¬
ately pointed. MA membranously connected to tegulum.
Epigyne and vulva: Epigynal hyaline area with anterior border
m-shaped (Fig. 105). Posterior sclerite protruding anteroven-
trally as dumbbell-shaped moderately sclerotized bar (Figs.
130, 131), posterior membrane moderately protruding ante-
riad (Fig. 134). CO anterolateral to posterior sclerite. Epigynal
‘pseudo teeth’ prominent (Fig. 131). Vulva consists of
combined narrowly convoluted duct, CD less sclerotized, with
distinct appendages, RC stronger sclerotized (Figs. 133, 134).
FD only represented by small, leaf-shaped appendages. Other
important characters: Cheliceral promargin with 4, retromar-
gin with7 small teeth, more proximally, the teeth become

JOURNAL OF ARACHNOLOGY

smaller. Colulus developed as trapezoidal plate with distal
margin moderately w-shaped. PMS bearing one conspicuously
prominent spigot. PLS with distal segment longer than basal
segment (4/3; Fig. 129). Tarsal trichobothria at leg I 8. Small
teeth on paired claws of leg I 11. Leg spination: male pedipalp
(3-0-0, 2-0-0, 1-1+lp-O), female pedipalp (2-0-0, 2-0-0, 2-1+lp-
0), leg femora (1-3- 1-0 or 1-1 -3-0 in male, 1-2- 1-0 or 1 -2-3-0 in
male, l-l-l-O or 1-4-3-0 in male, l-O-l-O or 1-2-1-0 in male),
patellae (all 2-0-0), tibiae (O-O-O-O or 1 -0-0-0 or 0-0-0- 1 in male,
l-O-O-O or l-l-O-l in male, 2-1-0-1 or 2-2-2-3p in male, 2-1-0-1
or 2-2-2- lp+l+2p in male), metatarsi (0-0-0-4p-(-l or 0-0-2-
2p+2+Ip+l in male, 0-0-0-4p+l or 0-2-0-4p4-l in male, 0-3-2-
4p+l, 0-3-2-4p+l or 0-4-4- 1 44 p+1 in male), tarsi (0, 0, 0-0- 1-0
or 0-0-2-0 in male, 0-0- 1-0 or 0-0-2-0 in male). Coloration:
Carapace with two longitudinal symmetrical dark bands,
irregularly expressed, margins narrowly to broadly darkened
(Fig. 126). Chelicerae frontally with indistinct dark patches
(Fig. 127). Sternum darkened, with pale median band, midway
interrupted, posteriorly with black patch (Fig. 128). Opistho-
soma grayish with dark spots, without reddish pigments,
indistinct grayish median band, anteriorly with dark fork¬
shaped patch, posteriorly with grayish chevrons (Fig. 126).
Legs distinctly annulated. Colulus medially and laterally with
dark patches. ALS basally darkened, basal segment of PLS
darkened, distal segment of PLS only proximal half darkened.

Distribution. — Reported from four states of East-Central
Mexico: Colima, Guanajuato, Jalisco and Michoacan.

Comments. — Male and female specimens were not collected
together and show moderate morphological differences (e.g.,
size, leg spination) and are therefore only tentatively placed in
the same species. The females collected close to Jacala and
Coalcoman show some morphological differences to the type
specimens. If these differences are part of intraspecific
variation or a closely related taxa cannot satisfyingly be
judged here. Therefore, the identification of these specimens
remains uncertain.

Eratigena mexicana (Roth, 1968), comb. nov.

Figs. 138-149

Tegenaria flexuosa Roth, 1952: 285, figs. 1, 2 (in part,
misidentified female).

Tegenaria mexicana Roth, 1968: 15, figs. 21-26.

Type material. — Holotype male. MEXICO: Guerrero: Tax-
co, in cave, 29 July 1956, V. Roth, W. Gertsch (AMNH).

Paratypes. 1 9 allotype, same data as holotype (AMNH); 2
6, same data (AMNH: AB1178); 5 <?, same data except 28
July 1956 (AMNH: AB1154, AB1179); 1 S, Taxco, 1 October
1945, L. Isaacs (AMNH: AB1177).

Other material examined (of E. mexicana s. s.). — MEXICO:
Guerrero: 1 6, Grutas de Acuitlapan, 10 mi. E of Taxco, 9
April 1968, W. Calvert (AMNH: AB1105); 1 c?, 4 2, 1 juv.,
Taxco de Alarcon, E of Taxco, 1611 m, tropical forest, at
stones and at rock faces near street (terrain break), 5 October
2014, A. Bolzern, E. Gonzalez Santillan (NMB-ARAN-2751 1
to 27514: ABI217, AB1232, AB1247, AB1216: accession-nr.
LN887153, LN887188); 4 9, 4 juv., same data except 1670 m,
at rock faces near street (terrain break) (FC-UNAM: AB1206,
AB1285); 1 9, same data except, W. of Colonia la
Quebradora, 1825 m, secondary oak-pine forest, open, reddish

stones, 1670 m (FC-UNAM: AB1273); 1 9, Taxco de
Alarcon, Parque el Huixteco, 2434 m, pine-oak forest, 22
August 2013, H.E. Leon (NMB-ARAN-27515: AB1323).
Mexico: 1 9, Tenancingo, Tenancingo de Degollado, 2050
m, 7 October 1946, H. Wagner (AMNH: AB1129).

Other material examined (of E. cf. mexicana). — MEXICO:
Morelos: 1 9, Cuernavaca, 1700 m, 1 September 1941, H.
Wagner (AMNH: ABl 128); 3 9, Cuernavaca, in shallow cave
near town, 31 July 1956, W.J. Gertsch, V. Roth (AMNH:
ABl 123); 6 9, San Sebastian, north of Oaxtepec, 1874 m, old
train tunnel, 16 December 2011, S. Huber (NMB-ARAN-
27516: AB1091).

Diagnosis. — Male E. mexicana specimens differ from all
Nearctic Eratigena species in having a distinctly elongated
palpal femur and tibia (Fig. 140). Females are similar to
E. xilitla sp. nov. and differ from other species in having the
posterior membrane (internal posterior limitation of genital
area) distinctly protruding anteriad (arrow in Fig. 148). They
can be separated from E. xilitla sp. nov. specimens by having
the posterior membrane without a notch and the AME
diameter equal or smaller than that of the PME.

Description. — Essential information was provided by Roth
(1968).

Distribution. — Reported from the states of Guerrero and
Mexico (Mexico). Reports from the state of Morelos are
uncertain (see comment).

Comments. — The specimens from Morelos differ from
typical E. mexicana specimens in having wider genital ducts
(Fig. 149). Due to their affinity to E. tlaxcala (Figs. 203, 206)
and the absence of males, the identification of these specimens
remains uncertain.

Eratigena queretaro sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.
org:act:853FD45F-18FF-4C3B-B2B4-F781C3CCC98F
Figs. 7, 150-161

Type material. — Holotype fen}ale. MEXICO: Queretaro:
Pinal de Amoles, at road 120 between Jalpan de Serra and
Pinal de Amoles, close to La Curva del Chuveje, 20 km W. of
Jalpan, 1288 m, transitional forest, subtropical to mountain¬
ous, in tube with running water under road, 13 October 2014,
A. Bolzern, E. Gonzalez Santillan (FC-UNAM: AB1302).

Paratypes. 1 (3, 1 9, same data as holotype (FC-UNAM:
ABl 302).

Other material examined. — MEXICO: Queretaro: 3 9 , Pinal
de Amoles, at road 120 between Jalpan de Serra and Pinal de
Amoles, close to Puerto del Perico, 34 km W. of Jalpan, 2075
m, oak-pine forest, roadside cave, 14 October 2014, A.
Bolzern, E. Gonzalez Santillan (NMB-ARAN-2751 7:
AB1258: accession-nr. LN887154, LN887177); 1 9, Penamil-
ler, at path from Ri'o Blanco to Cueva Puerto del Leon, 2014
m, oak forest, 14 October 2014, A. Bolzern, E. Gonzalez
Santillan (NMB-ARAN-2751 8: AB1218: accession-nr.
LN887155, LN887178).

Etymology. — The specific name is a noun in apposition
taken from the type locality.

Diagnosis. — Female specimens of E. queretaro sp. nov.
differ from all others of the group in having an hourglass¬
shaped posterior sclerite (Figs. 154, 155), and an anteriorly
distinctly arcuate genital duct with prominent appendages

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

119

(Figs. 157, 158). Males are similar to E. decora, E. giiamto sp.
nov., E. rothi and E. selva in having at least a moderately
pointed distal sclerite of the MA (Figs. 159, 161). They differ
from E. guanato sp. nov., E. rothi and E. selva in having a
relatively slim, finger-shaped and simply pointed dorsal
branch at the RTA (Fig. 161; rather than distally hook¬
shaped in E. quanato sp. nov., strong and basally bent in
E. rothi and E. selva), and from E. decora in having a
triangular and sharply pointed distal sclerite of the MA (rather
than subtriangular, moderately pointed).

Description, — Measurements: Male (paratype): CL 2.56,
CW 1.9, STL 1.2, STW 1.18, OL 2.77, OW 1.44. Leg I (4.3,
1.0, 4.2, 4.65, 2.55), II (3.7, 0.93, 3.25, 3.85, 2.1), III (3.43, 0.84,
3.0, 4.03, 2.0), IV (4.35, 0.93, 4.03, 5.4, 2.6), Pedipalp (1.1,
0.46, 0.58, 0.86), bulbL 0.4. Female (holotype): CL 3.17, CW
2.33, STL 1.42, STW 1.3, OL 3.85, OW 2.5. Leg I (3.88, 1.2,
3.87, 3.83, 2.17), 11 (3.53, 1.17,2.9,3.3, 1.9), III (3.18, 1.0,2.53,
3.2, 1.73), IV (4.0, 1.6, 3.75, 4.45, 2.23). Pedipalp (1.26, 0.5,
0.88, 1.3). FPL 0.38, EPW 0.58. Eyes: eye rows moderately
procurved (Fig. 151). PME 0.15, PLE 0.17 , AME 0.14, ALE
0.2. Eye distances: PME-PME 0.8 x PME, PME-AME 0.8 x
PME, PME-PLE 0.8-1 x PME, PME-ALE 1 x PME, AME-
AME 0.6 X AME, AME-ALE 0.4 x AME. CLYl 1.75 x
AME, CLY2 1 X ALE. Male pedipalp: RTA with two
branches, lateral branch broadly lobe-like, indistinctly pro¬
truding, dorsal branch strongly sclerotized, narrowly finger
shaped and sharply pointed (Figs. 160, 161). Short dorsal
spike on palpal tibia absent. Embolus length about 1.5 x CB,
originating at 9 o’clock position, distal tip at 3^ o’clock
position. Conductor lamelliform, distal portion only moder¬
ately elongated, lateral margin folded (Figs. 160, 161).
Terminal end of conductor strongly elongated, pointed.
Transversal ridge of conductor expressed as hyaline ridge.
Conductor membranously connected to tegulum. MA origi¬
nating at 6 o’clock position, protruding, longer than wide,
distal sclerite translucent, black, long triangular, pointed
(Figs. 159-161). MA membranously connected to tegulum.
Epigyne and vulva: Epigyne medially with a pale, narrow oval
hyaline area. Posterior sclerite protruding anteroventrally as
moderately sclerotized bar, hourglass-shaped, anterolaterally
not rounded (Figs. 154, 155), posterior membrane protruding
anteriad (Fig. 158). CO anterolateral to posterior sclerite.
Epigynal ‘pseudo teeth’ minute (Figs. 154-156). Vulva consists
of combined narrowly convoluted duct, anterior distinctly
arcuate, with distinct appendages (Figs. 157, 158), RC
stronger sclerotized. FD only represented by small, leaf¬
shaped appendages. Other important characters: Cheliceral
promargin with 4, retromargin with 8 small teeth, more
proximally, the teeth become smaller. Colulus developed as
trapezoidal plate with distal margin moderately w-shaped.
PMS bearing one conspicuously prominent spigot. PLS with
distal segment longer than basal segment (3/2; Fig. 153).
Trichobothria on cymbium and palpal tarsus absent. Tarsal
trichobothria at leg I 7-8. Small teeth on paired claws of leg I
9-10. Leg spination: male pedipalp (2-0-0, 2-0-0, 1- 1-1-1 p-0),
female pedipalp (2-0-0, 2-0-0, 2-l-(-Ip-0), leg femora (0-2-0-0 or
2-2-0-0 or 2-3-2-0, 2-1-1-0 or 2-1-0-0 or 2-3-2-0, 2-1-1-0 or 2-2-
2-0, 1 -0-2-0 or 2-1 -1-0), patellae (all 2-0-0), tibiae (0-0-0- 1 or 1-
0-0-2 or l-l-0-3p, 2-1-0-0 or 2-l-0-3p+l, 2-1-1-1 or 2-2-2-2+lp,
2-1-1-2 or 2-2-2-3-f-lp), metatarsi (0-0-0-4p-|-l , 0-0-0-4p-|-l or 0-

l-0-4i>fl, 0-2-2-4i>fl or 0-3-2-4f>fl, 0-3-2-4p+l or 0-3-3-
lp-|-!-|-2p+l), tarsi (0, 0, 0, 0-0- 1-0 or 0-1 -3-0). Coloration:
Carapace with two well expressed longitudinal symmetrical
dark bands, margins narrowly to broadly darkened, head
region dorsolaterally with two distinct, longitudinally curved
dark bands (Fig. 150). Chelicerae frontally with indistinct dark
patches (Fig. 151). Sternum darkened, with pale median band,
midway interrupted (Fig. 152). Opisthosoma grayish with
dark spots, median band with reddish pigments, anteriorly
with dark patch, posteriorly with chevrons (Fig. 150). Legs
annulated, ventrally more distinctly expressed as dorsally.
Colulus moderately darkened. ALS basally and laterally
darkened, basal segment of PLS distinctly darkened, distal
segment of PLS only proximal half darkened.

Distribution. — Reported from only a narrow area in the
state of Queretaro (Mexico).

Comments. — Specimens of E. queretaro sp. nov. were
collected at three different sites in a relatively narrow area.
The most natural habitat was at the path from Rio Blanco to
the Cueva Puerto del Leon in an old oak forest. There, in a
shady valley, the spiders built their webs between rocks near a
small creek. In contrast, representatives of this species were
also collected in a tube with running water under a road (Fig.
6) within an area with subtropical to mountainous transitional
forest.

Eratigena rothi (Gertsch, 1971), comb. nov.

Figs. 162-173

Tegenaria rothi Gertsch, 1971: 107, figs. 161-163.

Type material. — Holotype male. MEXICO: Hidalgo: Cueva
de El Ocote, 1.5 km N. of Palomas, 20 July 1956, V. Roth,
W.J. Gertsch (AMNH).

Other material. — MEXICO: Hidalgo: 1 cJ, 4 9, Jacala,
collected in cave at night, 20 July 1956, V. Roth, W.J. Gertsch
(AMNH: AB1173); 1 <5,5 9, same data except roadside cave,
1 mile N. of Palomas (AMNH: AB1157, AB1172); 3 9, 10-25
miles S. of Jacala, 1 July 1956, V. Roth, W. Gertsch (AMNH:
AB1170); 1 d, 6 9, same data except 20 July 1956 (AMNH:
AB1174, AB1175); 2 S, Tlanclnnol, 43 km SW. of Huejutla,
1500 m, cloud forest, 14 August 1983, S. & J. Peck (AMNH:
AB1138); 2 9,2 km N. of del Pinalito, El Cardenal, 2301 m,
coniferous forest, day catch, 16 October 2009, O. Francke, R.
Paredes, J. Cruz, T. Lopez, T. Palafox, C. Santibanez, A.
Valdez (CNAN: AB1201); 3 9, 7 juv., Zimapan, at street from
Morelos (Trancas) to Puerto de Piedra, 2405 m, pine forest, at
terrain break near street, at rocks, 10 October 2014, A.
Bolzern, E. Gonzalez Santillan (FC-UNAM: AB1211,
AB1221, AB1270); 2 9, Nicolas Flores, close to El Endnote,
at street from Morelos (Trancas) to Puerto de Piedra, 2557 m,
pine forest, at terrain break near street, at rocks, 10 October
2014, A. Bolzern, E. Gonzalez Santillan (FC-UNAM:
AB1257, AB1297); 1 9, same data except Puerto de Piedra,
Santa Cueva, 2498 m, at rocks at cave entrance (NMB-
ARAN-27519: AB1298: accession-nr. LN887156, LN887189);
3 <5,7 9, 4 juv., Chapulhuacan, between Puerto Chaballo and
Chapuluacan, near road 85, 1087 m, in tube with running
water under road, 10 October 2014, A. Bolzern, E. Gonzalez
Santillan (NMB-ARAN-27520 to 27524: AB1212, AB1228,

120

JOURNAL OF ARACHNOLOGY

AB1241, AB1263, AB1210: accession-nr. LN887157,
LN887190).

Diagnosis. — Male and female specimens of E. rothi are very
similar to E. xilitla sp. nov. and differ from all other West
Nearctic ErcUigena species in having the AME larger than the
PME. They differ from the sister species, E. xilitla sp. nov., in
their larger size (CL longer than 4.5 mm; rather than smaller
than 4 mm in E. xilitla sp. nov.). Males differ from E. xilitla
sp. nov. by the shape of the distal sclerite of the MA (Fig. 167;
long subtriangular and pointed, rather than spoon-shaped and
rounded), and females differ by the shape of the posterior
sclerite (Fig. 170).

Description. — Essential information was provided by
Gertsch (1971).

Distribution. — Reported from several localities in the north¬
western part of the state of Hidalgo (Mexico).

Comments. — Morphologically, the two species E. rothi and
E. xilitla sp. nov. are very closely related. This sister-species
relationship is supported by the maximum likelihood tree
based on mitochondrial sequences (Fig. 1). Specimens of
E. rothi were collected during an excursion in 2014 from three
different habitats: pine forest at terrain break, at a huge cave
entrance between rocks and in a tube with running water
under a road. E. xilitla sp. nov. seems to preferably inhabit
oak-pine forests, but otherwise similar habitats.

Eratigem selva (Roth, 1968), comb. nov.

Figs. 174-194

Tegenaria mexicana selva Roth, 1968: 23, figs. 28, 29.
Tegenaria selva Roth: Gertsch, 1971: 105.

Type material. — Holotype male. MEXICO: San Luis Potosi:
Cueva de la Selva, west of Xilitla on the Xilitla-Ahuacatlan
road, north of Tamazunchale, 10 April 1966, T. Raines
(AMNH).

Paratypes. 1 9 allotype, Sotano at Valle de los Fantasmas,
24 November 1966, J. Fish, J. Davis (AMNH: AB1131).

Other material examined. — MEXICO: Hidalgo-. 1 S, Sotano
de la Hoya de las Vigas, 1.5 km N Pinalito, 22 March 1981, J.
Reddell, T. Archery (AMNH: AB1107); 1 9, La Sotono del
Hondo de Pinalito, Hwy 85, 1 January 1976, C. Soileau, P.
Strickkand (AMNH: AB1144). San Luis Potosr. 19,5 km N
Valle de Los Fantasmas, 40 km E Zaragoza, Cueva de la
Laguna, 3000 m, 20 May 1972, Wm. Elliott, P. Lynn, R.M.
McEachern (AMNH: ABllOl); 1 6, Cueva Pueblo Ilamado,
58 km Mpio Villa de Zaragoza, 2298 m, pine-oak forest, date
unknown, A. Valdez, O. Francke, J. Cruz, C. Santibahez
(CNAN: AB1197); 1 9, Gruta de los Muertes, Xilitla Plateau,
28 March 1900, D. Pate (AMNH: AB1158); 3 9, Sotanade La
Silleta, 30 March 1980, D. Honea (AMNH, AB1109); 2 9,
Municipio de Zaragoza, Cueva de los Caballos, 30 km E San
Luis Potosi, 3000 m, 18 May 1972, Wm. Elliott (AMNH:
AB1146); 4 9, Sierra del Pina, Microwave Tower Road, La
Cueva de los Murcielagos, 29 December 1975, C. Soileau, P.
Strickkand (AMNH: AB1147); 1 9, Sotano de Abernathy, W.
of Valle de los Fantasmos, 30 January 1969, Wm. Elliott, D.
Honea, M. Abernathy (AMNH: AB1I60); 1 9, Sotano de
Aranas, W of Valle de los Fantasmas, 29 January 1969, R.
Harmon, J. Cepeda (AMNH: AB1132); 1 c?, 3 9, Sotano de
Golondrina, Puerto Altamira, 40 km E San Luis Potosi, 3000

m, 17 March 1972, Wm. Elliott, R. Mitchell, J.A.L. Cooke, G.
Campell, G. Graves, M. Brownfield (AMNH: ABl 151); 2 <S , 1
9, Sotano de Las Golondrinas, Valle de los Fantasmos, 29
November 1968, Wm. Elliott, J. Jarl, S. Cathey, M. Burk
(AMNH: ABl 159); 2 9, Sotano de Ojo de Agua, 4 km S San
Francisco, 30 November 1968, Wm. Elliott, J. Jarl (AMNH:
ABl 133); 2 9, Sotano Puerto de los Lobos, San Francisco, 14
September 1968, B. Elliott (AMNH: ABl 134).

Diagnosis. — Male and female specimens of E. selva differ
from all West Nearctic Eratigena species by their large size in
combination with very long legs (CL > 5 mm, patella-tibia
length leg I > 9 mm) and an indistinctly patterned abdomen
(grayish). In addition, females differ by having a very strongly
sclerotized epigynal posterior sclerite (Figs. 182, 191).

Description. — Essential information was provided by Roth
(1968).

Distribution. — Reported from the north-western part of the
state of Hidalgo and the state of San Luis Potosi (Mexico).

Comments. — Even though the type locality of E. selva
(Cueva de la Selva, now called Cueva del Salitre) was revisited
during an excursion in October 2014, no specimens or webs
could be detected there.

The specimens from Sotano de Golondrina (ABl 151) are
morphologically very similar to typical E. selva specimens, but
differ to some extent in epigynal and genital morphology
(Figs. 186-188, 191-194). However, due to the lack of more
information (more specimens with these features, DNA data)
these specimens are here treated as a variation of E. selva.

Eratigena tlaxcaia (Roth, 1968), comb. nov.

Figs. 195-206

Tegenaria mexicana tlaxcaia Roth, 1968: 24.

Tegenaria tlaxcaia Roth: Brignoli, 1974: 230.

Type material. — Holotype male. MEXICO: Tlaxcaia-. Tlax-
cala, in underground water conduit, 26 July 1956, V. Roth, W.
Gertsch (AMNH).

Paratypes. 1 9 allotype, same data as holotype (AMNH); 3

(J, 1 9, same data (AMNH: ABl 171).

Other material examined (of E. tlaxcaia s. s.). — MEXICO:
Distrito Federal-. 1 9 , Gustavo A. Madero, Canada hacia la
cueva del Fraile, 2614 m, 25 June 2009, H. Montano, A.
Valdez, R. Paredes, T. Garrido (CNAN: CNAN-Ar009746).
Mexico-. 1 (?, 58 km E. of Mexico City, pine forest, under log,
24 July 1956, V. Roth, W. Gertsch (AMNH: ABl 162). Puebla-.
1 (5, Huachinango, 4.4 miles SW. of Huachinango, 1700 m,
moist ravine oak forest, malt traps, 25 July 1969, collector
unknown (AMNH: ABl 110); 1 S, pyramid at Cholula, 20
August 1965, J. Reddell, J. Fish, W. Bell (AMNH: ABl 1 13); 1
9, San Pedro Cholula, at northern slope of hill NE. of
Cholula, 2221 m, pine forest, small cave, 8 October 2014, A.
Bolzern, E. Gonzalez Santillan (NMB-ARAN-27525:
AB1271: accession-nr. LN887159, LN887I91); 1 9, W. of
Rio Frio, 2956 m, 22 August 1964, W. & J. Ivie (AMNH:
ABl 111). Tlaxcaia-. 5 9, 2 juv., Tlaxcaia, Ocatlan, Barage
Chapitel, within city in an open space between houses, 2222 m,
old manmade tunnel/cave under the city, at the entrance
(which was a dump), 9 October 2014, A. Bolzern, E. Gonzalez
Santillan (FC-UNAM: AB1229, AB1242, AB1296; NMB-
ARAN-27526: AB1233: accession-nr. LN887158, LN887192).

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

121

Other material examined (of E. cf. tlaxcala). — MEXICO:
Guerrero: 1 6, Tetipan, 5 km E de Casahuates, 2275 m, pine-
oak forest, 4 June 2010, O. Francke, D. Barrales, J. Cruz, A.
Valdez (CNAN; AB1196).

Diagnosis. — Males of E. tlaxcala are similar to specimens of
E. caverna, E. fernandoi sp. nov., E. gertschi, E. selva
(variation) and E. xilitla sp. nov. and differ from other species
in having a spoon-shaped, distally rounded distal sclerite (Fig.
195,197; rather than subtriangular or triangular and distally
pointed). Males and females differ from E. caverna and
E. xilitla sp. nov. in having normally developed eyes with the
AME as large as the PME (Fig. 199; rather than all reduced in
E. caverna, PME larger than AME in E. xilitla sp. nov.).
Males differ from E. fernandoi sp. nov. and E. gertschi in
having a narrow, distinctly longer then wide distal sclerite of
the MA (Fig. 197; rather than broad), and from E. selva
(variation) in having a conductor with an unevenly curved
distal margin (arrow in Fig. 196; rather than evenly curved).
Females are very similar to E. gertschi, E. giianato sp. nov.,
E. rothi and E. xilitla sp. nov., but differ from E. rothi and
E. xilitla sp. nov. in having the AME as large as the PME,
differ from E. guanato sp. nov. in having the posterior sclerite
not distinctly dumbbell-shaped, and differ from E. gertschi in
having the distal segment of the PLS not twice as long as the
basal segment.

Description. — Essential information was provided by Roth

(1968).

Distribution. — Reported from three states: Distrito Federal,
Puebla and Tlaxcala (Mexico).

Comments. — The specimen from Guerrero differs to some
extent morphologically from typical E. tlaxcala specimens
(e.g. shape of RTA and median apophysis) and its identifica¬
tion remains doubtful.

Eratigena xilitla sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.
org:act:A9F274FD-3456-4D7F-8A21-B9DF2B9B5DB8
Figs. 207-218

Type material. — Holotype male. MEXICO: San Luis Potosv.
Xilitla, at road from Xilitla to Las Adjuntas, Cueva del Aqua,
451 m, near cave entrance at rock surfaces, 12 October 2014,
A. Bolzern, E. Gonzalez Santillan (FC-UNAM: AB1219:
accession-nr. LN887161, LN887I80)..

Paratypes. 19,2 juv., same data as holotype (FC-UNAM:
AB1219: accession-nr. LN887161, LN887180); 19,4 juv.,
same data (NMB-ARAN-27527: AB1301).

Other material. — MEXICO: Hidalgo: 2 9, 2.5 km N. of
junction Zacualtipan-Santiago Tianguistengo, 2101 m, pine
forest, 6 November 2010, A. Valdez, O. Francke, J. Cruz, C.
Santibanez, E. Miranda (CNAN: AB1195). Puebla: 2 9, 1
juv., Ajalpan, street between Pala and Nicolas Bravo, 2653 m,
oak-pine forest, at terrain break at steep slope near path in the
forest, 7 October 2014, A. Bolzern, E. Gonzalez Santillan
(NMB-ARAN-27528 to 27529: AB1278, AB1287: accession-
nr. LN887160, LN887179); 1 9, same data except 2619 m
(FC-UNAM: AB1246). San Luis Potosi: 1 9, Tamazunchale,
6 July 1941, L.I. Davis (AMNH: AB1125). Queretaro: 19,2
juv., Pinal de Amoles, at road 120 between Jalpan de Serra
and Pinal de Amoles, close to La Curva del Chuveje, 20 km W.
of Jalpan, 1288 m, transitional forest, subtropical to moun¬

tainous, in tube with running water under road, 13 October
2014, A. Bolzern, E. Gonzalez Santillan (FC-UNAM:
AB1235). Veracruz: 1 d, 4 9, 10 miles W. of Jalapa, volcanic
cave in pine forest, 26 July 1956, V. Roth, W. Gertsch
(AMNH: AB1164); 1 9, Acejete, 1 km NE. of La Joya, 2204
m, 30 October 2006, O. Francke, A. Valdez, C. Santibanez
(CNAN: AB1194); 1 d, 1 9, 2 juv., Coatepec, village park,
1265 m, 10 December 2010, S. Huber (NMB-ARAN-27530:
AB1090); 1 (?, Huatusco, 7 km E. of Huatusco, cloud forest,
22 June 1983, S. & J. Peck (AMNH: AB1136).

Etymology. — The specific name is a noun in apposition
taken from the type locality.

Diagnosis. — Male and female specimens of E. xilitla sp. nov.
are very similar to E. rothi and differ from all other West
Nearctic Eratigena species in having the AME larger than the
PME. They differ from the sister species, E. rothi, in their
smaller size (CL shorter 4 mm; rather than longer than 4.5 mm
in E. rothi). Males differ from E. rothi sp. nov. by the shape of
the distal sclerite of the MA (Figs. 207, 209; spoon-shaped and
rounded, rather than long subtriangular and pointed), and
females differ by the shape of the posterior sclerite (Fig. 215).

Description. — Measurements: Male (holotype): CL 3.13,
CW 2.47, STL 1.44, STW 1.36, OL 3.37, OW 1.7. Leg I
(5.48, 1.33, 5.35, 6.15, 3.15), II (4.4, 1.1, 4.15, 4.95, 2.65), III
(4.0, 1.0, 3.33, 4.5, 2.25), IV (5.35, 1.16, 4.75, 6.5, 3.33),
Pedipalp (1.6, 0.54, 0.82, 1.36), bulbL 0.5. Female (paratype):
CL 3.17, CW 2.4, STL 1.4, STW 1.36, OL 4.0, OW 2.9. Leg I
(4.2, 1.1, 3.85, 4.0, 2.15), II (3.45, 1.08, 2.83, 3.1, 1.75), III (3.3,
1.0, 2.47, 3.1, 1.63), IV (4.22, 1.03, 3.5, 4.2, 2.03). Pedipalp
(1.4, 0.54, 0.88, 1.32). EPL 0.31, EPW 0.67. Eyes: eye rows
moderately procurved (Fig. 212). PME 0.16, PLE 0.18, AME
0.18, ALE 0.18. Eye distances: PME-PME 0.8 x PME, PME-
AME 1 X PME, PME-PLE 1 x PME, PME-ALE 1.3 x PME,
AME-AME 0.3 x AME, AME-ALE 0.3 x AME. CLYl 1 .2 x
AME, CLY2 1 X ALE. Mcde pedipalp: RTA with two
branches, lateral branch subtriangular lobe-like, moderately
ventrad protruding, dorsal branch strongly finger shaped,
distally truncated (Fig. 209). Short dorsal spike on palpal tibia
absent. Embolus length about 1.4 x CB, originating at 9-10
o’clock position, distal tip at 3 o’clock position. Conductor
lamelliform, distal portion only moderately elongated, lateral
margin folded. Terminal end of conductor strongly elongated,
pointed. Transversal ridge of conductor expressed as indistinct
hyaline ridge. Conductor membranously connected to teg-
ulum. MA originating at 6 o’clock position, protruding, longer
than wide, distal sclerite spoon-shaped, rounded (Figs. 207-
209). MA membranously connected to tegulum. Epigyne and
vulva: Epigyne medially with a pale, hyaline area, long oval.
Posterior sclerite protruding anteroventral as moderately
sclerotized bar, dumbbell-shaped (Fig. 215), posterior mem¬
brane strongly protruding anteriad and notched (Fig. 218).
CO anterolateral to posterior sclerite. Epigynal ‘pseudo teeth’
prominent (Fig. 215). Vulva consists of combined narrowly
convoluted duct, CO less sclerotized with distinct appendages
(Figs. 217, 218). FD only represented by small, leaf-shaped
appendages. Other important characters: Cheliceral promargin
with 4-5, retromargin with 7-1 1 small teeth, more proximally,
the teeth become smaller. Colulus developed as trapezoidal
plate with distal margin moderately w-shaped. PMS bearing
one conspicuously prominent spigot. PLS with distal segment

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longer than basal segment (3/2; Fig. 214). Trichobothria on
cymbium and palpal tarsus absent. Tarsal trichobothria at leg
I 8. Small teeth on paired claws of leg I 8-9. Leg spination:
male pedipalp (3-0-0 or 3-1-0, 2-0-0, 1-1-flp-O), female
pedipalp (2-0-0, 2-0-0, 2-1+lp-O), leg femora (1-3-2-0 or 2-3-
2-0, 1 -3-2-0 or 2-3-2-0, 1 -2-2-0, 1 -2-2-0 or 2- 1-1-0), patellae (all
2-0-0), tibiae (l-l-O-O, or l-l-O-l, l-l-O-O, 2-1-1-1, 2-2-1-1 or 2-
2-2-1 or 2-2-2-2), metatarsi (0-0-0- 1 p+l+2p+l , 0-1-0-
lp+l+2p-hl, 0-2-2- lpj-l+2p+l or 0-3~2-4p+l, 0-3-2-4p-f-l or
0-3-3-4p+l), tarsi (0, 0, O-O-l-O or 0-0-2-0, 0-1-2-0 or 0-1-3-0).
Coloration: Carapace with two broad longitudinal symmetri¬
cal dark bands, margins narrowly to broadly darkened (Fig.
211). Sternum darkened, with pale median band with
moderately serrated lateral margins (sometimes indistinct),
posteriorly with indistinct black patch (Fig. 213). Opisthoso-
ma dark brown, with distinct reddish median band with black
spots, bordered by yellowish bands, posteriorly with yellowish
chevrons (Fig. 211). Legs distinctly annulated, dorsally
sometimes indistinct. Colulus laterally with dark patches.
ALS basally and distally darkened, basal segment of PLS
darkened, distal segment of PLS only proximal third darkened
(Fig. 214).

Distribution. — Reported from five states along the eastern
mountain range (Sierra Madre Oriental): Hidalgo, Puebla,
Queretaro, San Luis Potosf, and Veracruz (Mexico).

Comments. — The specimens collected by Roth and Gertsch
in 1956 were misidentified as T. tlaxcala (Roth, 1968: 25). See
also comment for E. rotlii.

DISCUSSION

The result that the endemic species in the Nearctic region
belong to both genera, Tegenaria and Eratigena, is surprising.
This hypothesis would imply that either both genera
originated earlier than the split of Laurasia (approximately
55 million years ago; Ellis & Stoker 2014), or that one or both
of them invaded the continent later.

The mexican Eratigena species can be split into the two
described, well diagnosable groups: the //e.vnos'a-group and the
mexicana-groxx^. Based on morphological data and mtDNA
sequences, the mexicana-gxou'p is more complex with some
very closely related species. Differences in male and female
genitalia between species are sometimes hard to detect. That is
why we even propose to use size as a character to separate
species (Bolzern et al. 2013a). However, most species are well

diagnosed by morphological and molecular data, although
some specimen identifications remain uncertain due to the low
number of available individuals (unknown magnitude of
intraspecific variation) and the absence of molecular data for
certain species.

In addition to the species groups in focus, it is mentionable
that (based on mtDNA sequences; Fig. 1) Textrix denticulata
(Olivier, 1789) is placed outside Textrix Sundevall, 1833, that
Agelena canariensis Lucas, 1838 is closely related to Agele-
scape gideoni Levy, 1996 (and only distantly related to other
Agelena Walckenaer, 1805 species), and that the Novalena
Chamberlin & Ivie, 1942 specimens from Mexico represent a
monophyletic clade apart from Novalena intermedia (Cham¬
berlin & Gertsch, 1930).

ACKNOWLEDGMENTS

For the loan of the specimens included in this work, we are
grateful to the institutions, curators and collection managers
mentioned in the Methods section. We are very thankful to
Siegfried Huber (Germany) and Leonel Perez Miguel (Mexico)
for the donation of relevant specimens. We are deeply grateful
to the Natural History Museum of Basel and its staff for the
provision of equipment and technical support. We are grateful
to Armin Coray for the generous loan of his microscope.
Special thanks go to Edmundo Gonzalez Santillan and
Fernando Alvarez Padilla (Mexico) for their great support
before, during and after the fieldtrip to Mexico in 2014. We are
also grateful to Enrique (last name unknown), Carmello
Salinas and Antioco Salinas Sanchez for their guidance to the
caves in Queretaro. We are thankful to Daniele Polotow,
Jeremy Miller and Michael Rix for constructive comments and
reviews of the manuscript. We are indebted to the following
foundations for financial support: “Stiftung Emilia Guggen-
heim-Schnurr der Naturforschenden Gesellschaft Basel” and
the “Kugler-Werdenberg-Stiftung des Naturhistorischen Mu¬
seum Basel”. Laboratory expenses were funded by the “Easier
Stiftung fiir Biologische Forschung”. The field expedition was
supported by the “Fritz-Sarasin-Stiftung der Freiwilligen
Akademischen Gesellschaft”, the “Stiftung Dr. Joachim de
Giacomi der Akademie der Naturwissenschaften Schweiz”,
and the Vincent Roth Research Foundation of the American
Arachnological Society. Funding for the collection of the new
species (in part) was provided by UNAM-DGAPA-PAPIIT
project IN2 13612.

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123

Figures 8-16. — Tegeiuiria chiricahucie Roth, 1968, female (8-12) and male (13-16). 8, epigynal area, ventral view; 9, vulva, dorsal view; 10,
same, dorsolateral view; 11, same, posterior view; 12, same, anterior view; 13, left male pedipalp, dorsoretrolateral view; 14, cymbium and bulb,
prolateral view; 15, same, ventral view; 16, same, retrolateral view. C = conductor; CD = copulatory duct; CO^copulatory opening; DB = dorsal
branch of RTA; E = embolus; FD = fertilization duct; PT = epigynal ‘pseudo teeth’; MA = median apophysis; R = lateroventral ridge of RTA;
RC = receptaculum; RTA = retrolateral tibial apophysis; T = tegulum; VB = ventral branch of RTA.

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Figures 17-29. — Evatigemi edmimdoi sp. nov., female (17-22) and male (23-29). 17, habitus, dorsal view; 18, epigynal area, ventral view; 19,
vulva, dorsal view; 20, same, posterior view; 21, same, anterior view; 22, same, dorsolateral view; 23, carapace and chelicerae, anterior view; 24,
sternum ventral view; 25, spinnerets, ventral view; 26, bulb, prolatera! view; 27, same, ventral view; 28, same, retrolateral view; 29, left male
pedipalp, retrolateral view. Scale of 1 7 = 2 mm; scale of 24 = 1 mm; scale of 23 = 0.5 mm; scale of other images = 0.2 mm. CD = copulatory duct;
DP = distal portion of conductor; DS = distal sclerite at MA; FD = fertilization duct; PM = Posterior membrane; PS = posterior sclerite; PT =
epigynal ‘pseudo teeth’; RC = receptaculum; TR = transversal ridge at conductor.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

125

Figures 30-41. — Eratigena flexuosa (F.O. Pickard-Cambridge, 1902), male holotype (30-36) and female (37-41). 30, habitus, dorsal view; 31,
sternum, ventral view; 32, spinnerets, ventral view; 33, cymbium and bulb, prolatera! view; 34, same, ventral view; 35, same, retrolateral view; 36,
chelicerae, posterior view; 37, carapace and chelicerae, anterior view; 38, epigynal area, ventral view; 39, vulva, posterior view; 40, same, anterior
view; 41, same, dorsal view. Scale of 30 = 2 mm; scale of 31, 37 = 1 mm; scale of 32, 36 = 0.5 mm; scale of other images = 0.2 mm.

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Figures 42-53. — Eratigena florea (Brignoli, 1974), female (42-46) and male (47-53). 42, epigynal area, ventral view; 43, vulva, anterior view;
44, same, dorsolateral view; 45, same, dorsal view; 46, same, variation (specimen labeled ""prope" florea by Brignoli); 47, habitus, dorsal view; 48,
carapace and chelicerae, anterior view; 49, sternum, ventral view; 50, spinnerets, ventral view; 51, cymbium and bulb, prolateral view; 52, same,
ventral view; 53, same, retrolateral view. Scale of 47 = 2 mm; scale of 48-49 = 1 mm; scale of 50 = 0.5 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

127

Figures 54-65. — Eratigena yarini sp. nov., female holotype (54-62) and male paratype (63-65). 54, habitus, dorsal view; 55, carapace and
chelicerae, anterior view; 56, sternum, ventral view; 57, spinnerets, ventral view; 58, epigynal area, ventral view; 59, vulva, anterior view; 60,
same, dorsolateral view; 61, same, dorsal view; 62, same, posterior view; 63, cymbium and bulb, prolateral view; 64, same, ventral view; 65, same,
retrolateral view. Scale of 54 = 2 mm; scale of 55-56 = 1 mm; scale of 57 = 0.5 mm; scale of other images = 0.2 mm.

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Figures 66-80. — Eratigem hlanda (Gertsch, 1971), female holotype (66-71), and Eratigena cf. gertschi, female (72-75), and male (76-80). 66,
carapace and cheiicerae, anterior view; 67, spinnerets, ventral view; 68, epigynal area, ventral view; 69, vulva, posterior view; 70, same, anterior
view; 71, same, dorsal view; 72, epigynal area, ventral view; 73, vulva, dorsal view; 74, same, dorsolateral view; 75, same, posterior view; 76,
carapace and cheiicerae, anterior view; 77, pedipalp, retrolateral view; 78, bulb, prolateral view; 79, same, ventral view; 80, same, retrolateral
view. Scale of 66, 76-77 = 1 mm; scale of 67 = 0.5 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

129

Figures 81-89. — Eratigena caverua (Gertsch, 1971), male holotype (81-84) and female allotype (85-89). 81, bulb, prolateral view; 82, same,
ventral view; 83, same, retrolateral view; 84, carapace and chelicerae, anterior view; 85, epigynal area, ventral view; 86, vulva, posterior view; 87,
same, dorsolateral view; 88, same, dorsal view; 89, same, anterior view. Scale of 84 = 1 mm; scale of other images = 0.2 mm.

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Figures 90-101. — Eraligena decora (Gertsch, 1971), male holotype (90-96) and female paratype (97-101). 90, habitus, dorsal view; 91,
carapace and chelicerae, anterior view; 92, spinnerets, ventral view; 93, bulb, prolateral view; 94, same, ventral view; 95, same, retrolateral view;
96, pedipalp, retrolateral view; 97, epigynal area, ventral view; 98, vulva, posterior view; 99, same, anterior view; 100, same, dorsal view; 101,
same, dorsolateral view. Scale of 90 = 2 mm; scale of 91, 96 = 1 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

131

Figures 102-113. — Eratigena femamioi sp. nov., female paratype (102-108) and male holotype (109-113). 102, carapace and chelicerae,
anterior view; 103, sternum, ventral view; 104, spinnerets, ventral view; 105, epigynal area, ventral view; 106, vulva, dorsal view; 107, same,
anterior view; 108, same, posterior view; 109, habitus, dorsal view; 110, pedipalp, retrolateral view; 1 11, bulb, prolateral view; 1 12, same, ventral
view; 113, same, retrolateral view. Scale of 109 = 2 mm; scale of 102-103 = 1 mm; scale of 104, 1 10 = 0.5 mm; scale of other images = 0.2 mm.

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123

124

125

Figures 1 14-125. — Eratigena gertsclii (Roth, 1968), male holotype (1 14-1 18) and female (119-125). 1 14, cymbium and bulb, prolateral view;
115, bulb, ventral view; 1 16, same, retrolateral view; 1 17, pedipalp, same; 118, spinnerets, lateral view; 1 19, sternum, ventral view; 120, abdomen,
dorsal view; 121, carapace, same; 122, epigynal area, ventral view; 123, vulva, anterior view; 124, same, dorsolateral view; 125, same, dorsal view.
Scale of 120 = 2 mm; scale of 1 17, 119=1 mm; scale of 1 14 = 0.5 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEG EN ARIA AND ERATIGENA

133

Figures 126-137. — Eratigena gucmato sp. nov., female paratype (126-134), and male (135-137). 126, habitus, dorsal view; 127, carapace and
chelicerae, anterior view; 128, sternum, ventral view; 129, spinnerets, ventral view; 130, epigynal area, ventral view; 131, same; 132, vulva,
anterior view; 133, same, dorsolateral view; 134, same, dorsal view; 135, bulb, prolateral view; 136, same, ventral view; 137, same, retrolateral
view. Scale of 126 = 2 mm; scale of 127-128 = 1 mm; scale of other images = 0.2 mm.

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Figures 138-149. — Eratigena me.xicana (Roth, 1968), male holotype (138-143) and female (144-148). Eratigena cf. mexicana, female (149) .
138, carapace and chelicerae, anterior view; 139, abdomen, dorsal view; 140, pedipalp, retrolateral view; 141, bulb, prolateral view; 142, same,
ventral view; 143, same, retrolateral view; 144, sternum, ventral view; 145, epigynal area, ventral view; 146, vulva, anterior view; 147, same,
dorsolateral view; 148, same, dorsal view; 149, same. Scale of 139-140 = 2 mm; scale of 138, 144 = 1 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

135

Figures 150-161. — Eiatigena queretaro sp. nov., female holotype (150-153, 155-158), female paratype (154), and male paratype (159-161).
150, habitus, dorsal view; 151, carapace and chelicerae, anterior view; 152, sternum, ventral view; 153, spinnerets, ventral view; 154-155, epigynal
area, ventral view; 156, vulva, posterior view; 157, same, anterior view; 158, same, dorsal view; 159, bulb, prolateral view; 160, same, ventral view;
161, same, retrolateral view. Scale of 150 = 2mm; scale of 151-152 = 1 mm; scale of 153 = 0.5 mm; scale of other images = 0.2 mm.

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Figures 162-173. — Eratigeiui rothi (Gertsch, 1971), male holotype (162-169), and female (170-173). 162, abdomen, dorsal view; 163, carapace,
same; 164, carapace and chelicerae, anterior view; 165, cymbium and bulb, prolateral view; 166, bulb, ventral view; 167, same, retrolateral view;
168, sternum, ventral view; 169, spinnerets, ventral view; 170, epigynal area, same; 171, vulva, anterior view; 172, same, posterior view; 173, same,
dorsal view. Scale of 162-164, 168 = 1 mm; scale of 165, 169 = 0.5 mm; scale of other images = 0.2 mm.

171

165

172

162

164

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

137

Figures 174-185. — Eratigena selva (Roth, 1968), male holotype (174-178), female (AB1158, 179-185). 174, bulb, prolateral view; 175, same,
ventral view; 176, same, retrolateral view; 177, carapace and chelicerae, anterior view; 178, pedipalp, retrolateral view; 179, sternum, ventral view;
180, abdomen, dorsal view; 181, spinnerets, ventral view; 182, epigynai area, ventral view; 183, vulva, posterior view; 184, same, anterior view;
185, same, dorsal view. Scale of 177-178, 180 = 2 mm; scale of 179 = I mm; scale of 181 = 0.5 mm; scale of other images = 0.2 mm.

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JOURNAL OF ARACHNOLOGY

Figures 186-194. — Variation of Eratigena selva (Roth, 1968), male (186-189), and female (190-194) from Sotano de Golondrina (AB1151).
186, bulb, prolateral view; 187, same, ventral view; 188, same, retrolateral view; 189, pedipalp, same; 190, spinnerets, ventral view; 191, epigynal
area, ventral view; 192, same, dorsolateral view; 193, same, anterior view; 194, same, dorsal view. Scale of 189 = 1 mm; scale of 190 = 0.5 mm;
scale of other images = 0.2 mm.

Figures 195-206. — Eratigena tlaxcala (Roth, 1968), male holotype (195-197), and female (198-207). 195, bulb, prolateral view; 196, same,
ventral view; 197, same, retrolateral view; 198, abdomen, dorsal view; 199, carapace and chelicerae, anterior view; 200, sternum, ventral view;
201, spinnerets, ventral view; 202, epigynal area, ventral view; 203, vulva, dorsal view; 204, same, posterior view; 205, same, anterior view; 206,
same, variation, dorsal view. Scale of 198, 200 = 2 mm; scale of 199 = 1 mm; scale of 201 = 0.5 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

204

205

203

Figures 207-218. — Eratigena xilitia sp. nov., male holotype (207-210), and female paratype (21 1-218). 207, bulb, prolateral view; 208, same,
ventral view; 209, same, retrolateral view; 210, pedipalp, same; 211, habitus, dorsal view; 212, carapace and chelicerae, anterior view; 213,
sternum, ventral view; 214, spinnerets, same; 215, epigynal area, same; 216, vulva, posterior view; 217, same, anterior view; 218, same, dorsal
view. Scale of 21 1 =2 mm; scale of 212-213 = 1 mm; scale of 210, 214 = 0.5 mm; scale of other images = 0.2 mm.

BOLZERN & HANGGI— NEARCTIC TEGENARIA AND ERATIGENA

141

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2016. Journal of Arachnology 44:142-147

The Mediterranean recluse spider Loxosceles vufescem (Dufour, 1820) (Araneae: Sicariidae) established

in a natural cave in Thailand

Narin Chomphuphuang*, Sureerat Deowanish', Chaowalit Songsangchote^, Varat Sivayyapram’, Panupong Thongprem^ and
Natapot Warrit’; 'Center of Excellence in Entomology and Department of Biology, Faculty of Science, Chulalongkorn
University, Bangkok, Thailand 10330. E-mail: natapot. w@chula.ac.th; “Spider Planet Research Center, 49/201
Sukhapiban 5 Soi 45 Rd., Orngean, Saimai district, Bangkok, Thailand 10220; ^Department of Biology, Faculty of
Science, Silpakorn University, Nakhon Fathom, Thailand 73000

Abstract. Loxosceles rufescens (Dufour, 1820), the Mediterranean recluse spider, is a cosmopolitan species with toxic
venom which can occasionally cause dermatological injuries in humans. Here, we report the finding of L. rufescens through
intensive survey and exploration of six natural limestone caves in the western region of Thailand. These data provide the
first direct evidence of L. rufescens living in large numbers in a natural habitat outside of their native Mediterranean range.

Although the currently known distribution of L. rufescens in Thailand is quite narrow (the spiders were only found in one
of the six caves explored), data on their biology and local habitat preferences are provided to better understand the
colonization requirements of this species in the target area.

Keywords: Cave, distribution, toxic, violin spider

The genus Loxosceles Heineken & Lowe, 1832, the recluse
or violin spiders, is one of two genera in the family Sicariidae
(Araneae), comprising 107 species distributed around the
world (Platnick 2015). They are notorious for their bites,
which occasionally produce a suite of symptoms known as
‘loxoscelism’, characterised by severe necrotic dermatologic
injuries (Swanson & Vetter 2006). One of the most well-known
recluse species is the Mediterranean recluse spider, L. rufescens
(Dufour, 1820). The original range of this species is in the
circum-Mediterranean region, however it has been spread
widely, most likely through accidental human transportation
(Vetter 2008). It is now found in many temperate and tropical
areas including the islands of the Atlantic, Madagascar, the
Hawaiian islands, Australia, Mexico, and the United States
(Gertsch & Ennik 1983). In Asia, L. rufescens has been
reported in India (Tikader 1963), China (Chen & Gao 1990;
Chen & Zhang 1991; Song et al. 1999), Russia (Dunin 1992),
Taiwan (Song et al. 1999), Japan (Yaginuma 1940, 1986;
Yoshikura 1987; Ono 2009), and South Korea (Namkung
2002). A recent molecular phylogenetic study of the L.
rufescens complex suggested that L. rufescens might have
occurred in Malaysia, although limited numbers of specimens
were used for the study (only one female and two males)
(Duncan et al. 2010).

Here, we report the finding of a large population of L.
rufescens in a natural habitat in Southeast Asia. We document
the presence of L. rufescens in western Thailand, provide
observations on its morphology, natural history, habitat and
identification, and propose how it may have arrived in
Thailand.

METHODS

We surveyed six natural limestone caves located in the Khao
Wang Khmer area of Kanchanaburi province in the western
part of Thailand (14°25'N, 98°52'E) on 18 April and 25 May
2015 (Figs. 1, 2). These caves are located in the conservation
area of the Plant Genetic Conservation Project under the

Royal Initiative of Princess Maha Chakri Sirindhorn (RSPG).
Spiders were hand collected and specimens were preserved in
95% ethanol and transferred to the Center of Excellence in
Entomology, Chulalongkorn University (Bangkok) for dis¬
section and identification.

Spiders that appeared to be L. rufescens were examined to
confirm their identity, and to provide morphological data on
the population found in Thailand. The genitalia of females
were dissected and cleared with a 3M KOH aqueous solution.
An Olympus SZ60 stereoscope coupled with an Olympus
digital camera (Camedia c-4040 zoom) was used to photo¬
graph the diagnostic features of the putative L. rufescens. All
measurements (in millimeters) were carried out under an
ocular micrometer in a stereomicroscope (Olympus: Zeiss
Stemi DV4). Descriptions are based on one specimen for each
sex; however, for the leg measurements (left legs), we also
displayed measurements for an additional 5? and AS
specimens (Table 1).

The following abbreviations are used throughout the text:
AME, anterior median eye/s; CL, carapace length; CW,
carapace width (measured at the widest point); ED, endite;
LA, labium; LE, lateral eye/s; SL, sternum length; SW,
sternum width (measured at the widest point); and TL, total
length.

Specimens were identified in accordance with Greene et al.
(2009). All voucher specimens (i.e., 10 9,4 S and 6 subadult
juveniles) are deposited at the Chulalongkorn University
Museum of Zoology (CUMZ), Bangkok (Thailand) for future
analysis.

SYSTEMATICS

Family Sicariidae Keyserling, 1880
Genus Loxosceles Heineken & Lowe, 1832

Loxosceles Heineken & Lowe, 1832 in Lowe, 1832: 321. Full
synonymy: see Gertsch & Ennik (1983) and Platnick
(2015).

142

CHOMPHUPHUANG ET AL.— RECLUSE SPIDER IN THAILAND

143

Figure 1. — The ‘Death Railway’ trail (black dashed line), which is in close proximity to Tum-Wangpra cave (red spot) where specimens of
Lo.xosceles rufesceiis were found. Yellow spots are caves that we explored without finding any L. rufescens. The inset picture on the top right is
indicative of what remains of the railway nowadays, whereas the inset picture on the bottom right shows the locality of Tum-Wangpra cave.

Figure 2. — A schematic outline of Tum-Wangpra cave located in
the Khao Wang Khmer area of Kanchanaburi province, Thailand.
Light green shading indicates areas where more than 100 L. rufescens
individuals were found (residing mostly under scattered rocks on the
ground), whereas yellow shading indicates areas with less than 100
individuals (and most specimens hiding in crevices on the cave walls).
Dark grey areas depict the limestone cave boundaries, and light grey
areas illustrate small limestone boulders that are accessible by
humans. The red spot corresponds with the red arrow in Fig. 6,
showing the exact location of the spider aggregation site.

Type Species. — Scytodes rufescens Dufour, 1820, by subse¬
quent designation of Bonnet (1957).

Remarks. — As is typical of the genus Lo.xosceles, all
specimens examined from Thailand possessed the following
suite of characters: carapace flattened, longer than wide with a
deep fovea (Fig. 5); clypeus porrect (Fig. 5); legs long and
slender; sternum longer than wide; and abdomen oblong
bearing spine-like setae (Lotz 2012). A diagnostic character for
the females of L. rufescens is the structure of the spermathecae,
which are characterized by the presence of closely-spaced
receptacles and wide, laterally brown, sclerotized copulatory
tubes (Fig. 3). These features were found in all 10 female
specimens examined. In addition, when we examined four
male specimens, the palpal tibia length to height ratio was less
than 2.0, the cymbium was about half to less than half the
length of the tibia, the length of the cymbium was similar to
that of the pulpal bulb, and the palpal bulb was globular with
a thin embolus (Fig. 4). These male characters are generally
diagnostic of L. rufescens (Lotz 2012), although not exclu¬
sively so (Duncan et al. 2010; Planas & Ribera 2015; Planas et
al. 2015).

Loxosceles rufescens (Dufour, 1820)

(Figs. 3-5, 8, 9)

Scytodes rufescens Dufour, 1820: 203, pi. 76, fig. 5. Full
synonymy: see Platnick (2015).

Material examined. — THAILAND: Kanchanaburi: 69, 3
subadult juveniles, Sai Yok district, Tum-Wangpra cave,
14°24'47"N, 98°51'43"E, 18 April 2015, hand collected, N.
Chomphuphuang (CUMZ-AR-ARA-Sic.2015.1-9); 4 c?, 49,

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JOURNAL OF ARACHNOLOGY

Table 1. — Left leg and palp measurements of a single representative adult female and adult male of Loxosceles rufescens from Thailand, and
averages and standard deviations of five additional females and four male specimens (all measurements are in millimeters). Leg formulas are
provided.

I

II

III

IV

Palp

Female (CUMZ-AR-ARA-Sic.2015.1); leg formula: 2413

Femur 5.04 5.40

4.8

5.52

1.35

Patella

1.11

1.17

0.99

0.90

0.33

Tibia

5.28

6.56

4.80

5.46

0.87

Metatarsus

5.70

6.00

5.10

6.60

-

Tarsus

1.38

1.25

1.00

1.25

1.08

Total

18.51

20.38

16.69

19.73

3.63

Male (CUMZ-AR-ARA-Sic.2015.10); leg formula;
Femur 5.12

2413

7.00

4.74

5.52

1.41

Patella

1.17

1.05

0.90

1.05

0.30

Tibia

6.80

8.40

5.90

5.92

0.90

Metatarsus

6.63

8.63

6.10

7.25

-

Tarsus

1.50

1.74

1.20

1.65

0.60

Total

21.22

26.82

18.84

21.39

3.21

Five adult females (CUMZ-AR-ARA-Sic. 201 5.2-6); leg formulas: 2143
Femur 4.29±0.99 4.56±0.99

3.96±0.86

4.38±0.99

0.93±0.32

Patella

0.90±0.21

0.87±0.36

0.92±0.19

0.86±0.17

0.37±0.05

Tibia

3.72±1.85

4.23±2.21

3.14±1.51

3.59±1.75

0.53±0.22

Metatarsus

4.28±1.28

4.70±1.36

4.06±1.07

4.44±1.37

-

Tarsus

1.38±0.16

1.39±0.13

1.20±0.20

1.23±0.24

1.00±0.22

Total

15.24±3.54

16.52±3.96

13.78±3.07

15.13±3.29

2.65±0.42

Four adult males (CUMZ-AR-ARA-Sic. 2015. 10-13); leg formulas: 2143
Femur 5.06±0.55 6.18±0.95

4.54±0.56

4.88±0.82

i.05±0.24

Patella

1.02±0.14

0.94±0.30

1.08±0.35

0.96±0.08

0.38±0.10

Tibia

6.15±0.86

7.40±1.16

4.85±0.87

5.28±0.76

0.55±0.26

Metatarsus

5.86±0.72

7.26±1.35

5.30±0.79

6.29±0.83

-

Tarsus

1.45±0.13

1.51±0.20

1.25±0.06

1.44±0.25

0.40±0.14

Total

14.51±8.78

17.11±10.84

12.91±7.97

14.05±8.60

1.95+1.21

3 subadult juveniles, same data except 25 May 2015
(CUMZ-AR-ARA-Sic.201 5. 1 0-20).

Description. — Carapace flattened, longer than wide, with a
deep fovea and porrect clypeus (Fig. 5); chelicerae joined
basally, with an immovable, thumb-like extension on the
medial apical surface and short fangs; six eyes in three diads;
legs long and slender, with two tarsal claws bearing serrated
bristles on a small onychium; female genitalia haplogyne, with
single broad opening and two spermathecae.

Female (CUMZ-AR-ARA-Sic.2015.1); TL = 8.1; CL = 3.6,
CW = 3.2; eye diameter 0.15; AME-LE = 0.27; eye row
strongly recurved; abdomen length = 4.26, width = 3.0; clypeus
height = 0.32, SL = 1 .88, SW = 1 .59; LA = 0.78 long, 0.66 wide;
ED = 1.29 long, 0.36 wide; leg formula: 2413; leg and palp
measurements shown in Table 1 . Carapace and chelicerae light
brown, anterior carapace with dark brown ‘violin’ pattern
(Fig. 5); legs and palps pale yellow to orange covered by short
black setae, female palp without claw; coxae and sternum pale

Figures 3-4.— Diagnostic characters of Loxosceles rufescens: 3, female spermathecae (dorsal view); 4, male pedipalp. Scale bars = 0.15 mm
(Fig. 3), 0.5 mm (Fig. 4).

CHOMPHUPHUANG ET AL —RECLUSE SPIDER IN THAILAND

145

Figure 5. — Adult female Lo.xosceles rufesceus carapace (dorsal
view). Scale bar = 1 mm.

yellow, labium and endites brown; abdomen yellowish,
covered with setae. Receptacles of spermathecae closely
spaced; copulatory tubes wide with lateral brown sclerotized
area (Fig. 3).

Female variation-. A single female specimen (CUMZ-AR-
ARA-Sic.2015.2) has two black spots on the posterior end of
the carapace; subadults or recently molted individuals with
pale pigment in the violin pattern.

Male (CUMZ-AR-ARA-Sic.2015.10): TL = 5.10; CL =
2.68, CW = 2.40; eye diameter 0.09; AME-LE = 0.21; eye row
strongly recurved; abdomen length = 3.00, width = 1.55;
clypeus = 0.27; SL = 1.23, SW = 1.35; LA = 0.45 long, 0.51
wide; ED = 0.90 long, 0.33 wide; leg formula: 2413 (see
discussion); leg and palp measurements shown in Table 1.
Overall body and coloration similar to female except for the
abdomen, which is distinctly smaller than that of the female.
Palpal tibia length to height ratio less than 2.0; cymbium half
of tibia length; cymbium as long as length of palpal bulb; bulb
globular and embolus thin (Fig. 4).

Eggs'. In the laboratory, we retrieved a silken egg sac from
one female nine days after the collection date. The sac
contained 24 eggs with egg diameters between 1.07-1.15 mm.
The eggs took 23 days after the egg sac was deposited to hatch

Figures 6-9. — Tum-Wangpra limestone cave, the locality where Lo.xosceles rufescens specimens were collected: 6, entrance to the cave (red
arrow indicates the area where numerous L. rufescens were found); 7, cave surface (red arrows indicate L. rufescens retreats on the limestone
ground); 8, flocculent web of L. rufescens on the crevice of the limestone ground; 9, L. rufescens clinging to the cave wall.

146

JOURNAL OF ARACHNOLOGY

into spiderlings under an ambient temperature of 27°C and
70% relative humidity. Only 21 individuals successfully
hatched (87% hatch rate). We measured the total body length
of the spiderlings two days after they emerged from the egg
sac, at which time they were between 1 .20-1 .68 mm (average
1.46 mm).

Habitat: Of the six limestone caves and peripheral urban
areas that we surveyed, L. nifescens were found in only one of
the caves locally known as “Tum-Wangpra” (Fig. 2). This
cave has two major areas that extended approximately 20-25
m from the cave entrance. The accessible area for humans is
about 340 m" with an average height of 5 m above ground.
The cave is partially accessible by sunlight at a distance of 5 m
from the cave entrance, which is devoid of the spiders. The
surface of the ground inside the cave is slightly warm (annual
average air temperature = 27°C and annual average relative
humidity = 81.6 %), and mainly covered with a dry loamy sand
and bat guano (Fig. 6). A population of the Old World hog¬
nosed bat, Craseonycteris thonglongyai Hill, 1974 is also found
inhabiting the cave. An intensive survey revealed that the
spiders were aggregated in similar microhabitats throughout
the cave (Figs. 7-9); they hid themselves in crevices on the
walls (Fig. 9), or under scattered rocks on the ground (Fig. 7-
8) where they made small retreats of flocculent silk. Some
individuals of L. nifescens also clung on rocks beneath other
spiders’ webs. By counting the visible spiders, we determined
that there might be approximately 500 or more L. nifescens
individuals living in the cave.

DISCUSSION

L. nifescens is a cosmopolitan species widely distributed
throughout the world (Platnick 2015). Although native to the
Mediterranean region, the species has been spread to other
areas by human activities (Harvey 1996). In Israel, L. nifescens
is moderately common in houses and basements (Shulov et al.
1962). Greene et al. (2009) suggested that populations of the
genus Loxosceles in the United States tend to be extremely
dense in favorable urban environments such as steam tunnels
and subterranean habitats. In Iran, L. nifescens is also found
inside buildings and under rocks and logs in urban areas
(Zamani & Rafinejad 2014). In the natural environment,
Loxosceles populations can be found in caves and/or cavern¬
like habitats (see Newlands 1975; Gertsch & Ennik 1983;
Griffin 1998; Ferreira et al. 2005; Gongalves-de-Andrade et al.
2007). Here, we report the anomalous discovery of L. nifescens
in a natural habitat in Thailand. Until now, L. nifescens has
never been reported in a natural habitat outside of its native
range in the Mediterranean region. Clearly, the description of
the physical parameters of the cave in which the spiders were
found, and an understanding of their basic biology, are
important considerations for determining the colonization
requirements of this species in the target area.

Although speculative, the occurrence of L. nifescens in this
part of Thailand might be explained by passive transportation
by humans during World War II. The area in which we
discovered L. nifescens was called the ‘Hellfire Pass’, when it
was a major route for the construction of the infamous “Death
Railway’’ or the Burma-Siam Railway along the Mae Klong
River in Kanchanaburi province (Waterford 1994). The
entrance of Tum-Wangpra cave is in close proximity to the

railway (Fig. 1); material for the construction of the railway
may have harbored the spiders, since specific railing material
had to be shipped from Japan during that period, and there
were already reports of L. nifescens present in Japan before
1940 (Bosenberg & Strand 1906; Strand 1918; Yaginuma
1940).

In North and South America, studies on the biology,
distribution, and medical aspects of Loxosceles are ongoing,
particularly for the brown recluse spider L. reclusa Gertsch &
Mulaik, 1940, which is endemic to the USA (Swanson and
Vetter 2005). In contrast, research on the genetic diversity and
venom potency of L. nifescens has only been extensively
studied in recent years (Planas & Ribera 2015; Planas et al.
2015). A protein expression analysis of the sphingomyelinase
D (SMase D) protein, which is considered to be the major
component of Loxosceles venom that causes dermatological
injuries (Binford et al., 2008), suggested that the SMase D
protein activity in L. rufescens venom is as high as in other
Loxosceles species (Planas et al. 2015). In 2014, it was reported
that a man in Phrae province in the northern part of Thailand
had been bitten by L. reclusa and died (Bangkokpost 2014).
The symptoms reported were very similar to loxoscelism, and
this news sparked much attention and in some cases hysteria
from the general public, much of it unwarranted. However, a
doctor later concluded that the man died as a result of a
secondary bacterial infection due to an unidentified spider’s
bite, and not loxoscelism as originally thought. This hypoth¬
esis agrees with our survey of the village perimeter near where
the man was bitten, which did not yield any L. reclusa. Thus,
in Thailand there is still no verified report of a human being
bitten by a Loxosceles spider.

Our next goal is to survey the distribution of L. nifescens
beyond the Khao Wang Khmer area to identify the true range
of the species. Molecular studies of the Thai L. nifescens are
equally important for determining the spider’s origins. Indeed,
since genetic diversity can be high within species of Loxosceles
that share similar spermathecal and palp morphologies
(Duncan et al. 2010; Planas & Ribera 2015; Planas et al.
2015), our samples need to be tested to determine whether they
are genetically consistent with L. rufescens s. s.

ACKNOWLEDGMENTS

We thank the Plant Genetic Conservation Project under the
Royal Initiative of Princess Maha Chakri Sirindhorn (RSPG),
Chong Khao Khat, Sai Yok, Kanchanaburi province and the
Royal Thai Army through Major Chanwit Prathomkamneard
for the access to collect the spider specimens. The first sighting
record of L. rufescens in Tum-Wangpra cave was initially
reported to CS via Mr. Kirati Kunya. Dr. Deborah Smith
from the Department of Ecology and Evolutionary Biology,
University of Kansas, USA, reviewed the manuscript and
provided invaluable comments. We also greatly appreciate two
anonymous reviewers for providing insights to the current
state of Loxosceles studies and many important corrections.
The assistance of Ms. Nungruthai Wichaikul is appreciated
for helping us collect the spiders. Dr. Thongchai Ngampra-
sertwong provided the authors with the temperature and
humidity data at the collecting site.

CHOMPHUPHUANG ET AL.— RECLUSE SPIDER IN THAILAND

147

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2016. Journal of Arachnology 44:148-152

Chromosomal analyses of Salticinae and Lyssomaninae reveal a broad occurrence of the 2nd =28, XjXiO

karyotype within Salticidae

Douglas Araujo', Mariana Bessa Sanches', Juliane da Silva Gonsalves Santana Lima”, Erica Vanessa Juliao do Nascimento^,
Andre Marsola Giroti"', Antonio Domingos BrescoviC, Doralice Maria Cella^ and Marielle Cristina Schneider^:
'Universidade Federal de Mato Grosso do Sul, UFMS, Setor de Biologia Geral, Centro de Ciencias Biologicas e da
Saude, Cidade Universitaria, Bairro Universitario, 79070-900, Campo Grande, Mato Grosso do Sul, Brazil. E-mail: d.
araujo@ufms.br; ^Universidade Estadual de Mato Grosso do Sul, UEMS, Unidade Universitaria de Ivinhema, Centro,
79740-000, Ivinhema, Mato Grosso do Sul, Brazil; '^Universidade Estadual de Mato Grosso do Sul, UEMS, Unidade
Universitaria de Mundo Novo, Universitario, 79790-000, Mundo Novo, Mato Grosso do Sul, Brazil; '^Instituto
Butantan, Laboratorio Especial de Colegoes Zoologicas, Av. Vital Brasil, 1500, 05503-900, Sao Paulo, Sao Paulo, Brazil;

meinoriam; ^Universidade Federal de Sao Paulo, UNIFESP, Departamento de Ciencias Biologicas, Av. Prof. Artur
Riedel, 275, 09972-270, Diadema, Sao Paulo, Brazil

Abstract. Brazil possesses the richest fauna of Salticidae in the world, including 560 species; however, no representative of
the Brazilian fauna has been cytogenetically analyzed up to now. It has been demonstrated that karyotype data are a useful
source for discussions on the phylogeny and chromosome differentiation of some salticid lineages. In this work, the first
chromosome study of salticid species from Brazil is presented, with the addition of five genera to the 38 previously
investigated worldwide. The analysis of mitotic and/or meiotic cells revealed 2ncJ =28, X1X2O in Asaracus sp., Coryphasia
sp., Chira sp., Frigga quintensis (Tullgren, 1905), and Lyssomanes pauper Mello-Leitao, 1945. This karyotype constitution
is the most common for Salticidae, occurring in species of distinct clades. The diploid number 2n9 =28 observed in
Hasariiis adansoni (Audouin, 1826) is unexpected, differing in one autosomal pair from the karyotype previously registered
for males of the same species. The cytogenetic information reported here reinforces the wide occurence of 2n(? =28, X1X2O
within Salticidae, including species belonging to different clades and biogeographical regions. This karyotype is a shared
character of Salticidae + Philodromidae, found exclusively in these families within Dionycha, suggesting its sister
relationship already proposed in the literature.

Keywords: Jumping spider, meiosis, sex chromosome system, diploid number, chromosome evolution

A hundred years has past since Painter (1914) cytogeneti¬
cally studied a salticid spider for the first time, Maevia
mclemens (Walckenaer, 1837) [under Maevia vittata (Hentz,
1846)]. Despite the huge contribution of Maddison (1982,
1996) and Maddison & Leduc-Robert (2013), which cytoge¬
netically analyzed 86 salticids, only 155 species belonging to 38
genera were karyotyped up to now (Araujo et al. 2016). This
number corresponds to only 2.65% of the 5,850 taxonomically
described Salticidae species (World Spider Catalog 2016).
Furthermore, many clades, mainly those predominantly
composed of Neotropical species (Amycoida, Marpissoida,
Euophryini and Freyina) or basal species (non-salticines)
(Maddison 2015) remain almost unknown from the karyolog-
ical point of view. Only six Neotropical Salticidae species,
belonging to the genera Bryantella Chickering, 1946, Den-
dryphantes C.L. Koch, 1837, Metaphidippus F.O.Pickard-
Cambridge, 1901 (Salticinae, Dendryphantini, Dendryphaiiti-
na) (Scioscia 1997) and Hahronattus F.O.Pickard-Cambridge,
1901 (Salticinae, Plexippini, Harmochirina) (Maddison &
Leduc-Robert 2013) were cytogenetically studied, and, among
these genera, only Bryantella is exclusively Neotropical (World
Spider Catalog 2016). Within non-salticines, only Holcolaetis
vellerea Simon, 1910 (under Holcolaetis vidua Lessert, 1927)
(Spartaeinae, Spartaeini, Holcolaetina) was karyotyped (Mit¬
tal 1961, 1964).

At a first glance, the salticids cytogenetically analyzed seem
to constitute a relatively homogeneous group, mostly com¬
posed of species with 2n(5' =28, XiXiO (Araujo et al. 2016). A

multiple sex chromosome system (SCS) of the X1X2O type is
rare in other animal groups but the most common in spiders
(see Araujo et al. 2012). In this SCS, the sex is not determinate
by the presence of an Y or W chromosome, as it occurs in
most mammals and birds. The number of copies of each X
chromosome determines the sex, a single copy of each one
(X1X2O) is characteristic of a male and two copies of each one
(X1X1X2X2), characterizes a female. The “0” after the X1X2
denotes the absence of a Y chromosome in male complement.
Thus, in this SCS, if the male diploid number is 2ii(? = 28 (26
autosomes plus X1X2), the female diploid number is 2n9 = 30
(26 autosomes plus X]XiX2X2). At the end of male meiosis I,
both X) and X2 segregate to the same cell pole and the
opposite pole contains no sex chromosomes. Recently, a study
showed that uncommon karyotypes in spiders, with a multiple
sex chromosome system including a Y chromosome (X1X2Y
and X 1X2X3 Y), occur in Hahronattus, and probably evolved
independently several times within this genus (Maddison &
Leduc-Robert 2013).

Cytogenetic studies on underrepresented salticid clades
could provide us with information about the homogeneity or
heterogeneity of some lineages and could be useful for
discussions concerning salticid systematics. Thus, the goal of
this study is to analyze the chromosomes of six Salticidae
species from Brazil: the Salticinae Asaracus sp., Chira sp. and
Frigga quintensis (Tullgren, 1905) (Aelurillini), Coryphasia sp.
(Euophryini), and Hasarius adansoni (Audouin, 1826) (Hasar-

148

ARAUJO ET AL.— BROAD OCCURRENCE OF 2Nc? = 28, X,X20 IN SALTICIDS

149

Table 1. — Salticid spiders investigated in this work with their respective samples and collection localities in Brazil. SP = state of Sao Paulo, MS
= state of Mato Grosso do Sul, PR = state of Parana. Classification according to Maddison (2015).

Taxa

Sample

Collection locality

Salticinae, Saltafresia, Aelurillini, Freyina

Asaracus sp.

Id

Margem da Lagoa Xambre, Parque Nacional de llha Grande,
54°00'01"W), PR

Altonia (23°52'2I"S,

Chira sp.

Id

Rio Claro (22°24'00"S, 47°34'19"W), SP

Frigga quintensis (Tullgren, 1905)

2d

Margem da Lagoa Xambre, Parque Nacional de llha Grande,
54°00'01"W), PR; Ivinhema (22°18'00"S, 53°49'16"W), MS

Altonia (23°52'21"S,

Salticinae, Saltafresia, Euophryini

Coryphasia sp.

Id

Rio Claro (22°24'00"S, 47°34'19"W), SP

Salticinae, Saltafresia, Hasariini

Hasarius adansoni (Audouin, 1826)

19

Ivinhema (22°18'00"S, 53°49'16"W), MS

Lyssomaninae

Lyssomanes pauper Mello-Leitao, 1945

4d,3$

Reserva Particular do Patrimonio Natural da UFMS (20°29'58
Campo Grande, MS

;"S, 54°36'48"W),

iini), and the Lyssomaninae Lyssomanes pauper Mello-Leitao,
1945.

Except for the genus Hasarius Simon, 1871, karyotyped by
Suzuki (1951, 1954), the remaining genera were not previously
cytogenetically analyzed (Araujo et al. 2016).

METHODS

The number of individuals and collection localities of the
species examined in this work are listed in Table 1. Collecting
permits were provided by the Instituto Brasileiro do Meio
Ambiente e dos Recursos Renovaveis - IBAMA and Instituto
Chico Mendes de Conservagao da Biodiversidade - ICMBio
(15382-1 and 15157-1). The voucher specimens were deposited
in the arachnological collection of the Laboratorio Especial de
Colegoes Zoologicas, Instituto Butantan (IBSP, curator A. D.
Brescovit), Sao Paulo, state of Sao Paulo, Brazil. The
chromosome preparations were obtained following Araujo et
al. (2008), i.e., the gonads were submitted to 2 hours in a
treatment with 0.16% colchicine solution (diluted on physio¬
logic solution: 7.5 g NaCl, 2.38 g Na2HP04, 2.72 g KH2PO4,
in 1 1 of distilled water), 15 minutes in a hypotonic treatment
with tap water, and fixation with methanol/acetic acid (3:1).
Later, the gonads were dissociated in 45% acid acetic solution
on the surface of a microscope slide that was heated to 35 °C/
40 °C, and standard stained with 3% Giemsa solution (3% of
commercial Giemsa and 3% of phosphate buffer pH 6.8 in
distilled water) for 12 min. At least 30 cells were considered in
the analysis of each species. The chromosome morphology
was determined following the nomenclature proposed by
Levan et al. (1964). Difficulties in obtaining certain stages of
cell division occurred due to the low number of specimens, for
most species, or the development stage of the individuals, as in
the case of L. pauper.

RESULTS

Male diplotene/metaphase I cells of Asaracus sp., Corypha-
sia sp., dura sp. and F. quintensis are composed of 13
autosomal bivalents and two sex univalents (X] and X2). Thus,
the meiotic formula of these species is I3II-I-X1X2O. The
number of chiasmata per bivalent is usually one, localized on
terminal, interstitial or proximal regions, but some bivalents

with two terminal chiasmata can be observed in some cells.
The sex chromosomes can be easily identified due to their
positive heteropycnosis and/or peculiar disposition, since they
normally appear side by side or at least close to each other
(Fig. lA-D). The positive heteropycnosis of the sex chromo¬
somes is detected even at pachytene, in which it is possible to
observe the X] and X2 closely packed, being difficult to
establish their limits (Fig. IE), or clearly separated from each
other (Fig. IF). Male metaphase II cells of Asaracus sp. and F.
quintensis exhibited n = 13 or n = 15, confirming the regular
segregation of the Xj and X2 chromosomes to the same pole at
anaphase I (Fig. IG, H). In some metaphase II nuclei, the sex
chromosomes cannot be distinguished from the autosomes
(Fig. IG), but in others, these elements presented a positive
heteropycnosis (Fig. IH).

Spermatogonial prometaphase/metaphase cells of Corypha-
sia sp., F. quintensis and L. pauper showed 2n<? == 28, X1X2O
(Fig. II, K), which is compatible with the meiotic findings and
allow us to conclude that all salticid species above mentioned
possesses 2n = 28, X1X2O in males and 2n = 30, X|X]X2X2 in
females. Unfortunately, only female specimens of H. adansoni
were collected. The analysis of oogonial metaphases of L.
pauper and II. adansoni revealed the diploid number of 2n = 30
and 2n = 28, respectively (Fig. IL, M). The chromosomal
morphology was not established due to the irregular shape of
the elements and/or absence of mitotic plates, except for the
chromosomes of L. pauper and H. adansoni, which are clearly
telocentrics.

DISCUSSION

The data presented in this work are the first for salticid
species collected in Brazil. This seems to be fundamental
considering that Maddison & Hedin (2003) and Maddison et
al. (2008) suggest a biogeographical division between Old
World and New World salticid fauna. In addition, almost all
new world salticids cytogenetically analyzed are from North
America (Pinter & Walters 1971; Maddison 1982, 1996;
Tugmon et al. 1990; Maddison & Leduc-Robert 2013), despite
the fact that Brazil possesses the richest salticid fauna in the
world, with 560 species (Metzner 2016). All Salticidae species
analyzed here, except H. adansoni, revealed meiotic and/or
mitotic features that are consistent with 2n6 =28, X1X2O, the

150

JOURNAL OF ARACHNOLOGY

Figure 1. — Meiotic and mitotic cells of the salticid species. A-D. Diplotene/metaphase I spermatocytes of Asaracus sp. (A), Coryphasia sp. (B),
Chira sp. (C) and Frigga qidntensis (D) with 13 autosomal bivalents plus two sex univalents (Xi and X2). For exemplification, the arrow indicates
one autosomal bivalents with one interstitial chiasma, the empty arrowheads show autosomal bivalents with one terminal chiasma, and the full
arrowheads point to two terminal chiasmata in one autosomal bivalent. E, F. Pachytene cells of Chira sp., showing the positive heteropycnosis of
the sex chromosomes, that appear together (E) or separated (F). G, H. Metaphase II cells of Asaracus sp. (G) and Frigga cpdiUensis (H) with n =
13 in the right and n = 13 + X1X2 in the left. In (H) it is possible to identify the positive heteropycnotic sex chromosomes. I-K. Spermatogonial
prometaphase/metaphase cells of Coryphasia sp. (I), Frigga qidntensis (J), and Lyssomcmes pauper (K), with 2ncJ =28. L, M. Oogonial metaphase
cells of Lyssomcmes pauper (L), with 2n9 = 30 and Hasarius adansoni (M), with 2n? = 28. Scale = 10 pm.

most common chromosome constitution observed in the
family (Araujo et al. 2016).

Despite the fact that no quantitative analysis of chiasmata
was carried out, the species analyzed here seem to show a
tendency to have only one chiasma per bivalent, a common
characteristic in spiders (White 1973). However, in contrast to
the observations of White (1973) suggesting that the chiasmata
are primarily proximal in spiders, the nuclei of the species
analyzed herein show a diversity of chiasma position (distal,
interstitial and proximal). This pattern was already described
for Hahronaltus species with a X1X2O system, contrasting to
the Y-possessing species of the same genus, which showed a
tendency to have distal chiasmata (Maddison & Leduc-Robert
2013). Chiasmata in other positions than distal could act
against the occurrence of chromosome fusions that form
metacentric elements, including metacentric neo-Ys (for a
more detailed discussion see White 1973 and Maddison &
Leduc-Robert 2013).

Based on our results, Asaracus, Chira and Frigga (2n(J = 28,
X]X20) are the only genera, along with Aelurillus Simon, 1884,
with described karyotypes within the tribe Aelurillini (Maddi¬
son 201 5). The karyotype data of Aelurillus politiventris (O. P-
Cambridge, 1872), from Israel, is 2nc? = 21, XO (Gorlova et al.
1997). This difference in diploid number and sex chromosome
system can reflect the biogeographical distribution of these
genera. Instead of belonging to the same tribe Aelurillini, the
first three genera are part of the Neotropical subtribe Freyina,
and Aelurillus belongs to the Afro-Eurasian subtribe Aelurillina
(Maddison 2015). Thus, within Aelurillini, the karyotype seems
to be subtribe-specific, but it is important to emphasize that
most genera, including the entire African subtribe Thiratoscir-
tina, are cytogenetically unknown.

Euophryini, which encompasses Coryphasia (Maddison
2015), possesses two other species that were cytogenetically
studied: Euophrys pseudogamhosa Strand, 1915, from Israel
(Gorlova et al. 1997) and Jolus minulus L. Koch, 1881 (Suzuki
1951), both also with 2nS — 28, X1X2O. In the phylogenetic

ARAUJO ET AL.— BROAD OCCURRENCE OF 2Nc? =28, XiXjO IN SALTICIDS

151

hypothesis of Zhang & Maddison (2015), Coryphasia,
Euophrys C.L. Koch, 1834 and Jotus L. Koch, 1881 belong
to distinct clades within Euophryinae (currently Euophryini,
see Maddison 2015). However, despite the phylogenetic and
geographical distance, all three genera share the same diploid
number and sex chromosome system. Neon Simon, 1876,
classically placed in Euophryinae (Proszyhski 2012; Metzner
2016), was relocated to a new group, Astioida, by Maddison et
al. (2008, 2014), which possesses only one cytogenetically
analyzed genus, Myrmaracline MacLeay, 1839. A close
relationship between Neoii (Astioida, Neonini) and Myrmar-
achne (Astioida, Myrmarachnini) is proposed by the cytoge¬
netic data, because Neon reticulatus (Blackwall, 1853), from
Turkey, exhibits 2nc? =21, XO (Kumbigak 2014) and four of
the six Myrmaracline species analyzed cytogenetically showed
2n6 = 23, XO (Hackman 1948; Bole-Gowda 1958); these
karyotypes are much more similar to each other than to the
2n6' = 28, X1X2O found in the Euophryini Coryphasia,
Euophrys and Jotus.

Some considerations should be addressed regarding the
female specimen of H. adansoni (Hasariini) with the unex¬
pected 2n$ = 28. . Taking into account that Suzuki (1954)
described 2nS = 28, X1X2O in H. adansoni, it was supposed
that females of this species would have a diploid number of
2n$ = 30, X]XiX2X2. However, this female specimen has a
diploid number lower than the expected. Even though the sex
chromosome system was not identified in the present work, if
we consider the same system found by Suzuki (1954), the
female specimen analyzed here probably has the constitution
2n$ = 28, X1X1X2X2. Thus, this could be a case of
polymorphism in the number of autosomes, that is, 26
autosomes in the Japanese population studied by Suzuki
(1954) and 24 autosomes in the population from Ivinhema,
state of Mato Grosso do Sul, Brazil (present work).
Polymorphism in the number of autosomes was already
described in another salticid species, Habronattus viridipes
(Hentz, 1846), in which Maddison (1982) found 2nc? = 28,
X)X20 in most males, and 2n3 = 30, X]X20 in only one
specimen. Both cases, show a discrepant chromosome number
in only one specimen. Nevertheless, the presence of this
unusual diploid number within the //. adansoni population
from Ivinhema, Brazil, needs still to be confirmed. Without a
more thorough collection effort, a distinction between a
population polymorphism (Japan x Brazil) or an individual
chromosome variation cannot be made. In fact, the 2x\S =28,
X1X2O observed in other cytogenetically analyzed Hasariini,
Habrocestum rubroclypeatum Lessert, 1927 (Mittal, 1964),
reinforces the hypothesis that the 2n 9 =28 encountered in the
female specimen of H. adansoni from Ivinhema is a
populational variation of the karyotype 2nc? = 28/2n9 = 30,
which is commonly described for Salticidae.

Lyssomanes pauper is the first Lyssomaninae karyotyped, but
the occurrence of 2n6 = 28, X1X2O in this species and the other
non-salticine cytogenetically studied, Holcolaetis vellerea (Mit¬
tal 1961, 1964) (Spartaeinae, Spartaeini), both basal salticids,
points to a broad distribution of this karyotype within salticids.
Moreover, the presence of 2nS — 28, X]X20 only known for
Salticidae and Philodromidae within Dionycha (see Araujo et
al. 2016), suggests a sister relationship between these families, as
already proposed by Ramirez (2014).

The data presented here reinforce the karyotypic homoge¬
neity within salticids, with the 2n3 =28, X1X2O widespread in
species of several clades and biogeographical regions. How¬
ever, in many clades, only one or a few species were
karyotyped so far, a situation that blurs an unrevealed
chromosome heterogeneity, as shown for Habronattus, a
genus that has around 66% of its 99 species already
karyotyped (Araujo et al. 2016; World Spider Catalog 2016).
This genus exhibited four different diploid complements,
including the X1X2Y, X1X2X3Y and XO sex chromosome
systems, besides the classical 2nS = 28, X1X2O (Maddison &
Leduc-Robert 2013).

ACKNOWLEDGMENTS

This research has financial support from Conselho Nacional
de Desenvolvimento Cientifico e Tecnologico, CNPq Univer¬
sal 471821/2008-0 grant to DA, CNPq 303028/2014-9 grant to
ADB, and Funda9ao de Amparo a Pesquisa do Estado de Sao
Paulo, FAPESP 2011/21643-1.

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2016. Journal of Arachnology 44:153-164

The hemolymph vascular system in Amneus diademalus with special focus on intraspecific variability in

artery systems

Jens Runge and Christian S. Wirkner: Universitat Rostock, Allgemeine & Spezielle Zoologie, Institut fiir
Biowissenschaften, Universitatsplatz 2, 18055 Rostock, Germany. E-Mail: jens.runge@uni-rostock.de

Abstract. The European garden spider, Araneus diademalus Clerck, 1757, is one of the most common spiders in central
Europe. However, despite its abundance, comparatively little is known about its internal anatomy. We therefore conducted
an examination of the hemolymph vascular system (HVS) of A. diademalus, as part of a comparative survey on the
circulatory system in spiders. The HVS of A. diademalus was investigated using micro-computed-tomography and serial
sectioning and visualized using 3D-reconstruction software. In order to examine the HVS for intraspecific variability, over
30 specimens were studied in detail. The HVS of A. diademalus consists of a tubular heart, which is situated along the
dorsal midline of the opisthosoma. Anteriorly, the heart gives rise to the anterior aorta and posteriorly to the posterior
aorta. Three pairs of cardiac arteries originate from the dorso-latera! section of the heart and the branching pattern of
these arteries in A. diademalus is visualized and described here for the first time. The anterior aorta runs through the
pedicel into the prosoma where it branches to supply the muscles and organs with hemolymph. The central nervous system
in particular is supplied by a large number of arteries, which show some interesting branching patterns, e.g., a unilateral
origin of the transganglionic arteries in the subesophageal ganglion, and the supply of the lower lip by the first of these
transganglionic arteries. Furthermore, there are a number of arteries in the HVS with unilateral (asymmetrical) origins.

Some cases of intraspecific variability are demonstrated, e.g., for the arteries of the upper and the lower lip. The data
presented here are discussed in reference to information on the HVS in Araneae available in existing literature.

Keywords; 3D imaging, micro-CT, right-left asymmetry

The earliest descriptions of the hemolymph vascular system
(HVS) in Araneae can be traced back to the beginning of the
19'*’ century (e.g., Cuvier 1810, pp. 257-263; Treviranus 1812)
and, in the decades which followed, there was a marked
increase in the interest this organ system generated (Brandt
1840; Grube 1842; Blanchard 1849; Schimkewitsch 1884).
Although there has been a lot of interest in the opisthosomal
part of this organ system, the prosomal HVS has received less
attention (Claparede 1863; Causard 1896; Petrunkevitch 1911;
Willem 1917; Crome 1952). However, the particularly detailed
results presented by Schneider (1892) and Causard (1896) have
had a significant influence on our perception of the vascular
system in spiders.

In essence, the circulatory system of Araneae, as in other
arthropods, can be divided into two parts; a) the HVS,
consisting of the heart and arteries and, b) the hemolymph
lacunar system, comprising sinuses and lacunae (Wirkner
2009; Wirkner et al. 2013). The hemolymph is distributed
throughout the body by the pumping heart via artery systems.
At the ends of the arteries, the hemolymph leaves the HVS and
enters the hemolymph lacunar system. Within this latter
system, the hemolymph is channeled to the book lungs and
from there the oxygenated hemolymph is channeled - via
sinuses - to the pericardial sinus. Here the hemolymph
reenters the heart lumen via ostia, slit-like openings in the
wall of the heart (see e.g., Wirkner & Huckstorf 2013).

In the context of a comprehensive survey on the circulatory
system in Araneae, we present here the results of a detailed
study on the HVS in the species Araneus diademalus Clerck,
1757 using up-to-date morphological analysis and visualiza¬
tion methods. Morphological descriptions are usually gener¬
alized by species and, after a series of intermediate steps, it is
possible to attain an evolutionary analysis of organ systems
(Richter & Wirkner 2014). However the problem of intraspe¬

cific variability has often been neglected in morphological
studies. In Crustacea, it is known that different arterial
patterns can occur within one species (Keiler et al. 2013) and
even within genetically homogenous populations as in
Procambarus fallax (Hagen, 1870) f. virginaiis (Vogt et al.
2009). To be able to effectively generalize morphological
results with respect to one species, a large data set must be
obtained. We studied 33 specimens of A. diademalus using
micro-computed-tomography (micro-CT) and 3D-reconstruc-
tions to provide a detailed morphological description of the
HVS, with special focus on intraspecific variability of arterial
patterns.

METHODS

Animals. — The specimens of A. diademalus were collected in
the urban area of Rostock (Mecklenburg-Western Pomerania,
Germany) in October 2010, and between August and
November 2011. Around 100 adult and penultimate females
and seven adult males were obtained. The body length of the
females ranged from 8-18 mm; in the males the range was
between 5 and 6 mm. Only males were used for semi-thin
sectioning (see below). Additionally, one specimen of Araneus
quadratus Clerck, 1757 was studied. This sample was collected
in September 2010 on the island of Hiddensee (Mecklenburg-
Western Pomerania, Germany).

The spider taxonomy employed follows ‘The World Spider
Catalog’ (2015).

Ninety-two of the female specimens were treated using the
injection method - as detailed in the following section. To
verify the quality of the injection, injected specimens were
checked using micro-CT. Ultimately, 33 specimens (injected
and uninjected) were scanned and used for further 3D
investigation. Table 1 shows the different procedures used to

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Table 1. — The applied treatments and methods for the considered 33 specimens. Ad: Araneus diadeinatus', Aq: Araneus cjiuidratus; CPD:
critical point dryer; KOH: potassium hydroxide.

Specimen-ID

Method

Sex

Injection

(resin)

Tissue treatment

Contrasting treatment

AdOl, Ad03 Adl2, Adl3

Adl4,

micro-CT

f

PU4ii

KOH (maceration)

LugoFs solution

Adl5 Adl6, Adl7
Ad04, Ad05 Ad07, AdlO

Adll

micro-CT

f

Dubosq-Brasil

alcoholic Iodine-solution

Ad06

micro-CT

m

-

Dubosq-Brasil

alcoholic Iodine-solution

Ad08

micro-CT

f (penultimate)

-

Dubosq-Brasil

alcoholic Iodine-solution

Ad30

semi-thin

m

-

Dubosq-Brasil

azure II and methylene blue

Ad39, Ad40

sections

micro-CT

m

Dubosq-Brasil

alcoholic Iodine-solution

Ad41, Ad43 Ad44, Ad45

Ad46,

micro-CT

f

PU4ii

Bouin

PU4ii mixed with 1% iodine enriched

Ad47 Ad48

Ad69

micro-CT

f

PU4ii

Bouin

Butanon; LugoFs solution

LugoFs solution; alcoholic Iodine-solution

Ad72, Ad86

micro-CT

f

PU4ii

Bouin

LugoFs solution

Ad92, Ad94

micro-CT

f

Mercox II

Bouin

alcoholic Iodine-solution; CPD

Ad95

micro-CT

f

Mercox II

Bouin

alcoholic Iodine-solution

AdlOS

micro-CT

f

Mercox II

Dubosq-Brasil

alcoholic Iodine-solution

AqOl

micro-CT

f

PU4ii

Bouin

alcoholic Iodine-solution

treat each of the 33 specimens. When referring to a specific
specimen, the assigned Specimen-ID (see Table 1) is given,
e.g., in figure legends.

Injection of resin. — To prepare casts of HVS, we followed
the methods as described by Wirkner & Richter (2004).
Spiders were anesthetized with a lethal dose of chloroform and
then injected with resin. We used the casting resin Mercox II
(Ladd Research Industries, USA) or the polyurethane Pu4ii
(vasQtec, Univ, of Zurich, Switzerland). Just before being
injected, the resin was mixed with a hardener (Pu4ii) or a
catalyst (Mercox II) and put into a 5 ml syringe (Braun
Injekt®, Luer Solo) that was locked onto a medical infusion
set, 0.4 X 20 mm in size (servoprax. Wing Flo®, Luer Lock).
The injection setup was finely adjusted using a mechanical
micromanipulator. The legs of the spiders were removed
during the injection procedure so the resin could leave the
body. To allow the resins to polymerize and temper, the
specimens were left for several minutes after the injection.
Mercox Il-injected and uninjected specimens were fixed in
Duboscq-Brasil fixative (10 parts; saturated picric acid
dissolved in 96% ethanol, 4 parts; 37% formaldehyde, 1 part;
glacial acetic acid), while Pu4ii-injected specimens were fixed
in Bouin’s fluid (15 parts; saturated aqueous picric acid, 5
parts; 40% formaldehyde, 1 part; acetic acid). To ensure
enhanced contrast, preparations later used for micro-CT were
stored for at least 12 hours in alcoholic iodine-solution or in
Lugol’s solution (aqueous iodine potassium iodide solution)
(Metscher 2009). The prosomata and the opisthosomata of the
animals were separated at the pedicel to aid the fixing and
contrasting process. This method was exclusively applied to
female specimens.

Specimens with a good injection result were critical-point-
dried (EMITECH K850, UK) to further ameliorate contrast.

Mlicro-computed-tomography (micro-CT). — X-ray scans
were performed using the Phoenix Nanotom® (Phoenix |X-
ray, GE Sensing & Inspection Technologies) high-resolution
micro-CT system in high-resolution mode (target; molybde¬
num, mode; 0-1; performance; ca. 3-6 W; number of images;
1440; detector-timing; 1500-5000 ms; voxel size; ca. 4-8 pm).

The surrounding medium of the specimens during the scan
was distilled water, ethanol, or air depending on the initial
fixing and contrasting procedure.

Semi-thin section series. — Histological semi-thin section
series were carried out for male specimens. For this purpose,
the prosoma was removed from the opisthosoma and fixed in
Bouin’s fixative. It was then dehydrated in ethanol and, after
an intermediate step of epoxypropane, embedded in araldite
epoxy resin under vacuum. Serial semi-thin sections (1 pm)
were made with a Leica Ultracut UCT microtome using a
diamond knife. The sections were stained with a mixture of 1%
azure II and 1% methylene blue in aqueous 1% borax solution
for approximately 5-25 s at 80-90°C.

3D reconstruction. — The image stacks produced using
micro-CT were loaded into the software Imaris 7.0.0 by
Bitplane to create all 3D-reconstructions. A scene was created
in the program module ‘Surpass’. To visualize whole data sets,
the volume rendering function was chosen. The contours of
the studied morphological structures were marked with a
polygon on virtual cross sections.

Images/Illustration. — All figure plates were arranged using
the Corel Graphics Suite X3. Images were embedded into
Corel Draw X3 files and edited in Corel PhotoPaint X3 using
general imaging enhancement tools (contrast, brightness).

The PDF version of the current study is equipped with
interactive 3D-content. Three-dimensional objects resulting
from the aforementioned 3D-reconstruction process were
converted using Adobe 3D-Reviewer (version; 9.0.0.107). To
activate the 3D-content, click on the desired image and use the
mouse buttons to navigate.

RESULTS

The following section predominantly comprises a descrip¬
tion of the HVS in A. diadematus and, as the heart constitutes
the central organ of the whole system, it is a logical starting
point. The terminology employed follows the ontology of
arthropod circulatory systems (OArCS; 'Wirkner unpubl.
data). In accordance with this ontology, only single un-

RUNGE & WIRKNER— HEMOLYMPH VASCULAR SYSTEM IN ARANEVS DIADEM ATUS

155

branched arteries are termed ‘artery system’, such as ‘first
cardiac artery system’. Complete vascular trees, i.e., arteries
and all their branches are termed ‘artery system’, for example
‘first cardiac artery system’. An artery system is named after
the stem artery, to which all its branches can be traced back.

The morphological descriptions that follow are divided into
two parts. The first part comprises the description of stable
morphological patterns, which were observed in all specimens.
The second part, ‘Variability’, considers morphological
patterns that were found to differ between specimens. The
numerical ratios by which certain patterns were observed are
given in this context.

HVS of the opisthosoma. — The heart: The heart of A.
diadematus lies in the dorso-medial region of the opisthosoma.
It runs in an anterior direction from the third pair of the
dorso-ventral muscles to the posterior end of the pedicel. In
the anterior part of the opisthosoma, the heart bends (in a
slight s-shape) ventrally toward the pedicel but not directly
adjacent to the anterior cuticle (the opisthosoma overhangs
the pedicel here) (Figs. lA & C, 2A).

At its posterior end, the heart merges into the posterior
aorta. The posterior aorta runs towards the spinnerets where it
splits at their distal end. The posterior aorta system does not
show any further ramifications (Fig. IB, D, E). Anteriorly, the
heart is extended by the anterior aorta, which supplies the
whole prosoma.

Three pairs of ostia are located on the lateral side of the
heart and three pairs of cardiac arteries originate on the
ventro-lateral side of the heart (Fig. 2). The first pair of ostia is
situated on a dilatation of the myocard which is situated
dorsally to the bending of the heart toward the pedicel. These
ostia are oriented in a dorso-ventral direction. The second and
the third pairs of ostia are located in the dorso-lateral region
of the heart. They lie dorsally to the origins of the first and
second cardiac artery systems, respectively. The second pair of
ostia is located in the heart region between the most anterior
pair of dorsal muscles and the first pair of dorso-ventral
muscles. The third pair of ostia lies between the first and
second pair of dorso-ventral muscles. The third pair of cardiac
artery systems arises laterally to the origin of the posterior
aorta system at the posterior end of the heart.

Cardiac artery systems: Three pairs of cardiac artery
systems originate from the heart. The first and second pairs
of cardiac artery systems originate ventro-laterally to the
second and third pair of ostia, respectively (Fig. 2A). The third
pair of cardiac artery systems originates at the posterior end of
the heart, laterally to the origin of the posterior aorta system.
All three pairs of cardiac artery systems run to the periphery of
the opisthosoma (Fig. IB, interactive 3D content).

The first pair of cardiac artery systems mainly supplies the
antero-lateral part of the opisthosoma (Fig. IB, C). The
second system supplies the central region of the opisthosoma
(Fig. 2B, C) and it includes a prominent stem artery, the
second cardiac artery, which runs to the ventral muscle cord.
Numerous smaller arteries with anterior or posterior orienta¬
tion originate from the second cardiac artery, which also
divides into two equally sized arteries dorsally to the ventral
muscle cord.

The third pair of cardiac artery systems supplies the
postero-lateral part of the opisthosoma (Fig. IB, C).

Variability; There is one artery of the third cardiac artery
system (Fig. ID, E; ca3*) that splits off medially and runs
postero-ventrally to the stercoral pocket. Every specimen
displayed a unilateral occurrence of this artery, however in six
specimens the origin was observed to be at the right third
cardiac artery, and at the left cardiac artery in a single
specimen.

It should be noted that here we describe variable patterns of
major arteries. Variability in smaller arteries is not in the scope
of this study and should be a topic of further investigations
(see also Discussion).

Ligaments of the heart: The heart is suspended by ten pairs
of dorsal ligaments, nine pairs of lateral ligaments and three
pairs of ventral ligaments (Fig. 2). The dorsal ligaments are
attached to the dorsal midline of the heart at regular intervals.
They are attached almost rectangular to the heart from where
they run to the dorsal cuticle of the opisthosoma. Only the
tenth dorsal ligaments run at a more oblique angle: in this case
the dorsal attachment points are in a posterior direction. Each
pair of dorsal ligaments comprises two single ligaments, which
lie close to each other at the attachment sites at the heart and
diverge dorsally.

The lateral ligaments originate at the dorso-lateral side of
the heart and run laterally to the cuticle. The third, seventh
and ninth lateral ligaments attach at the apodemes of the
dorsal muscle (dm), the second dorso-ventral muscle (dvm2)
and third dorso-ventral muscle (dvm3), respectively (Fig. 2A,
B).

The ventral ligaments originate at the ventro-lateral sides of
the heart. The first pair of ligaments runs postero-ventrally
between the dorsal muscles. The second pair of ventral
ligaments originates medially to the origins of the second
cardiac artery system and runs ventrally between the ovaries.
The third pair of ventral ligaments originates medially to the
third cardiac artery system and runs ventrally along the
ventral side of the stercoral pocket (Fig. 2A).

HVS of the prosoma. — Anterior aorta system: The anterior
aorta system supplies the whole prosoma (Fig. 3). The anterior
aorta (Fig. 3B; aa) emanates anteriorly from the heart, which
runs dorsally through the pedicel. On reaching the stomach,
the anterior aorta splits into two aortic arches which flank the
stomach laterally (Fig. 3B; interactive 3D content; aoa).
Shortly after this first split, the paired third dorsal prosoinal
artery system (Fig. 3A, B; dpa3) splits off dorsally and
supplies the dorsal musculature of the posterior prosoma by
numerous ramifications. Anterior to the third dorsal prosoinal
artery system, the peribuccal artery system (Fig. 3B; pba)
originates. This artery system supplies the dorsal dilatator
musculature of the sucking stomach.

Posterior to the supraesophageal ganglion, the aortic arches
bend ventrally and run along the dorsal surface of the
subesophageal ganglion. In this region, the aortic arches give
rise to four pairs of leg arteries (11, 12, 13, 14), an unpaired first
median transganglionic artery (mtgal) and an unpaired
supraneural artery (sa). The pedipalpal arteries (pa) originate
from the first leg arteries in close proximity to where they split
off the aortic arches (Fig. 3A; interactive 3D content).

Cheliceral artery systems: The cheliceral artery systems (Fig.
3A, B; cha) originate in the dorsal bend of the aortic arches.

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JOURNAL OF ARACHNOLOGY

Figure 1. — Hemolymph vascular system in the opisthosoma of A. diadematus. PDF version contains interactive 3D content. To activate click
on Fig. 1C in Adobe Reader. Rotate object with mouse and see further functionalities in content menu. A. Volume rendering of the opisthosoma
showing the position of the heart (red), digestive organs (green) and ovaries (beige). Virtual section through the median sagittal plane; lateral
view. Specimen-ID: Adll. B, C. Surface rendering showing the hemolymph vascular system in the opisthosoma. Right-sided cardiac artery
systems are hidden but are included in the digital 3D content of Fig. 1C. Specimen-ID: AdOl. B. Antero-lateral view. C. Lateral view. D, E.
Surface rendering of the posterior aorta system and third cardiac artery systems showing the varying origin of the asymmetric artery (pink);

RUNGE & WIRKNER HEMOLYMPH VASCULAR SYSTEM IN ARANEUS DIADEM ATUS

157

(D

Figure 2. — Schematic drawings of the heart, cardiac artery systems and ligaments in spatial relation to the opisthosomal dorsal and dorso-
ventral muscles in A. diaclematus. A. Lateral view. B. Dorsal view, cal-3: first-third cardiac artery; dll 10: first-tenth dorsal ligament; dm: dorsal
muscle; dvml-3: first-third dorso-ventral muscle; h: heart; 111-9: first-ninth lateral ligament; osl-3: first-third ostium; vll-3: first-third ventral

ligament.

They supply the anterior region of the prosoma and the
chelicerae. Before the cheliceral arteries enter the chelicerae,
some major artery systems originate. The optic artery systems
(Fig. 3A, B; oa) run dorsally and bend anteriorly towards the

eyes. After reaching the median eyes, the optic arteries arch
laterally towards the lateral eyes (Fig. 3B, C). The optic artery
systems also supply the venom glands (Fig. 3A-C; vg) and the
dorsal musculature of the anterior prosoma.

posterior view. D. Asymmetric artery originating from the left third cardiac artery system (ca3*). Specimen-ID: Ad03. E. Asymmetric artery
originating from the right third cardiac artery system (ca3*). Specimen-ID: Ad45. cal-3: first-third cardiac artery system; g: gut; h: heart; o:
ovary; poa: posterior aorta system; sp: stercoral pocket.

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Figure 3. — Hemolymph vascular system in the prosoma of A. diadematus. PDF version contains interactive 3D content. To activate click on
Fig. 3B in Adobe Reader. Rotate object with mouse and see further functionalities in content menu. A-C. Surface rendering of the hemolymph
vascular system (red) and the venom glands (cyan) in the prosoma (volume rendering). Specimen-ID: Ad69. A. Antero-lateral view; dorsal
branching artery systems of the cheliceral artery system are hidden but are included into the digital 3D content of Fig. 3B. B. Dorsal view. C.
Anterior view. D. Surface rendering of the hemolymph vascular system and the central nervous system; ventro-lateral view. Specimen-ID: AqOl.

cha dpa3

vg

cha

mtga1

cha

dpa3

aoa

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The dorsal musculature of the medial portion of the
prosoma is supplied by two paired arteries: the first (dpal)
and the second dorsal prosomal artery systems (dpa2). They
originate from the proximal part of the cheliceral arteries (Fig.
3A, B).

The unpaired upper lip artery system (Fig. 3C; ula)
originates from one (see ‘Variability’ below) cheliceral artery.
The upper lip artery runs postero-ventrally before it bends
ventrally into the upper lip.

Variability: We observed that the origin of the upper lip
artery system was not consistent. In five specimens, the upper
lip artery originated in the right cheliceral artery and, in
another five specimens, it originated in the left cheliceral
artery.

Leg artery systems and pedipalpal artery systems: The aortic
arches split radially into the four leg artery systems, running
dorsally to the subesophageal ganglion (Fig. 3 A, D; 11, 12, 13,
14); the first leg artery systems run in an antero-lateral
direction, the second leg artery systems in a lateral direction,
the third leg artery systems in a postero-lateral direction and
the fourth leg artery systems in a posterior direction. There are
just a few arteries that branch off in the proximal regions of
the leg arteries. Exceptions to this are the pedipalpal artery
systems at the first leg arteries, and the lateral transganglionic
arteries at each leg artery. The majority of small ramifications
at the leg arteries are located inside the leg. These secondary
arteries supply the leg musculature. All leg arteries run
dorsally along their respective leg nerves (Fig. 3D).

The pedipalpal arteries (Fig. 3A; pa) originate in the
proximal region of the first leg arteries and pass the supra-
esophageal ganglion laterally, nearly at the same level as the
esophagus penetrates the supraesophageal ganglion (Fig. 3D).
The pedipalpal arteries are located dorsally on the pedipalpal
neuropils. The pedipalpal arteries divide and these arteries
supply the large coxal podomere and the distal pedipalpus,
respectively (Fig. 3A; interactive 3D content).

Supraneural artery system: The unpaired supraneural artery
system originates at the medial side of one aortic arch, or in
the transient area to the most proximal region of one fourth-
leg artery. The supraneural artery runs posteriorly along the
median line of the body resting on the dorsal side of the
opisthosomal nerve. The supraneural artery represents the
origin of the posterior group of the median transganglionic
arteries (mtga) (further details on mtga are provided below in
“Supply of the central nervous system”). This artery traverses
the pedicel ventrally to the opisthosomal nerve, however, it
seem that it does not enter the main part of the opisthosoma,
as the injected resin stopped abruptly at the posterior end of
the pedicel.

Variability: The origin of the supraneural artery system can
either be found on the left (in six specimens) or the right (in
three specimens of A. diadematus and in the specimen of A.
quadratus) aortic arch. No correlation was found between the
originating sides of the first median transganglionic artery and

the supraneural artery; all four possible combinations were
present in Araneus (Fig. 4).

Supply of the central nervous system (prosomal ganglion):
The supraesophageal ganglion is supplied by two pairs of
ventral branching arteries originating at the ventro-medial side
of the cheliceral arteries (Fig. 5; vbl, vb2). They run postero-
ventrally into the supraesophageal ganglion and end there.

The subesophageal ganglion is supplied by two groups of
arteries: the median transganglionic arteries (mtga) and the
lateral transganglionic arteries (Itga).

The unpaired median transganglionic arteries penetrate the
subesophageal ganglion along the midline in a regular pattern.
They run to the ventral side of the subesophageal ganglion
where they end openly (Fig. 3D; mtga). The number of
observed, i.e., injected median transganglionic arteries varied
from 5 to 12 (in 4 specimens of A. diadematus and the
specimen of A. quadratus', for a more detailed analysis, see
‘Discussion’). Despite the variety in the number of median
transganglionic arteries, our study detected differing sites of
origin: the arteries branch off the first median transganglionic
artery, off one aortic arch or off the supraneural artery.

The first median transganglionic artery (Fig. 3A; interactive
3D content; mtgal) originates from the medial side of one
aortic arch dorsally to the subesophageal ganglion. This artery
penetrates the prosomal ganglion and runs ventrally along the
esophagus (Fig. 3D). Posterior to the pharynx, the first median
transganglionic artery bends ventrally and enters the lower lip
with numerous ramifications. A varying number of median
transganglionic arteries originate from the first median trans¬
ganglionic artery (for further details see ‘Variability’ below).

Variability: The origin of the first median transganglionic
artery system is either on the left (in four specimens of A.
diadematus and in the specimen of A. quadratus) or the right
aortic arch (in seven specimens).

A more detailed account of the median transganglionic
arteries of two specimens is now presented, because a more
generalized description was not possible:

Specimen Ad86 (with twelve detected mtga): The first
median transganglionic artery originates from the right aortic
arch. The second to seventh anterior median transganglionic
arteries originate from the larger first median transganglionic
artery. The subsequent five posterior median transganglionic
arteries originate from the supraneural artery. The supra¬
neural artery originates from the left aortic arch (Fig. 5A).

Specimen AqOl (with eleven detected mtga): The first
median transganglionic artery originates from the left aortic
arch. The second to third anterior median transganglionic
arteries originate from the larger first median transganglionic
artery. The subsequent two median transganglionic arteries
originate from the right aortic arch. The subsequent six
median transganglionic arteries originate from the supraneural
artery (Fig. 5B). The supraneural artery originates from the
right aortic arch.

The six pairs of lateral transganglionic arteries (Itga) arise at
the leg arteries and pedipalpal arteries. They run ventrally

I-IV: first-fourth walking leg neuropil; aa: anterior aorta system; aoa: aortic arch; cha: cheliceral artery system; dpal -3: first-third dorsal
branching artery system; pba: peribuccal artery system; 11-4: first-fourth leg artery system; Itga: lateral transganglionic arteries; mtgal: first
median transganglionic artery system; oa; optical artery system; pa: pedipalpal artery system; vg: venom glands; pn: pedipalpal neuropil; sa;
supraneural artery system; spg: supraesophageal ganglion; ula: upper lip artery system.

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Figure 4. — Schematic drawings illustrating intraspecific variability in the origin (blue) of artery systems (red) in the region of the central
nervous system (yellow); dorsal view. The four schemes show all possible combinations of origins of the first median transganglionic artery and
the supraneural artery system. The lower row of schemes also shows the different origins of the lateral transganglionic arteries. I-IV: first-fourth
walking leg neuropil; 11-4: first-fourth walking leg artery; Itga: lateral transganglionic arteries; mtgal: first median transganglionic artery; og:
opisthosomal ganglion; pa: pedipalpal artery; sa: supraneural artery; spg: supraesophageal ganglion.

between the lateral processes of the subesophageal ganglion
(Fig. 3D; Itga). At the ventral side of the subesophageal
ganglion, all lateral transganglionic arteries bend medially and
end openly. The lateral transganglionic arteries arising from
the pedipalpal and first to third leg arteries always have their
origin at the anterior side. Thus the first lateral transganglionic
arteries (originating from the pedipalpal arteries) lie anterior
to the pedipalpal neuropil. The subsequent lateral trans¬
ganglionic arteries originating from the first to third walking
leg arteries conform to the same pattern. The last two pairs of
lateral transganglionic arteries are an exception to this pattern,

surrounding the fourth leg neuropil anteriorly and posteriorly.
So the most posterior lateral transganglionic arteries, between
the fourth leg neuropil and opisthosomal ganglion always
originate at the posterior side of the fourth leg artery (Fig. 4).

Variability: The lateral transganglionic arteries running
between the third and fourth leg neuropils can either originate
at the posterior side of the third leg artery (in one specimen) or
at the anterior side of the fourth leg artery (in six specimens
and one specimen of A. quadratus), but always symmetrically
in each specimen (Fig. 4).

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161

Figure 5. — Schematic drawings illustrating intraspecific variability (blue) concerning the median transganglionic arteries in the hemolymph
vascular system (red) in the region of the central nervous system (yellow); lateral view. Exemplified on the specimens A. Ad86 and B. AqOl. aoa:
right aortic arch (partially); cha: cheliceral artery system (partially); dpal-2: first-second dorsal branching artery; mtga: median transganglionic
arteries; sa: supraneural artery system; vbl-2: first-second ventral branching artery.

There are further arteries supplying the region between the
subesophageal ganglion and the sternum, which are not in
direct contact with the central nervous mass. These arteries
stem from all four leg arteries before they enter the legs, and
from the first median transganglionic artery before it enters
the lower lip (not shown).

DISCUSSION

Comparative morphology of the HVS in the prosoma. — Only
a handful of studies describing arteries in Araneus diadematus
currently exist, and there is indeed only one study (Schneider
1892) that describes prosomal artery systems. Generally within
Araneae, the course of the anterior aorta and its two aortic
arches is corresponding (Schneider 1892; Causard 1896;
Gerhardt & Kaestner 1938; Crome 1952; Huckstorf et al.
2013, 2015). However, in the following section, differences in
the branching patterns of prosomal vascular systems in
Araneae are discussed.

Our study indicates that the dorsal prosoma in A.
diadematus is supplied by five paired artery systems: the optic
artery system (oa), three dorsal prosomal artery systems
(dpal-3) and the peribuccal artery system (pba). This indicates
two more dorsal artery systems than previously described by
Schneider (1892). As Schneider (1892) correctly identified, the
anteriormost off-branching artery system (her “nv”, fig. 28)
supplies the venom glands. However, we found differences
with respect to the orientation of this artery system and the
region it supplies. A large artery of both artery systems bends
anteriorly by flanking main parts of the venom gland (see Fig.
3B), and not posteriorly as described by Schneider (1892).
Indeed, only a small part of the venom glands is supplied by a
separate, posteriorly-running artery. Furthermore, this artery
system supplies the whole anterior region on each side of the
prosoma from the median eyes to the lateral eyes and is
therefore termed the optic artery system here. No such
description of this course of the optic artery system in A.
diadematus existed until now. However, similar optic artery
systems have been described for other species {Tegenaria sp.:

Schneider 1892; Agelena lahyrinthica (Clerck, 1757): Causard
1896).

Apart from the optic artery system, two further artery
systems are described by Schneider (1892); a dorsal prosomal
artery system posterior to the optic artery system (which
cannot definitely be equated to the first or the second dorsal
prosomal artery system described in the present study), and an
artery system supplying the dorsal dilatator muscles of the
stomach, which can be matched to the herein described
peribuccal artery system. Our study therefore identifies two
additional dorsal prosomal artery systems in A. diadematus,
and describes them here for the first time; one originates from
the cheliceral artery (Fig. 3B; 3D content; dpal or dpa2), and
the other - the third dorsal prosomal artery system (Fig. 3A,

B, 3D content; dpa3 - originates from the anterior aorta after
its first division. A comparable number of dorsal prosomal
arteries with comparable supplied regions were described in
Cupiennius salei (Keyserling, 1877) (Huckstorf et al. 2013). In

C. salei, two paired dorsal prosomal arteries (their “dorsal
branches of the cheliceral artery”) originate from the cheliceral
artery, as in A. diadematus. The third dorsal prosomal artery
system in C. salei (their “dorsal artery of the anterior aorta”)
exhibits notable differences as it is unpaired, and originates
posteriorly to the first ramification of the anterior aorta.

Furthermore, we can demonstrate that the origin of the
upper lip artery system lies anterior to that of the optic artery
system on the cheliceral artery (6 specimens). These results
throw new light on Schneider’s (1892) study, which previously
described the origin of the upper lip artery system to be
posterior to the optic artery system.

Arterial supply of the prosomal ganglion: Two pairs of
arteries, which have not been identified before in A.
diadematus, supply the supraesophageal ganglion. In C. salei,
however, three pairs of arteries originating from the cheliceral
artery have been shown to supply this region of the brain
(Huckstorf et al. 2013). It remains unclear however, whether
the number of supraesophageal arteries contains phylogenetic
information, or whether it correlates with size of the supra¬
esophageal ganglion.

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With regards to the subesophageal ganglion, the lateral
transganglionic arteries have been described for A. lahyrintli-
ica by Causard (1896), C. salei by Huckstorf et al. (2013) and
for Kukulcania hihernalis (Hentz, 1842), Austrochilus fovstevi
Grismado, Lopardo & Platnick, 2003 and Hickmania troglo¬
dytes (Higgins & Petterd, 1883) by Huckstorf et al. (2015). The
origin of the lateral transganglionic arteries in these species
exhibit the same pattern: namely one lateral transganglionic
artery that originates anteriorly in the proximal region of the
pedipalpal artery, one lateral transganglionic artery that
originates anteriorly in the first to fourth walking leg artery,
respectively and - at the fourth walking leg artery - one lateral
transganglionic artery that originates posteriorly. All lateral
transganglionic arteries run between adjacent lateral neuro¬
pils. This pattern was not consistent in A. diadematiis since the
origin of the fifth lateral transganglionic arteries is subject to
intraspecific variability (Fig. 4). The alternating pattern where
the fifth lateral transganglionic arteries originates posteriorly
from the third walking leg artery has not been described in
Araneae.

The median transganglionic arteries in Araneae generally
originate from five arteries connecting the right and the left
aortic arches or the supraneural artery (Schneider 1892;
Causard 1896; Huckstorf et al. 2013). This pattern has also
been described in scorpions (KluBmann-Fricke et al. 2012,
2014), Amblypygi and Uropygi (KluBmann-Fricke & Wirkner
in press). In Araneus, connecting arteries are missing so the
origin of the median transganglionic arteries can be found at
one aortic arch, the first median transganglionic artery or the
supraneural artery.

Another variation found in median transganglionic arteries
in different specimens (see results) points to a different rather
technical problem. The injection method itself may lead to
different results concerning the median transganglionic
arteries due to their relative small diameters, i.e., in some
specimens some of the median transganglionic arteries may
have not been filled with resin. We therefore generally
refrained from taking numerical variation into account in this
study. All variation accounted here relates to structural
differences. We therefore postulate that 12 median trans¬
ganglionic arteries (the maximal number counted) occur in A.
diadematiis, but this figure is still lower than the results
presented by Schneider (1892), who counted 13 median
transganglionic arteries (including her “labiale post.” (Ip)
supplying the lower lip, see below). Taking other investiga¬
tions into account, it appears that the number of median
transganglionic arteries varies between spider species from, for
example, Tegenaria sp. (Schneider 1892) and A. labyiinthica
(Causard 1896) with 13 to 12 median transganglionic arteries
in C. salei (Huckstorf et al. 2013). The lowest number hitherto
presented is for Argyroneta aquatica (Clerck, 1757) with six
median transganglionic arteries (Crome 1952). Whether or not
quantitative intraspecific variability also occurs in median
transganglionic arteries still needs to be tested in a future
study.

The arterial supply of the lower lip in A. diadematus (Fig.
3A) and in A. quadrat us is peculiar, since the corresponding
artery originates from one aortic arch and not from the
cheliceral artery, as in other spiders (Causard 1896; Crome
1952; Huckstorf et al. 2013). This artery represents the

elongated first median transganglionic artery (Schneider
1892). This can be deduced from its origin and its course
through the subesophageal ganglion on the ventral side of the
esophagus. We can therefore conclude that the lower lip artery
(originating from the cheliceral artery) has been reduced in
Araneus, while the hemolymph supply is taken over by the first
median transganglionic artery.

The first median transganglionic artery and the supraneural
artery can originate from the right or the left aortic arch,
respectively. As illustrated in the schematic drawings (Fig. 4),
all four possible combinations were present in Araneus (see
Fig. 4). No correlation was found between the originating
sides of the first median transganglionic artery and the
supraneural artery.

Comparative morphology of the HVS in the opisthosoma. —

Heart: The posterior aorta of most spiders is unbranched
except for a terminal bifurcation close to the spinnerets (Fig.
ID, E) (Causard 1896; Huckstorf et al. 2013, 2015), and A.
diadematus is no exception to this. Indeed, the only recorded
account of any variation is in Dysdera, which displays a more
anteriorly branched posterior aorta, a shortened heart and a
lower number of cardiac artery systems (Schneider 1892;
Causard 1896).

Ostia: Spider taxa show notable differences when compar¬
ing both the number and arrangement of ostia, and the origin
of cardiac arteries. With regards to the ostia, studies by
Petrunkevitch (1933) have shown how the number of pairs of
ostia can vary from five (in Liphistiidae) to two. In the
Mygalomorphae and Hypochilidae, the heart is equipped with
four pairs of ostia. Within the Mygalomorphae, the number of
ostia is reduced to three pairs in the taxa Barychelidae and
Migidae. There are more independent reductions to three pairs
of ostia in Filistatidae, Synspermiata and Entelegynae (see
Huckstorf et al. 2015). Several taxa independently exhibit even
fewer ostia - down to two pairs in taxa such as Cybaeidae,
Dysderidae, Segestriidae, etc. (see Petrunkevitch 1933 for a
comprehensive list). The number and position of the three
ostia found in A. diadematus fits the ground pattern of
Entelegynae and reinforces available data (Causard 1896;
Willem 1917).

Cardiac artery systems: Available literature is less expansive
on the subject of the cardiac arteries. However, we can find
evidence of a reduction from five pairs in Liphistiidae (Millot
1933) down to two pairs in A. aquatica (Crome 1952) and
Segestria sp. (Schneider 1892; Causard 1896). In Hypochilidae
and Austrochilioidea the number of ostia is reduced to four, of
which the posterior three are associated with a cardiac artery
system (Huckstorf et al. 2015). The heart of A. diadematus
possesses three paired ostia and three paired cardiac arteries
(Fig. 2). The origin of the first and second cardiac arteries is
located ventrally to the subsequent two ostia. The third
cardiac artery originates in the posterior region of the heart
and is not associated with a pair of ostia. This arrangement of
ostia and cardiac arteries corresponds to that exhibited in
Filistatidae, Synspermiata and Entelegynae (Causard 1896;
Willem 1917; Huckstorf et al. 2013, 2015). The results of this
study regarding the position of ostia and origin of cardiac
arteries confirm the results of Causard (1896) and Willem
(1917) for diadematus and contradict former descriptions of
six pairs of cardiac arteries (Blanchard 1849). In comparison

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163

the evolutionary reduction in number of ostia and of cardiac
arteries show different patterns; for a detailed discussion see
Huckstorf et al. (2013).

The most complex aspect of the opisthosomal HVS in
spiders, the branching pattern of the cardiac artery systems,
has so far been illustrated in just a few cases (Schneider 1892;
Causard 1896; Crome 1952; Huckstorf et al. 2013, 2015). The
only results previously available for cardiac arteries in A.
diadematus (Blanchard 1849) have to be dismissed: the number
of cardiac arteries stated makes it unreliable. The present
study therefore constitutes a novel and up-to-date insight into
the branching pattern of the three cardiac artery systems in A.
diadematus.

The main artery and most of the smaller arteries of all three
cardiac artery systems in A. diadematus run in close proximity
to the cuticle to the ventral side of the body. There is only a
small amount of overlap in the body regions (anterior-
posterior axis) supplied by each cardiac artery system. This
picture differs in Agelena labyrinthica where the second
cardiac artery system mainly remains in the interior opistho-
soma (Causard 1896). The periphery of the mid-opisthosoma
is supplied by arteries extending anteriorly from the third
cardiac artery system (Causard 1896). Differences can also be
found in C. salei when comparing the regions supplied by the
cardiac artery systems. In this species, the second cardiac
artery system supplies the bulk of the ventral opisthosoma.
Conversely, the ventral extension of the first and third cardiac
artery system in the anterior and posterior part of the
opisthosoma is reduced, respectively (Huckstorf et al. 2013).
To date, only a few studies are available that illustrate the
cardiac artery systems in detail. Therefore, the identification of
morphological or phylogenetic patterns is hampered and has
to be left open at this point.

Ligaments: Forming a coherent account of the ligament
pattern attached to the heart within the opisthosoma in A.
diadematus is not easy. Existing literature details differing
numbers for each of the three different types of ligaments
(Schimkewitsch 1884; Schneider 1892; Causard 1896; Willem
1917). The most extensive study so far on ligaments by
Causard (1896) describes 11 dorsal ligaments, eight lateral
ligaments and five ventral ligaments. Considering the dorsal
and lateral ligaments this description differs from our findings
of ten dorsal ligaments and nine lateral ligaments. However, it
is obvious that there is an equal number of ligaments overall in
both studies (19 dorsal and lateral ligaments). By comparing
both sequences, it appears likely what we describe as the
eighth lateral ligaments, was interpreted as the tenth dorsal
ligaments by Causard (for further descriptions of the set of
ligaments in other species see Causard 1896; Millot 1933;
Crome 1952; Huckstorf et al. 2013). Because the results are
too varied and are taken from an insufficient number of
species, we are unable to undertake an expedient discussion
here. However, further investigations on this topic are
obviously needed as the ligament patterns seem to reveal
information with regards to the shortening of the heart
(Huckstorf et al. 2013, 2015).

Intraspecific variability. — One of the aims of our study was
to highlight intraspecific variability within the HVS of A.
diadematus by studying 33 specimens. This number far exceeds
common morphological practice, in which just a few

specimens are analyzed. Comparing our study with other
investigations into the HVS in arthropods (Keiler et al. 2013,
2015 a, b; Huckstorf et al. 2015; Gopel & Wirkner 2015), we
can conclude that there are four major patterns of intraspecific
variability:

i. Variability of the course of arteries

ii. Variability of the number of off-branching arteries

iii. Variability of arterial origins

iv. Variability of the occurrence of anastomoses

Apart from the last phenomenon, all of the above-listed
patterns of variability occur in the HVS of A. diadematus.
However, in comparison to other spiders, we found that the
HVS in Araneus differs remarkably by the tendency to reduce
paired origins of arteries arranged along the body midline to
an unpaired, unilateral origin. These unilateral origins are
regions in which we frequently observed intraspecific variabil¬
ity.

Although these forms of intraspecific variability occur, there
are many features and patterns that are consistent across the
specimen range. These stable patterns can reliably be used for
character conceptualization for phylogenetic and evolutionary
analyses (Richter & Wirkner 2014).

ACKNOWLEDGMENTS

We like to thank Kerstin Schwandt for preparing histolog¬
ical sections and Katarina Huckstorf, Torben Gbpel, Bastian-
Jesper KluBmann-Fricke and Stefan Richter for fruitful
discussions and technical support. Helen Johnson improved
the English of this paper. Comments by Martin Ramirez and
an anonymous reviewer helped to improve the manuscript
further. CSW is supported by the Deutsche Forschungsge-
meinschaft DFG (WI 3334/4-1).

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2016. Journal of Arachnology 44:165-170

Limb loss and limb regeneration of crab spiders Misumena vatia

Douglass H. Morse: Department of Ecology and Evolutionary Biology, Box G-W, Brown University, Providence, RI
02912 USA. E-mail; d_morse@brown.edu

Abstract. Limb loss presents an interesting paradox: although it may permit escape from a potentially lethal situation, it
may result in subsequent fitness-lowering consequences. Some studies have found costs of limb loss; others have not. If
costs are high, they may dictate against retaining the ability to drop an appendage. I use a large data set derived from a
long-term study of the crab spider Misumena vatia (Clerck, 1757) to investigate the role of several size- and time-related
factors in evaluating the cost of losing variable numbers of legs, as well as of growing replacements. Specifically, do limb
loss and regeneration affect condition, and do the results differ with sex and age? I focused on adult males because of their
high frequency of forelimb loss, including loss of multiple limbs. Numbers of missing adult male forelimbs were correlated
with date captured and mass (corrected for number of missing forelimbs), suggesting that the spiders lost forelimbs
continually over the summer and that they reached progressively poorer condition than intact individuals, judged by a
disproportionate loss in body mass. The frequency of forelimb loss by penultimate males matched that of adult males, but
females and juveniles lost less than 1/10*’’ as many forelimbs as males. Males possessed many fewer partially regenerated
forelimbs than missing forelimbs, but these frequencies significantly exceeded those for females and younger juveniles.

Some information suggested that additional costs arose from the regeneration of forelimbs.

Keywords: Male-female difference, male-juvenile difference, predation, sexual dimorphism, Thomisidae

Considerable controversy exists over the consequences of
limb loss among many-limbed invertebrates such as spiders
and crustaceans. Although some studies have found little or
no measurable effect of limb loss on survival or fitness (Guffy
1998; Johnson & Jakob 1999); others have demonstrated such
a link, which may have major effects, including both prey
capture and escape from predators (Maginnis 2006a; Steffen-
son et al. 2014). Most limb losses in these forms result from
autotomy, a voluntary, nervously mediated defensive response
induced by external stimuli (Wilkie 2001; Fleming et al. 2007).
A widespread phenomenon, members of at least five
invertebrate phyla, 14 classes and 36 orders readily shed limbs
(Fleming et al. 2007), and many have retained the ability to
regenerate lost parts under certain conditions (Bely & Nyberg
2009; Foelix 201 1). Shedding a limb provides a convenient way
to escape predation or other contingencies, but the act may
have negative future consequences for an individual (Brueseke
et al. 2001; Lutzy & Morse 2008). In response to loss, some
species regenerate limbs, but this ability is not universal
(Randall 1981; Maginnis 2006a), and the utility of this trait is
open to question in some species (Maginnis 2006b; Lutzy &
Morse 2008). In particular, many of these studies provide little
detail that might provide broader insight into the overall
advantages of the ability to shed a limb. Although shedding a
limb, as opposed to shedding a life, is clearly advantageous in
the short term, what success do the survivors enjoy? Do they
obtain any fitness benefits that would separate them from
those preyed upon, and if so, how much? The data set
assembled here allows in-depth evaluation of this question.
For instance, do losses of one, two or three limbs change the
probability of future success?

Here I present data on limb loss and subsequent regener¬
ation from a large sample of crab spiders, Misumena vatia
(Clerck, 1757) (Thomisidae) gathered over 13 seasons, which
provide insight into the possible relationship of several
potentially key variables to limb loss. More specifically, do
size (carapace width), mass, mass corrected for limb loss, date

of capture, date of death, year, or collection site vary with limb
loss? And, what relationship does regeneration have to these
variables, any of which might provide insight into the fitness
consequences of shedding limbs and attempts to regenerate
them? I chose these variables because they allow key insight
into the possible effects of size on limb loss and how limb loss
affects body condition. The high frequency also provides the
opportunity to gather adequate numbers of individuals for
analysis. Do these relationships change or do they remain
predictable and constant? And, is it worth trying to regenerate
a limb?

Male Misumena Latreille, 1804 and related species lose
limbs with a high frequency (Dodson & Schwaab 2001; Lutzy
& Morse 2008). High mobility is an important trait, and the
loss of forelimbs seriously compromises the spiders’ locomotor
capacity (Lutzy & Morse 2008). Although focusing on adult
males, I compare them where possible with penultimate males,
adult and penultimate females, and younger juveniles. One of
the most highly sexually dimorphic of terrestrial animals
(LeGrand & Morse 2000; Morse 2007), Misumena provides an
excellent opportunity to make both intrasexual and intersex-
ual comparisons. Forces acting on individuals of such
strikingly different sizes may constrain the opportunity for
further change within one sex or the other. To the best of my
knowledge, no similar comparisons in extremely sexually
dimorphic species have been made (Maginnis 2006a; Fleming
et al. 2007). This work also expands on the results of an earlier
three-year study (Lutzy & Morse 2008) of adult males. I then
comment on the significance of these results for interpreting
the present pattern of forelimb loss and regeneration in
Misumena.

METHODS

Study area. — I collected Misumena in an old field at the
Darling Marine Center in South Bristol, Lincoln Co., Maine
(43°57'N, 69°33'W), and at several other locations along

165

166

JOURNAL OF ARACHNOLOGY

roadsides within 5 km of the Darling Center. These locations
are not managed except for being mown yearly. The sample
from the Darling field is a probable composite of the other
collection sites, because egg masses of females taken from the
other sites include those used to rear spiderlings for a wide
range of field experiments conducted over the period
encompassed by this study (2000-2012). Numbers of individ¬
uals from three other collection sites permitted individual
treatment as separate samples; those from 14 other locations
were pooled into a last sample. These five samples were
numbered 1-5.

The sites contain large numbers of flowers upon which
Misumena hunt as sit-and-wait predators. I inspected flowers
and collected spiders as I found them. Principal flower species
included common milkweed Asclepias syriaca, oxeye daisy
Leucantlieimon vulgare, common buttercup Ranimcidus acris,
black-eyed susan Rudbeckia liirta, common St. Johnswort
Hypericum perforatum, early goldenrod Solidago juncea and
Canada goldenrod S. canadensis. Although this study focuses
on adult males, I simultaneously captured penultimate males,
penultimate females, adult females and earlier instars. I used
many of these spiders in a wide variety of experiments not
central to the present study. The data set includes individuals
from 2000 and 2001 in Lutzy & Morse (2008), but does not
include results from 1999 because those data lack several
variables treated here.

The species. — Misumena vatia is a highly sexually dimorphic
sit-and-wait predator that frequents hunting sites in flowers
(described in detail in Morse 2007). Males are small and
extremely mobile, weighing from 2.5 to 8.0 mg with two pairs
of long anterior limbs (forelimbs) over twice the length of the
hind limbs. Males do not gain significant amounts of mass as
adults; hence, initial weighing of these individuals provides an
adequate measure of adult mass. The highly mobile adult
males search for unmated females and often guard large
penultimate females that will soon molt into adults, at which
point they will mate (Hainsworth & Morse 2000).

Penultimate males generally resemble adult males, though
with somewhat shorter forelimbs and larger abdomens. Both
penultimate and adult females are much larger and more
sedentary than the adult males. Large penultimate females
weigh 25 mg or more, and gravid adult females weigh as much
as 400 mg or more. Most juveniles (early instars) sampled
weighed 5-10 mg, near the size range of the males.

Procedure. — I captured the spiders during visits to flowers at
the various collection sites. I inspected flowers for the presence
of crab spiders, captured these individuals and placed them in
7-dram vials (5 cm tall, 3 cm diameter). I used the spiders in a
wide variety of experiments, which I have reported elsewhere
(e.g., Morse 2007, 2014). I retained approximately half of the
adult males in 7-dram vials through senescence, feeding them
every 2-3 days with small moths, flies and mosquitoes. I
subsequently used the remainder in field experiments in other
projects, and often lost these individuals in the process. I
gathered all the data presented, other than date of death, at
the time of capture; thus, they are not subject to potential
changes brought about by retention in the laboratory.

I first categorized males in terms of their number of missing
forelimbs, taking care not to include any individuals that had
lost limbs during capture. Then I measured the carapace width

of each individual to obtain a measure of overall size
independent of mass. I also weighed each individual with a
Denver Microbalance (Denver Instrument Company Model
A-200DS: Arvada, Colorado, USA), then calculated an
estimated mass for individuals missing forelimbs, using an
earlier measure of the mass of forelimbs, 8.2% of the body
mass (Lutzy & Morse 2008). Thus, I added 8.2% to the
measured mass of individuals missing one forelimb, 16.4% to
those missing two forelimbs, and 24.6% to those missing three
forelimbs. Because the mass of the first and second pairs of
limbs was similar in an earlier study (Lutzy & Morse 2008), I
added the same mass for each missing forelimb. In all, I
obtained a sample of 609 adult males with normal-sized
forelimbs, including those with one or more missing forelimbs.
Seventy (11.5%) of these individuals lacked one forelimb, 32
(5.3%) lacked two forelimbs, and 8 (1.3%) lacked three
forelimbs. I also obtained information about capture site,
year, date of capture and date of death. I compared forelimb
loss of the adult males with that of penultimate males,
penultimate females, and adult females captured at the same
times as the adult males and used in a wide range of unrelated
experiments.

Additionally, 24 adult males had one or more small
regenerating forelimbs no more than half the length of
normal-sized forelimbs. I collected the same data for these
individuals as for those lacking forelimbs. I also calculated
expected intact masses for the individuals with short forelimbs,
assuming that the mass of these limbs weighed one-fourth of
their “normal” weight. Only two individuals lost one of their
small posterior limbs, and I did not separate these individuals
from the others.

To evaluate the costs of forelimb loss and regeneration I
used the above-noted data on date of capture, date of death,
year, and capture site. These variables all provide insight into
the consequences of forelimb loss. Date of capture provides an
opportunity to determine whether losses continue as the
season progresses. Date of death provides the opportunity to
compare individuals from the field with those spared
predation, competition and other contingencies in the
laboratory. A decline in numbers infers mortality in the field.
Significant differences in year or capture site would complicate
the process of pooling data sets.

Analysis. — I analyzed the relation of limb loss and presence
of short limbs to carapace width and mass with one-way
ANOVAs. I also tested capture site, year, date of capture and
date of death (in the laboratory) in relation to forelimb loss
with ANOVAs. I used analysis of covariance (ANCOVA) to
evaluate further the effects of capture site, year, date of
capture and date of death on the size of males missing varying
numbers of forelimbs. I compared frequencies of forelimb loss
and short forelimbs among the different age and sex
combinations using G tests and the relationship of carapace
width and mass with linear regression. Because I lacked data
for every variable measured or tested on each individual, ns
differ in some of the analyses. Analyses were carried out in R
Version 2.13.0 (R Development Core Team 2011).

RESULTS

Missing forelimbs. — A substantial proportion of the adult
male spiders lost one or more forelimbs (1 10 of 609: 18.1%), a

MORSE— LIMB LOSS AND REGENERATION OF MISUMENA

167

Table 1. — Characteristics of adult male Misiunena vatia missing 0-3 forelimbs (mean ± SD), and results of ANOVAs. ' Predicted mass of
individual assuming it still possessed all forelimbs; ^ Julian days (30 June = 180); ^ Halfway between 2004 and 2005 = 4.5, etc.; Collection sites
numbered 1-5 (see Methods), with non-significance denoting no difference among sites.

Variable

F

df

P

Number of missing forelimbs («)

0 (491)

1 (70)

2 (32)

3 (8)

Carapace width (mm)

1.47±0.149

1.46±0.151

1.43±0.132

1.47±0.144

2.09

1,599

0.15

Mass (mg)

4.58± 1.540

4.20±1.415

3.44±1.010

3.06± 1.066

26.45

1,599

<0.0001

Mass corrected'

4.58±1.540

4.54±1.533

4.01±L182

3.76±1.357

5.1

1,599

0.024

Date of capture^

172.1±10.66

173.2±13.24

173.8±]0.59

181.6±15.05

4.53

1,599

0.034

Date of death^

203.4±18.11

201.5±21.96

201.6 + 16.48

207.6±25.42

0.11

1,300

0.74

Year"*

4.5±4.20

4.4±4.25

5.6±4.65

5.4±4.57

1.4

1,599

0.24

Collection site"*

2.7±1.57

2.9±1.56

2.9±1.68

3.3±1.91

1.56

1,599

0.21

result that did not significantly exceed the

proportion of

Neither date

of death, carapace

width.

year, nor

site of

penultimate males missing a forelimb (6 of 52: 11.5%; G\ =
1.84, P > 0.1). The adult male proportion greatly exceeded
those of both penultimate (7 of 336: 2.1%; Gi = 64.35, P <
0.0001)) and adult females (14 of 862: 1.6%; G| = 97.13, P <
0.0001). The frequency of forelimb loss in the penultimate
males also exceeded that of both penultimate and adult
females (G, = 8.27, P < 0.01; adults: G, = 11.72, P < 0.001).
Numbers of missing forelimbs of unsexed earlier instars
closely resembled those of females (4 of 227: 1.8%),
significantly less than both adult (Gj = 88.89, P < 0.0001)
and penultimate {G\ = 9.60, P < 0.01) males.

Body size (carapace width) did not differ significantly with
the loss of one or more forelimbs, with the four forelimb-loss
classes (0 - 3) exhibiting similar carapace widths at all of the
sites (Table 1). As predicted, individuals missing progressively
more forelimbs weighed less than those with fewer missing
forelimbs (Table 1). However, after correcting masses for
forelimbs lost, those with missing forelimbs were still
significantly lighter than intact ones (Table 1). Although
differences in mass between intact individuals and those with a
single missing limb (corrected for estimated mass of that
missing forelimb) were modest, suggesting a relatively minor
effect, those between one and two missing limbs showed a
several-fold decrease in mass, which was further extended in
those missing three limbs (Table 1). Carapace width and mass
(corrected) were nevertheless highly correlated (linear regres¬
sion: R~ — 0.643, F] 599 = 1083, P < 0.0001), accounting for
roughly two-thirds of the total variance.

Among other variables measured, proportions of individu¬
als missing forelimbs increased as the season progressed (date
captured) (Table 1), although the majority of forelimb loss had
already occurred by the first measure, early in the season.

collection differed significantly in relation to forelimb loss
(Table 1), allowing me to pool these data sets. Use of date of
capture, date of death, year and collection site as covariates
resulted in only one change in the relationship between size
and forelimb loss: site had a moderately significant effect
(i^3,298 = 2.81, P= 0.040).

Short forelimbs. — Individuals with short forelimbs arise
from ones that lost these forelimbs earlier in ontogeny. I
encountered individuals with short forelimbs far less frequent¬
ly than ones missing forelimbs (24 vs. 1 10; 3.8% vs. 18.1%; G]
= 127.08, P < 0.0001, goodness of fit, n = 633).

Carapace width did not differ significantly with the presence
of short forelimbs (Table 2). Neither mass (Table 2) nor mass
corrected for the short limbs (Table 2) differed significantly,
the result of a single anomalously large individual regenerating
two forelimbs. Of the other variables measured (date of death
carapace width, year, site of collection), none was significant
(Table 2).

I obtained one penultimate male with a short forelimb (1 of
52 = 1.9%, not differing significantly in frequency from adult
males (G] = 0.50, P > 0.3). I have also reared additional
penultimate males in the laboratory that lacked a forelimb and
subsequently molted into the adult stage with half-length
forelimbs similar to those seen on the 24 adult males reported
here. I only obtained occasional penultimate and adult females
with short forelimbs in much larger samples (penultimate: 1 of
336 = 0.8%; adult: 2 of 862 = 0.2%). These frequencies fall
significantly below those of the adult males (Gi = 13.85, P <
0.001; G\ = 28.84, P < 0.001, respectively). I found no early
instars with missing forelimbs (0 of 227).

Survival in field and laboratory. — Many confined male
Misumena lived for periods considerably longer than the

Table 2. — Characteristics of adult male Misumena vatia with 0-2 short forelimbs (mean ± SD), and results of ANOVAs. Superscripts 1^ as
in Table 1; ^ After removal of an anomalously large individual of 8.9 mg, mass = 2.93 ± 0.847 mg and mass corrected = 4.08 ± 0.777 mg.

Variable

F

df

P

Number of short forelimbs («)

0 (491)

1 (17)

2(7)

Carapace (mm)

1.47±0.149

1.45±0.140

1.46±0.178

0.23

1,513

0.63

Mass (mg)

4.58±1.540

4.06±1.134

4.21 ±2.222 ’

1.71

1,513

0.19

Mass corrected*

4.58±1.540

4.32± 1.208

4.77±2.516 ’

0.03

1,513

0.87

Date of capture^

172.1±10.66

175.1±12.98

170.9±9.10

0.2

1,513

0.65

Date of death^

203.4±18.11

204.1 ±18.54

215.1 ±28.27

0.63

1,262

0.43

Year’

4.5±4.20

4.9±4.40

2.3±3.90

0.73

1,513

0.39

Collection site'*

2.7±1.57

3.4±1.52

2.4±1.81

0.27

1,513

0.60

168

JOURNAL OF ARACHNOLOGY

maximum dates I have recorded in the field. During one year
that I weekly censused Misumena in all flowers at eight sites in
the vicinity of the Darling Center (total = 0.72 ha), I failed to
record adult males after 28 July, a pattern consistent with less
systematic observations made over the 2000-2012 period, in
which I have rarely found adult males in the field after that
date. However, I have frequently maintained males in the
laboratory until late August and early September, and 74.6%
of the males retained in this study (n = 303) survived past 28
July. I did not find significant differences in longevity in the
laboratory between intact individuals and those that had lost
forelimbs (ANOVA: F,,3oo = 0.11, P = 0.742).

DISCUSSION

Possible sources of forelimb loss. — Male Misumena lose
forelimbs at a highly significantly greater rate than either
penultimate or adult females. Forelimb loss of penultimate
males is more comparable to that of the adult males than of
females or juveniles and provides insight into the frequency of
loss among the adult males. Unfortunately, the sample of
penultimate males is relatively small, since virtually all of the
males since 2002 have molted into the adult stage before our
field season begins in early June, part of a pervasive shift in the
phenology of several species monitored since 1995 in the main
study area (D.H. Morse, unpubl. data).

Adult males engage in aggressive interactions when near
each other, but penultimate males, which have a comparable
rate of forelimb loss, generally do not undertake high-level
interactions (Holdsworth & Morse 2000). A relatively low rate
of forelimb loss of adult males even in staged encounters
between males at the sites of late-stage penultimate females (3
in 90 encounters, 3.3%: Hu & Morse 2004) and in mating
experiments (Morse 2010) makes the putative role of adult
male aggression unlikely as a sole or principal source of
forelimb loss. Opportunities for these interactions are rela¬
tively infrequent in the field, much lower than in the closely
related Misumenoides formosipes (Walckenaer, 1837) (Dodson
& Beck 1993; Dodson et al. 2015). The similar size (carapace
width) of individuals with different numbers of missing
forelimbs is also inconsistent with aggressive interactions
playing the major role in limb loss. Otherwise, one might
expect large dominant males to produce higher limb losses in
combat than smaller ones, because large individuals initiate
attacks more frequently than smaller ones, a pattern seen in
other species as well (Jakob 1994; Hu & Morse 2004). Hence,
the high frequency of missing limbs in adult males is unlikely
to result solely from male aggression.

Mating is a dangerous experience for male spiders, with
some species even dying after mating, including instances in
which they are killed by their mates (Andrade 1996; Foellmer
& Fairbairn 2003; Schneider et al. 2006). Mating takes a less
frequent toll in Misumena, but aggressive females do regularly
capture courting males. However, I have found that in
virtually all instances the females strike directly at the body,
and the male either escapes intact or is captured and killed
(Morse & Hu 2004; Morse 2010). Predators vary in their
tendency to strike at a spider’s body or limbs (Formanowicz
1990). Thus, male-female interactions also appear unlikely to
account for a major part of the observed limb loss.

Male forelimbs are especially long and slender and thus
potentially vulnerable to loss. Although not as long as those of
adults (Morse 2007), penultimate forelimbs are nevertheless
extremely long and slender relative to those of females (Morse

2007) and are thus potentially vulnerable to predators or to
entanglement in the vegetation (Maginnis 2006b). On the basis
of occasional observations made while collecting males in the
field, I predicted that the long, gracile form of male forelimbs
would enhance their vulnerability to entanglement in the
vegetation (petiole-stem interstices, etc.), especially if suddenly
attacked. Analogously, web-building spiders may autotomize
limbs if tangled in webs (Johnson & Jakob 1999).

Although spiders may experience difficulty in extracting
their limbs from their old molt (Maginnis 2006a; Foelix 201 1),
male Misumena molt successfully in the laboratory, as long as
I maintain adequate humidity. Problems of low humidity are
probably even less likely to occur in the field. Thus, this
potential problem appears unlikely to account for a major part
of forelimb loss of the males. The frequency of missing
forelimbs in the penultimate males appears adequate to
suggest that many of the adults missing forelimbs suffered
the loss earlier in ontogeny. Pasquet et al. (2011) and others
have reported losses of less than 1% in penultimate males of
other species.

Short forelimbs. — Adult males with short forelimbs experi¬
enced an even greater disadvantage under natural conditions
than those completely lacking limbs, in that they performed a
number of movements more slowly than even those missing
limbs, and most of them also lost condition (Lutzy & Morse

2008) . Some spiders held partially regrown limbs away from
the body, which therefore did not contribute to locomotion
(Vollrath 1990) or prey capture (Wrinn & Uetz 2008). The
spiders also expend considerable energy growing these new
limbs (Maginnis 2006a; Bely & Nyberg 2009). Thus, it appears
that these males would profit from losing their regenerative
ability, especially in the later instars. Indeed, one could
imagine such selective pressures having driven the loss of
regeneration in groups that have lost this ability. Losses of the
ability to regenerate limbs have occurred many times among
groups that exhibit autotomy and appear related to conditions
routinely experienced in the field (Bely & Nyberg 2009).

Since regeneration only produces external limbs at molts,
none of the small replacement forelimbs result from losses in
the adult stage. Most likely the adult males with partially
regenerating forelimbs lost their forelimbs early in their
penultimate stage. In some species of spiders, regeneration
will occur if limb loss takes place during the first quarter of an
individual’s penultimate instar (Foelix 2011, citing Bonnet
1930). This prediction is consistent with the statistically similar
level of forelimb loss seen in adult and penultimate males.

Male survival. — The difference between apparent adult male
survival in the field and laboratory suggests that they seldom
reach their maximum potential life span in the field. In the
field, males spend most of their adult lives vigorously
searching for females (LeGrand & Morse 2000). Although
adult males do feed (vs. males of many spiders: Foelix 2011)
and largely retain their weight over time, their high level of
activity (Holdsworth & Morse 2000) presumably takes its toll.
Male spiders regularly die before their females (e.g., Dodson &
Schwaab 2001). The similar survival in the laboratory between

MORSE— LIMB LOSS AND REGENERATION OF MISUMENA

intact individuals and those lacking forelimbs was initially
surprising, because individuals lacking forelimbs were signif¬
icantly lighter (after correction for missing forelimbs) and in
poorer condition than intact individuals when captured. This
result is probably a consequence of the easily captured food in
the laboratory, which permitted feeding to satiation. Thus, the
poor condition of individuals lacking one or more forelimbs in
the field may result from decreased success in prey capture
under complex conditions (Brueseke et al. 2001), as well as
compromised locomotor capabilities (Lutzy & Morse 2008).
The laboratory-retained individuals lacking one or more
forelimbs would presumably have performed poorly in a set
of locomotor tasks comparable to those they would experience
in the field. In fact, field-captured individuals could not travel
on lines as rapidly as intact ones (Lutzy & Morse 2008). If they
followed the pattern observed in intact individuals (Lutzy &
Morse 2008; Morse 2014), they probably would score poorly
in other locomotor activities (running, climbing) as well as in
line-crossing. Thus, forelimb loss results in compromised body
condition and performance.

Regeneration and forelimb loss. — Bely & Nyberg (2009)
identify the question of why regeneration persists where it
appears to be irrelevant as a major unaddressed question in
regeneration studies. Although regeneration of forelimbs
appears disadvantageous to penultimate and adult male
Misumena, it may be advantageous in early instars. However,
the earlier Misumena instars exhibit significantly lower
frequencies of forelimb loss than the penultimate and adult
males, which would suggest that selection could not operate as
strongly as on the older males. Regeneration might also be
advantageous for the females; however, they have strikingly
lower frequencies of lost or short forelimbs than the males,
making the explanation problematic.

Thus, the results leave a major unanswered question: why
are male and female forelimb losses so different? Results
presented here and in the literature suggest that this difference
involves a conflict between robustness and speed: the
difference between the ability of females to manipulate large
prey and the ability of males to move quickly when searching
for females.

ACKNOWLEDGMENTS

A long series of Brown University undergraduate students
assisted in the collection and maintenance of these spiders.
They are individually acknowledged in the papers cited here.
The US National Science Foundation supported the work on
Misumena over the 2000-2005 period. I thank the Darling
Center of the University of Maine for use of the study site and
K.J. Eckelbarger, T.E. Miller, L. Healy, and other staff
members for facilitating fieldwork on the premises.

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Brueseke, M., A. Rypstra, S. Walker & M. Persons. 2001. Leg

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Dodson, G.N. & M.W. Beck. 1993. Pre-copulatory guarding of
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Dodson, G.N. &. A.T. Schwaab. 2001. Body size, leg autotomy, and
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Dodson, G.N., A.G. Anderson & L.M. Stellwag. 2015. Movement,
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Steffenson, M.M., D.R. Formanowicz & C.A. Brown. 2014.
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Manuscript received 8 December 2015, revised 25 February 2016.

2016. Journal of Arachnology 44:171-175

Female feeding history impacts gonad development and reproductive timing in the wolf spider

Schizocosa ocreata (Hentz, 1844)

Brian Moskalik*'“ and George W. Uetz': 'University of Cincinnati, Cincinnati, OH 45221; "^Department of Natural
Sciences, University of St. Francis, Joliet, IL 60435; E-mail: BMoskalik@stfrancis.edu

Abstract. In mating systems that include semelparous reproduction and/or scramble competition, synchronous
maturation of the sexes is vital for success. However, food limitation may alter the onset of maturation or the overall
quality of the mature individuals and affect reproductive success. We examined the role of feeding history (well-fed vs.
long-term deprivation) on female reproductive timing and its correlation with temporal patterns of receptivity behavior in
the wolf spider Schizocosa ocreata (Hentz, 1844). We found that feeding history influenced developmental time and
delayed maturation in long-term food-limited females. There was no significant difference in relative condition between
treatments, yet well-fed females showed higher rates of receptivity. Peak receptivity behavior was correlated with the
estimated overall mass of female ovaries/eggs, with females that possess larger ovaries and eggs showing more receptive
behavior. This supports the hypothesis that while a food-limited female may attain maturity, the limiting factor underlying
reproductive success is gonad maturation.

Keywords: Fecundity, diet, receptivity, egg development

Understanding the interactions between nutrition and the
development of reproductive anatomy is important when
addressing mate choice, sexual selection, and sexual conflict
(Arnqvist & Rowe 2005; Uetz & Norton 2007). For
semelparous organisms with well-defined seasonal reproduc¬
tion, many factors may impact the onset and maintenance of
receptivity and courtship within a species (Lehrman 1965;
Barth & Lester 1973). This ontogeny may be linked to the
natural seasonality of the environment and impose intrinsic
control over the ability to acquire food and allocate sufficient
energy to reproductive efforts. Throughout the animal
kingdom, sexually reproducing species are constrained by
the maturation of reproductive structures and the initiation of
sex specific behaviors, as these behavioi's are often associated
with hormones released by the developing gonads (Lehrman
1965; Barth & Lester 1973; Ringo 1996).

Maintenance of physiological condition and gametes is also
important to reproductive success, and conflict exists between
mating age, egg maturity and egg maintenance (Moore et al.
2007). Research has demonstrated links between female
reproductive state, egg maturity, female receptivity and
aggression in both solitary and subsocial spiders and some
insects (Trabalon et al. 1988,1992; Wilgers & Hebets 2012). In
spiders, females have been shown to produce different levels of
hormones in relation to egg maturation, which contribute to
female tolerance of conspecifics (Trabalon et al. 1988,1992).
Even so, no research on spiders has yet examined the
condition-dependent nature of female ovary development
and its relationship to behavior, or the temporal consequences
of diet on reproductive physiology. There is, however,
evidence for compensatory development in spiders, based on
recovery of body size and mass after nutritional stress
(Jespersen & Toft 2003). Additional support suggests that
during times of stress (i.e., food deprivation) there should be
compromise in physiological processes (Gustafsson et al. 1994;
McNamara & Houston 1996). It is well-established that diet
has direct consequences for reproductive investment of
females (Enders 1976; Simpson 1995; Parker & Begon 1996;

Toft & Wise 1999; Kreiter & Wise 2001). Thus, while much
speculation exists about egg development/investment in
spiders (Foelix 1996), little else is known about the direct
effects of diet on gonad development. By examining the
interactions between diet and gonad development, we will
better be able to see how physiological limitations imposed by
long-term diet restrictions impact mate choice decisions of
females.

For many spider taxa, there are age-related differences in
mate choice and female receptivity, frequently accompanied
by differential male courtship investment (Norton & Uetz
2005; Uetz & Norton 2007; Wilgers & Hebets 2012; Rundus et
al. 2015). In the wolf spider Schizocosa ocreata (Hentz, 1844),
females have a predictable receptivity cycle after maturity, in
which they are initially highly resistant to male advances
(Week 1) followed by a level of high receptivity (Week 3) and a
return to resistance (Week 5 and later); high levels of
resistance are also demonstrated after mating (Norton & Uetz
2005; Uetz & Norton 2007). Uetz & Norton (2007) suggested
this pattern might be related to potential availability of males,
as phenology of maturation in this species is typically
asynchronous (i.e., males tend to mature before females,
leading to a male-biased sex ratio early in the breeding
season). However, long-term observations (Uetz & Roberts,
unpubl.) have shown that maturation synchrony does vary
from year-to-year with weather factors. Additional research
has demonstrated that this cycle is also condition dependent
(Moskalik & Uetz 2011), suggesting the possibility that
concomitant factors such as gonad development might be
driving receptivity. The analysis of egg maturity during phases
of this behavioral cycle may therefore shed light on what may
be driving female receptive and aggressive behavior.

Here, we tested two hypotheses: (1) diet will affect
development of and total investment in reproductive struc¬
tures of female spiders for both penultimate (follicle number,
size) and adult S. ocreata (fecundity, egg size, volume, total
clutch volume) and (2) female ovary and egg maturation in
well-fed spiders will coincide with the previously described

171

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JOURNAL OF ARACHNOLOGY

Figure 1. Image of ovulated follicles (oocytes) from a well-fed
ultimate female 21 days mature. F shows an individual follicle, the
arrow indicates pedicle/funiculus, and O indicates a fragment of the
ovary.

receptivity curve (Uetz & Norton 2007) and influence behavior
in a predictable manner. We predicted that if diet impacts
reproductive structure development, then the food-limited
group should demonstrate smaller average gonad size and
reduced numbers of ovulated follicles and/or mature ova
compared to the well-fed group. Also, if female ovary
development impacts receptivity, a relationship between egg
maturation (size) and previously observed temporal patterns
of receptivity will be seen, with female peak receptivity
coinciding with the time that eggs become larger because of
the accumulation of yolk.

METHODS

Spider maintenance. — Hatchling spiders were obtained from
field collected females with egg sacs and maintained in deli-
style containers (9 cm height x 5 cm width). Spiders were held
in the laboratory on a 14:10 lightidark cycle at 23°C and
randomly assigned to well-fed or food-limited dietary proto¬
cols (Uetz et al. 2002; Balfour et al. 2003). Both treatments
received gut-loaded crickets (Aclieta domesticus) with well-fed
individuals receiving 100% of their mass twice per week and
food-limited individuals receiving 50% of their body mass once
per week. Diet implementation and growth were followed
from hatching to adulthood. Individuals were then randomly
assigned to assay groups (penultimate; week 1 post ultimate
molt, hereafter mature week 1; week 3 post ultimate molt,
hereafter mature week 3).

Assessment of female growth. — We assessed all females and
compared mass, cephalothorax width (CW) and developmen-

Figure 2. — Biramous ovary with follicular development from well-
fed penultimate female. F indicates follicle clusters and U the oviduct/
uterine tube.

tal time (total days to maturity and number of instars)
between starvation and well-fed treatment groups with a one¬
way ANOVA. To assess female body condition between diet
treatments, we used an ANCOVA of weight x treatment, and
adult cephalothorax width (CW) as the covariate (Marshall et
al. 2000; Garcia-Berthou 2001; Schulte-Hostedde et al. 2005).

Assessment of female receptivity. — We assessed female
receptivity with behavioral assays at three designated times
(penultimate, mature week 1, mature week 3) matching those
of earlier studies of female behavior (Uetz & Norton 2007).
Females were placed with well-fed, lab-reared males in an
arena for 5 minutes and observed for receptive behavior and
male mounting behavior. The 5-minute duration has been
shown to be an effective time frame to determine female choice
(Moskalik & Uetz 2011). Males were mature for three to five
weeks and in apparently good condition. To control for male
variation, if no mating occurred, the first male was removed
and a second male was placed in the arena and allowed to
court for another 5 minutes. If mounting occurred, the pair
were immediately separated and the female was euthanized
under CO2, preserved in AAF fixative (10 ml concentrated
formalin: 5 ml glacial acetic acid: 75 ml 96% ethyl alcohol: 10
ml distilled water) and dissected to examine egg development.

To examine the effect of diet treatment on spider
development, we compared well-fed and food-limited treat¬
ment females (« = 75 and 46, respectively) using one-way
ANOVA. The effect of treatment on behavior was assessed by
using a Chi square comparison of proportion of receptive

MOSKALIK & UETZ— FEMALE FEEDING HISTORY AND GONAD DEVELOPMENT

173

Table 1. — Female development. One-way ANOVA analyses of
female development. Bold P value indicate significant differences
between diet treatments

Source

df

F ratio

P

Mature weight

1

8.534

0.004

Mature cephalothorax

1

7.871

0.006

Mean instar length

1

0.024

0.877

No. of molts

1

8.762

0.004

Total development duration

1

6.513

0.012

adult females at two points in time after maturity (Week 1 and

Week 3).

Images of eggs and oocytes were taken with an Olympus
digital camera (Figs. 1, 2). Measurements of egg development
included total egg number and mean egg diameter. Mean
diameter was generated by measuring the width of 10 eggs at
random or 10% of the clutch, whichever was greater. Using the
standard equation for the volume of a sphere, we estimated the
volume of the eggs using the mean diameter. A measurement
representing total investment (total clutch volume) was
derived by multiplying the mean volume by the total number
of eggs. Egg number and total estimated clutch volume were
analyzed with a two-way ANOVA, with treatment (starved vs.
well-fed) and female age (penultimate, mature week 1 , mature
week 3) as factors.

RESULTS

Spider development. — Stadia did not differ significantly
between treatments, but number of instars, total duration of
development, cephalothorax width, and adult mass varied
significantly with diet (Table 1). Tukey post hoc examinations
revealed that mean stadia did not differ significantly between
treatments (Well-Fed; 27 ± 4.5 vs. Starved: 27 ± 4.8 days;
Fi,9i =0.024, F = 0.89) but there was a significant effect of diet
on number of instars, with well-fed females requiring fewer
molts to attain maturity (Well-Fed: 6.68 ± 0.14 vs. Starved:
7.32 ± 0.17 molts respectively; Fi gj =8.76, P = 0.004). Total
developmental time varied significantly with well-fed females
maturing fastest (Well-Fed: 179 ± 4.9 vs. Starved: 196 ± 4.5
days: Fj 91 =6.51, /’ = 0.012). Well-fed females were also larger
and heavier than the long-term starvation groups (Table 1).
Results of female body condition indicate that relative
condition (i.e., mass scaled to body size) did not vary by
treatment when cephalothorax was accounted for (F149 =
0.94, F = 0.35).

Behavior. — In week 1, females within each treatment were
equally likely to be receptive to males {n — 20, X~ — 0.02, P =

Figure 3. — Graph showing the decline and divergence of mean egg
number ± standard error between diet treatments. The gray solid line
represents the well-fed group and the dashed line represents food-
limited group. * Indicates a significant difference.

0.89; <20% of females). However during week 3, treatment
significantly impacted the proportions of females receptive to
males {n = 20, X~ — 6.54, P = 0.011) with 60% of well-fed
females showing receptivity, versus 20% of limited females
becoming receptive.

Comparisons of egg development. — Egg number (Table 2)
per female was normally distributed and was analyzed with a
multifactor ANOVA, revealing whole model significance, a
significant difference between weeks, and a significant
interaction between diet and week (Table 3). Post hoc analyses
revealed that egg numbers declined uniformly between
treatments but by week 3, low diet treatments were
significantly lower than well-fed (Table 2, Fig. 3). Egg
diameter was not normally distributed (Shapiro-Wilks W =
0.92, P = 0.012) and was examined for outliers. Grubbs outlier
test (JMP 8) confirmed two outliers that were removed and the
subsequent distribution was then normal (Shapiro-Wilks W =
0.95, P = 0.19). The same multifactor ANOVA was applied
and indicated whole model significance and demonstrated that
week and diet both impacted egg diameter but there was no
interaction (Table 4). As expected, well-fed females had larger
eggs than food limited females (Tables 2, 4) and egg diameter
significantly increased with time (Tables 2, 4). Penultimate
females had the smallest diameter with eggs growing
successively larger each week (0.13 ± 0.006 mm vs. 0.!4 ±
0.003 mm vs. 0.16 ± 0.003 mm: penultimate, weeks 1 and 3
respectively). The total clutch investment showed significant
right skew (Shapiro-Wilks W = 0.80, P < 0.0001) and was Box
Cox Y transformed for best fit (Shapiro-Wilks W = 96, P =
0.19; JMP 8). The multifactor ANOVA showed whole model
significance and that diet and a diet x week interaction

Table 2. — Egg developmental characteristics based on treatment.

Treatment

Age

No. of egg
follicles

Follicle diameter
(mean, mm)

Follicle diameter
(range, mm)

Estimated clutch volume
(mnu^, investment)

Well-fed

Penultimate

127

0.133

0.125-0.148

0.165

Week 1

86

0.152

0.137-0.179

0.168

Week 3

81

0.189

0.157-0.233

0.305

Food-limited

Penultimate

123

0.130

0.122A).138

0.114

Week 1

91

0.121

0.115-0.127

0.105

Week 3

51

0.151

0.145-0.157

0.091

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JOURNAL OF ARACHNOLOGY

Table 3. — Two-way ANOVA analyses of mean number of eggs.
Bold P values indicate significant differences.

Source

df

F ratio

P

Whole model

5

11.79

<0.0001

Treatment

1

2.1011

0.16

Week

2

24.6155

<0.0001

Treatment*Week

2

3.9543

0.03

significantly impacted total investment (Table 5). Tukey post
hoc analyses revealed that initial clutch volume was equal in
both groups and there was a divergence for both groups in
week 1; in week 3, the divergence increased (Fig. 3).

DISCUSSION

Our results support previous findings that females who were
fed more grew faster and produced more eggs. As a
consequence, development and maintenance of follicles and
eggs was compromised in starvation treatments. The impacts,
however, were not significant until later in maturity (week 3).
This suggests that female feeding history can impact egg
maintenance, which could potentially affect receptivity behav¬
ior and subsequent mating or mate choice decisions. As
predicted, follicular development mirrored the behavioral
tendencies first observed by Uetz & Norton (2007). We
observed that a reduced female feeding history significantly
reduced the likelihood of female receptivity over time. From
these results, we can infer a correlation between female age,
diet, ovary/follicle development and receptivity. The initial
results suggest that females invest equal amounts of resources
into follicles regardless of treatment during their penultimate
stage until the onset of adulthood. Then as maturity and
adulthood ensue, females diverge in their ability to 1) maintain
overall egg numbers and 2) invest in egg nutrition that would
subsequently support the post-embryonic spiderling.

This research also highlights the impact that diet has on
spider developmental plasticity, as S. ocreata demonstrated
marked plasticity in developmental time. Well-fed females
were able to mature in fewer instars and still attain a
population mean adult size. On the other hand, food-limited
females were delayed in maturation by the addition of one or
two more instars, thus passing through 9-10 post emergent
instars, which has been previously reported for this species
(Amaya & Klawinski 1996). These late maturing females were
significantly smaller than the rest of the laboratory popula¬
tion. However, an ANCOVA based on mass scaled to body
size revealed no differences. This finding raises questions
regarding the sensitivity of ANCOVA vs. a ratio or residual
body condition index (BCI) when examining development and

Table 4. — Two-way ANOVA analyses of mean egg diameter. Bold
P values indicate significant differences.

Source

df

F ratio

P

Whole model

5

12.61

<0.0001

Treatment

1

11.26

0.002

Week

2

14.97

<0.000!

Treatment* Week

2

1.30

0.29

Table 5. — Two-way ANOVA analyses of mean total clutch
volume. Bold P values indicate significant differences

Source

df

F Ratio

P

Whole model

5

9.16

<0.0001

Treatment

1

14.99

0.0006

Week

2

1.08

0.35

Treatment* Week

2

4.09

0.03

plasticity within a population subjected to varying food
abundance.

Previous research on spider ovary development has
described the developmental stages eggs go through as they
mature and how these correlate with female ecdysteroid levels
in the spiders Coelotes terrestris (Wider, 1834) and Tegenaria
domestica (Clerck, 1757) (Trabalon et al. 1988, 1992). There
were several distinct differences between these results and our
wolf spider population. Seemingly, the development of
follicles in these agelenid spiders occurs after maturation,
whereas in our lycosid species it begins during the penultimate
stage. Additionally, the growth of the agelenid spiders was
very robust, with each individual requiring a fixed number of
instars to reach adulthood. Our lycosid spiders, even in the
absence of diet variation, showed some plasticity in matura¬
tion time and could mature earlier than the expected
population maxima (8*'’ instar post emergence).

Research presented here demonstrates the importance of
female diet and how it affects growth, behavior, reproduction
and fecundity of female S. ocreata wolf spiders. While growth
shows plasticity, certain other developmental aspects do not.
Female follicular ovulation seems to be fixed with respect to
the number of initial oocytes generated. There are clearly more
oocytes ovulated than can be supported by these females, as
even the well-fed group had an intrinsic “rate of decay” within
the first week of maturity. The loss of eggs may be a timing
event that eventually signals the maximum clutch amount for
the female, thus representing an “internal clock”. This
observation generates many interesting avenues of inquiry
that deserve future attention. Female behavior appears to be
regulated by feeding; perhaps food limited females have a

1.0t

Deprived Well-fed

Figure 4. — Estimated total volume of clutch in mm'^ (bars indicate
standard error). Letters indicate significant differences within and
between treatments [R = penultimate instar; 1, 3 indicate weeks post¬
maturity) based on Tukey post hoc test.

MOSKALIK & UETZ— FEMALE FEEDING HISTORY AND GONAD DEVELOPMENT

175

continuous rate of egg loss and ideally benefit from
cannibalism, not mating, in order to support fecundity.
However once egg loss slows or stops, mating should ensue,
regardless of feeding, as maximal fecundity has been reached
for that individual. Future studies should address potential
trade-offs and physiological relationships between fecundity,
survival and maturation timing.

ACKNOWLEDGMENTS

This work was submitted in partial fulfillment of the
requirements for completion of the Ph.D. degree in Biological
Sciences at the University of Cincinnati. This research was
supported by the National Science Foundation (Grants IBN-
0239164 and IOS1026995 to GWU), the American Arachno-
logical Society (BM) and the University of Cincinnati (Wei-
man-Wendell Fellowship to BM). We thank the Cincinnati
Nature Center for permission to collect spiders on their Rowe
Woods property and Dr. Elke Buschbeck for use of her scope
and her support with dissections. We would also like to thank
M. McMullen, J. Allen and A. Ficker for help in rearing
spiders, J. Rutledge, J. Johns, S. Gordon, and A. Orton for
other assistance, and A. Rypstra, E. Maurer, and S. Matter for
comments on an earlier draft of the manuscript.

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Manuscript received 17 September 2015, revised 14 February 2016.

2016. Journal of Arachnology 44:176-181

Influence of predator cues on terminal investment in courtship by male ScMzocosa ocreata (Hentz, 1844)

wolf spiders (Araneae: Lycosidae)

Benjamin Nickley\ Diana Saintignon^ and J. Andrew Roberts^: 'Department of Evolution, Ecology, and Organismal
Biology, The Ohio State University, 318 W. 12'*^ Ave, Columbus OH 43210; "School of Environment and Natural
Resources, The Ohio State University, 2021 Coffey Rd, Columbus OH 43210; ^Department of Evolution, Ecology, and
Organismal Biology, The Ohio State University at Newark, 1179 University Drive, Newark OH 43055. E-mail:
roberts.762@osu.edu

Abstract. Sexual signals play a critical role in mate attraction, but fitness benefits of signal production depend on a
number of external and internal influences. Sexual signaling can be energetically expensive, and has potential to attract
unwanted attention from predators. Male brushlegged wolf spiders, Schizocosa ocreata (Hentz, 1844) (Araneae:
Lycosidae), actively signal to females in the leaf litter habitat during their spring breeding season, but face a tradeoff
between current and future reproduction as the season progresses. The terminal investment hypothesis predicts that with
fewer available females, increasing risk of predation, and stronger influence of senescence as the season progresses, males
should take greater risks to secure mating. We explored this idea by exposing males of increasing ages to female cues alone
or female cues combined with predator cues. We found little or no direct evidence to support the terminal investment
hypothesis in this species, in that males across all ages essentially ceased active courtship in the presence of predator cues,
that is, there was no age related increase in courtship investment in the presence of predator cues. However, we found
distinct evidence of senescence in males based on age-related changes in behavior, which has not previously been directly
explored in this species. While males maintained similar levels of active courtship across all age classes (in the absence of
predator cues), older males increased their relative investment in maintenance behaviors (grooming) and decreased non-
courtship display behaviors such as tapping and leg raises. These findings suggest that studies of male behavior in this
species should be carefully designed to control for age-related variation in behavioral response.

Keywords: Senescence, predation, age effects, chemical cues, context dependence

Sexual signaling is known to be critical for mate attraction
in many species. Individuals produce signals that have been
shaped over evolutionary time to maximize transmission,
reception, and receiver response (Andersson 1994; Johnstone
1996; Rowe 1999; Bradbury & Vehrencamp 2011). Male
sexual signals are often elaborate and conspicuous, potentially
indicating male quality to females through size and/or
symmetry of traits or degree of courtship vigor (Clutton-
Brock & Albon 1979; Parker 1983; Kodrick-Brown & Brown
1984; Hebets & Uetz 1999; Byers et al. 2010). However, sexual
signals do not evolve in a vacuum and the fitness benefits
associated with signaling are contingent upon both external
(ecological/environmental) and internal (physiological) fac¬
tors. Many studies have shown that male traits favored by
females through mate attraction impose energetic costs and/or
increased the risk of predation on males (Andersson 1986;
Magnhagen 1991; Zuk & Kolluru 1998; Roberts et al. 2007;
Cady et al. 2011), but far fewer studies have investigated the
combined effects of physiological condition, such as age-
related performance declines (i.e., senescence), and external
influences (e.g., predator cues) on courtship behavior.

Selection should favor males who respond to internal and
external influences in a way that maximizes potential fitness
benefits associated with signaling (Bradbury & Vehrencamp
2011; Reichard & Anderson 2015). This is especially true for
males that face a declining chance of reproduction due to
senescence and/or increasing predation. The terminal invest¬
ment hypothesis suggests that males who face a tradeoff
between current and future reproduction, especially where
chances of future reproduction are small, should increasingly

invest effort in high risk/high reward behaviors like active,
complex signaling and courtship (Clutton-Brock 1984; Part et
al. 1992). Such an investment might increase mortality and/or
the influence of senescence (Bonduriansky et al. 2008), but
would raise the chances of successful reproduction even when
obstacles to reproduction are ever increasing (Clutton-Brock
1984; Bonduriansky et al. 2008).

The brushlegged wolf spider, Schizocosa ocreata (Hentz,
1844), has been a rewarding model for the study of sexual
signaling and mate choice (Uetz & Roberts 2002; Hebets &
Papa) 2005), and can be used as a model for investigating
issues of behavioral plasticity and context-dependent signaling
(Hebets 2011; Clark et al. 2012). Schizocosa ocreata is a
common ground-dwelling wolf spider abundant in leaf litter of
eastern deciduous forests of North America (Dondale &
Redner 1990). Females are cryptic and relatively sedentary
within the leaf-litter environment, while males traverse the
forest floor and actively seek and court hidden females by
displaying complex, multimodal signals (Aspey 1976; Cady
1983; Uetz & Roberts 2002; Uetz et al. 2013). Females select
males based on size and symmetry of morphological
characters (tufts) as well as aspects of courtship vigor (Uetz
& Roberts 2002; Hebets & Papaj 2005; Byers et al. 2010).
Female receptivity to male courtship increases until females
reach approximately three weeks post adult molt, after which
receptivity begins a steady decline with advancing age (Uetz &
Norton 2007). Males will mate multiple times given the
opportunity in this scramble-competition polygyny system
(Norton & Uetz 2005; Uetz & Norton 2007), but females
typically mate only once after which they become highly

176

NICKLEY ET AL.— PREDATOR INFLUENCE ON COURTSHIP SENESCENCE

177

aggressive toward further mating attempts, attacking and
often cannibalizing the male (Uetz & Norton 2007).

The silk draglines and associated chemical cues deposited by
females as they move through the environment play a critical
role in eliciting male courtship. The cues of a female
conspecific elicit male courtship responses even in the absence
of the female (Stratton & Uetz 1981), and provide valuable
information to males including species identity, female age,
and mating status (Roberts & Uetz 2004a, b, 2005). Males can
also detect and discriminate heterospecific, potentially preda¬
tory spider species, and aggressive, mated female conspecifics
by their silk and chemical cues, and have shown a decreased
courtship response to potentially dangerous congeners,
especially predators (i.e., Tigrosa spp., see Persons et al.
2002; Roberts & Uetz 2004b; Fowler-Finn & Hebets 2011).
The breeding season of Schizocosa ocreata occurs for a
relatively brief, 5-8 week period in the spring (May/June), and
the relative proportion of available, unmated females decreas¬
es while the number of potentially cannibalistic, mated females
increases (Roberts unpubl.). Males, therefore, have a decreas¬
ing chance of mating and increasing chance of being eaten by
aggressive females or heterospecific predators as the season
progresses.

Here we investigate the terminal investment hypothesis for
male S. ocreata by exploring the interaction between
physiological condition (age/senescence) and suppression of
courtship induced by environmental predator cues. If the
terminal investment hypothesis is valid in this species, then
males should exhibit plasticity in their courtship behavior in
response to external and internal conditions. Males decrease
investment in conspicuous courtship behavior in the presence
of predator cues in general (Roberts & Uetz 2004b; Fowler-
Finn & Hebets 2011), but if males suffer reduced reproductive
potential as they age, older age classes will be more likely to
engage in risky courtship behavior, that is, courtship in the
presence of predator cues. We compared the courtship
behavior of males from four different age groups exposed to
female cues alone or to combined female and predator cue
treatments to determine whether males exhibit a plastic
courtship response to either ecological or physiological
factors.

METHODS

Spider collection and maintenance. — Juvenile Schizocosa
ocreata were collected from The Dawes Arboretum, Licking
County, Ohio, USA (39.97849°N, 82.41 614°W) in late April
2010. Female sub-adult and adult Tigrosa helluo (Walckenaer,
1837) were collected from Waterman Farm at The Ohio State
University, Franklin County, Ohio, USA (40.01220°N,
83.03937°W) in October 2009 and May 2010. Only female T.
helluo were used in experiments, as females of this species are
considerably larger and generally more likely to attack prey
than males or juveniles (Walker & Rypstra 2002), and are
known to readily accept Schizocosa as prey (Roberts, personal
observation). Schizocosa were housed individually in plastic
containers (540 ml, round), with ~20 mm moistened peat moss
as a substrate and ad libitum water source, and Tigrosa were
housed similarly in larger containers (950 ml) with more
substrate (~50 mm) to allow burrowing. All individuals were
maintained at room temperature (22-25°C), stable humidity.

and a 13:1 Ih light:dark cycle to simulate spring lighting
conditions. Tigrosa helluo were fed a bitypic diet once a week
that included one to two adult crickets and one to two
mealworms. Schizocosa ocreata were fed twice weekly with
three to four fruit flies {Drosophila melanogaster) or two to
three 1 -week-old cricket nymphs (Acheta domesticus) as
appropriate for their size. All S. ocreata were checked daily
for ecdysis to determine date of maturation for tracking adult
age following the ultimate molt.

Silk collection and substrate preparation. — Wolf spiders
deposit silk and chemical cues as they traverse their
environment, and female cues, even in the absence of females
themselves, are known to induce males to court (Stratton &
Uetz 1981; Roberts & Uetz 2005; Foelix 2011). Further, silk
and chemical cues of Tigrosa spp. are known to elicit anti¬
predator behaviors in this and other wolf spider species
(Roberts & Uetz 2004b; Bell et al. 2006; Fowler-Finn &
Hebets 2011). In order to induce S. ocreata male courtship
and/or anti-predator responses, we collected silk and associ¬
ated cues from conspecific females, and from predatory female
T. helluo. Prior to each trial, we placed an individual female S.
ocreata on a clean sheet of filter paper (Fisherbrand, 90 mm
diameter, round) in an opaque plastic container (90 mm
diameter) and using a small brush, gently induced her to make
50 laps around the outside of the filter paper to standardize the
amount of cue material deposited. Female conspecifics used
for cue deposition were unmated and ranged in age from two
to four weeks post-ultimate molt (period of peak receptivity,
see Uetz & Norton 2007). Filter papers used in the predator
trials were first laden with conspecific female cues as above,
after which we placed individual T. helluo on each filter paper
and induced them to make 50 laps in the same manner as S.
ocreata females, depositing their cues on top of the S. ocreata
cues. Preliminary experiments showed no difference in male
signaling behavior resulting from order of cue deposition in
predator trials. Individual spiders were used only once for silk
deposition and no spider was fed within 24 hours of trials, to
both standardize hunger and reduce fecal contamination of
cues. All trials occurred within 10 minutes of completing the
silk deposition stage.

Experimental design. — To test the hypothesis that differenc¬
es in male age are correlated with differences in courtship
behavior in the presence of predator cues, we conducted a two-
way MANOVA design experiment with male age (one to four
weeks of maturity) and predator cues (present/absent) as
factors, individuals as replicates, and behaviors (Table 1) as
multiple dependent variables. The cohort of males available
for this study all matured within a five day period in order to
synchronize age effects and the timing of trials as closely as
possible. We selected 90 male S. ocreata from the lab
population as they molted to maturity and randomly assigned
each to one of the eight, age-by-predator cue treatment groups
(final sample sizes were approximately 1 1 per treatment
group). We used each male only once within 48 hrs of
reaching the appropriate age post adult molt such that males
“one week old” were six to eight days post maturity when used
in experiments, males two weeks old were 13 to 15 days post
maturity, etc.

We conducted behavioral assay trials in clear plastic arenas
(250 X 100 X 100 mm) where we placed filter paper disks

178

JOURNAL OF ARACHNOLOGY

Table 1. — Ethogram of male Schizocosa ocreata behaviors (adapted from Stratton and Uetz 1986; Delaney et al. 2007).

Behavior

Description

Jerky Tap

Tap

Leg Raise

Chemoexplore

Grooming

Locomotion

Stationary

Active, visual and seismic courtship where the male locomotes with rapid jerky movements while tapping the forelegs, and
occasionally the ventral body surface, on the substrate. Seismic signals in the form of percussion and stridulation are
also produced.

Sometimes called double tap, one or both forelegs actively tapped on the substrate.

Also called “arch” and/or “wave”, one or both forelegs is raised above parallel to the substrate then lowered without
striking the substrate.

Exploratory behavior where the anteriolateral palp surfaces are rubbed on the substrate while slowly locomoting.

The legs or pedipalps are drawn through the chelicerae, or lateral pairs of legs are brushed together rapidly.

Walking, includes wall climbing.

Motionless.

containing cues of female conspecifics, and predators as
appropriate, silk side up on the bottom of the arena
immediately prior to the onset of each trial. We then carefully
deposited males into the arena from above and video-recorded
their response to cues for 300s. Following each trial, we
removed and discarded the cue disks, cleaned the arena using
70% ethanol and a Kimwipe to remove any residual chemical
or silk cues, and allowed the arena to air dry. All recorded
trials were later scored for total duration (s) and frequency
(number/300s) of male courtship (Jerky Tap), display (Tap
and Leg Raise), exploratory (Chemoexplore), antipredator
(Stationary) and other, less common behaviors (Table 1),
using a freely available behavioral analysis program, JWatcher
(vers. 1.0). We transformed the resulting data appropriately
(log total duration and square root frequency), removed
outliers, and ran correlation matrices on all possible combi¬
nations of dependent variables to meet the assumptions of
both MANOVA (Tabachnick & Fidell 2001), and subsequent
ANOVA (Martin & Bateson 2007), then analyzed using JMP
(vers. 9; SAS Institute).

RESULTS

Frequency and total duration of behaviors were initially
analyzed using MANOVA. The overall model in each case
was highly significant (Frequency - Wilks’ Lambda F49 339 49 =

5.785, P <0.0001; Total Duration - Wilks’ Lambda F49,339 49
= 4.779, P <0.0001). There was a significant effect of both
male age (Wilks’ Lambda F2i,i9o.o7 = 3.645, P <0.0001) and
the presence of predator cues (Fy gg = 30.611, P <0.0001) on
the frequency of male behaviors, and the interaction was
significant (Wilks’ Lambda F21, 190.07 = 2.300, P = 0.0017).
Results were similar for the total duration data where there
were significant effects of male age (Wilks’ Lambda F21, 190.07 =
4.379, P <0.0001) and predator cues (Fyge = 37.398, P
<0.0001) on the total duration of male behaviors, also with a
significant interaction (Wilks’ Lambda F21, 190.07 = 3.044, P
<0.0001). The MANOVA analysis should be interpreted with
caution as we found high negative correlation between the
behavior “stationary” and other behavioral states. The
accepted solution would be to remove the redundant variable
(stationary) from analysis (Tabachnick & Fidell 2001), but
since this behavior is also an important antipredator response,
we felt strongly that it should be included. Further, the highly
significant interaction terms make interpretation of the
analysis difficult. For these reasons, we also analyzed each
behavior independently using two-way ANOVA with Bonfer-
roni adjustment (Tables 2, 3) (Tabachnick & Fidell 2001).

The presence of predator cues had a strong negative effect
on frequency and total duration of active courtship behavior
(Jerky Tap) of male S. ocreata, irrespective of male age (Tables
2, 3; Fig. 1). Frequency and total duration of Tap, a common

Table 2. — ANOVA results for mean frequency of behavioral bouts (number/300s trial) for male Schizocosa ocreata. (* Indicates significance
after Bonferroni correction (o(=0.007))

Display Behaviors

Source

df

Jerky Tap

Tap

Leg Raise

F

P

F

P

F

P

Male Age

3,72

0.082

0.970

7.634

<0.001*

10.413

<0.001*

Predator Cues

1,72

22.148

<0.001*

18.770

<0.001*

66.929

<0.001*

Age X Cues

3,72

0.875

0.458

4.201

0.009

6.117

<0.001*

Other Behaviors

Chemoexplore

Grooming

Locomotion

Stationary

Source

df

F

P

F

P

F

p

F

P

Male Age

3,72

1.278

0.289

5.324

0.002*

2.669

0.054

4.264

0.008

Predator Cues

1,72

5.080

0.027

0.000

1.000

17.092

<0.001*

8.599

0.005^

Age X Cues

3,72

0.847

0.473

0.383

0.766

2.734

0.050

2.993

0.036

NICKLEY ET AL.— PREDATOR INFLUENCE ON COURTSHIP SENESCENCE

179

Table 3. — ANOVA results for mean total duration (s) of behaviors for male Schizocosa ocreata. (* Indicates significance after Bonferroni
correction (o(==0.007))

Display Behaviors

Source

df

Jerky Tap

Tap

Leg Raise

F

P

F

P

F

P

Male Age

3,72

0.084

0.968

7.324

<0.001*

12.381

<0.001*

Predator Cues

1,72

22.401

<0.001*

5.045

0.028

83.359

<0.001*

Age X Cues

3,72

1.031

0.384

2.253

0.089

7.425

<0.001*

Other Behaviors

Chemoexplore

Grooming

Locomotion

Stationary

Source

df

F

P

F

P

F

p

F

P

Male Age

3,72

0.810

0.487

6.544

<0.001*

2.246

0.090

1.684

0.178

Predator Cues

1,72

0.894

0.348

0.000

1.000

8.362

<0.005*

10.513

0.002*

Age X Cues

3,72

1.094

0.357

1.007

0.395

1.711

0.172

1.315

0.276

male display trait correlated with active courtship, also
declined significantly with male age, but was only slightly
negatively impacted by the presence of predator cues (Tables
2, 3; Fig. 2). Leg Raises were significantly affected by both
increasing male age and predator cues such that the behavior
was performed almost exclusively in the presence of predator
cues, but declined in both frequency and duration with
increasing male age (Tables 2, 3; Fig. 3). The number and
duration of bouts of Chemoexploratory behavior was largely
unaffected by either predator cues or male age (Tables 2, 3),
and while there was no detectable influence of predator cues
on grooming, males groomed significantly more often and for
longer periods as they aged (Tables 2, 3; Fig. 4). Neither
locomotion nor time spent stationary was influenced by male
age, but males spent more and longer periods stationary and
fewer, shorter periods locomoting in the presence of predator
cues (Tables 2, 3).

DISCUSSION

First, and importantly, the frequency and total duration of
active, mate-seeking exploratory behavior (Chemoexplore)
was consistent across trials, unaffected by advancing male age
or the presence of predator cues (Tables 2, 3), so males were
clearly able to detect the presence of conspecific female cues
even under the influence of predator cues. All subsequent
results, then, are unlikely to be a consequence of “masking” of
conspecific female cues by predator cues. As suggested in
previous studies of this species (Roberts & Uetz 2004b;
Fowler-Finn & Hebets 2011), our results support that male S.
ocreata are able to detect and respond to cues of potential
predators by drastically modifying their behavior, even when
no predator is physically present and predator cues are
presented along with conflicting conspecific female cues.
Further, increasing male age has a strong effect on some,
but not all, male behaviors performed in response to female

50

^ 40

3,

o

'I 30

3

•o

S

2

c

re

as

1

20

10

Predator cues
No predator cues

Male age (weeks)

Figure 1. — Mean total duration (s) (-I-SE) of jerky tap behavior
(active courtship) for male Schizocosa ocreata exposed to the silk and
chemical cues of females in the presence or absence of predator cues.

16 1

14 -

2

12 -

c

o

m

10 -

3

■s

8 -

3

©

4-8

6 -

c

m

4 -

2 -

Predator cues
No predator cues

il i\

12 3 4

Male age (weeks)

Figure 2. — Mean total duration (s) (+SE) of tapping behavior for
male Schizocosa ocreata exposed to the silk and chemical cues of
females in the presence or absence of predator cues.

180

JOURNAL OF ARACHNOLOGY

Male age (weeks)

Figure 3. — Mean total duration (s) (+SE) of leg raise behavior for
male Schizocosa ocreata exposed to the silk and chemical cues of
females in the presence or absence of predator cues.

Male age (weeks)

Figure 4. — Mean total duration (s) (+SE) of grooming behavior for
male Schizocosa ocreata exposed to the silk and chemical cues of
females in the presence or absence of predator cues.

cues. Counter to our terminal investment predictions, we
found no meaningful interaction between increasing male age
(senescence) and presence of predator cues, suggesting that
male S. ocreata may not compensate for reduced reproductive
potential by increasing use of risky, complex courtship
behavior as they age.

Male S. ocreata exhibited equivalent levels of active
courtship across all age categories when exposed to
conspecific female cues alone (Fig. 1), suggesting that male
courtship vigor may not measurably senesce with increasing
age. Alternatively, and perhaps more likely, males may invest
additional resources into active courtship to meet some
threshold of vigor generally acceptable to receptive females
(Delaney et al. 2007; Shamble et al. 2009; Byers et al. 2010),
which is in line with predictions of terminal investment
(Clutton-Brock 1984). In stark contrast to the effects of
increasing age, males were unlikely to perform the prominent
“Jerky Tap” courtship display behavior when cues of
predatory T. lielluo were present (Fig. 1). This does not
support terminal investment under influence of predation,
but does confirm similar findings of two previous studies.
Roberts and Uetz (2004b), as part of an exploration of the
species-specificity of female S. ocreata chemical cues, found
that while males would occasionally court in response to silk
and chemical cues of female spiders within, and even far
outside, the wolf spider family (Lycosidae), they would not
court in response to female T. helhio cues. Fowler-Finn and
Hebets (2011), using number of body bounces as a proxy for
male courtship, found that courtship was greatly reduced in
the presence of Tigrosa spp. cues. Altogether, the results of
these three studies suggest that a significant reduction in
active courtship is an anti-predator response in this species.
Complex, multimodal courtship by male S. ocreata, per¬
formed in this context-dependent manner, may benefit males
in reproduction but must be severely costly in terms of
increased predation risk (Roberts et al. 2007; Roberts & Uetz
2008).

While active courtship may be reduced or extinguished in

the presence of predator cues across all age groups, younger
males (one to two weeks post adult molt) instead adopted
other, less “active” display traits (Figs. 2, 3). Leg Raise
behaviors were performed almost exclusively in the presence of
predator cues (Fig. 3), but were also clear indicators of male
senescence with frequency and duration declining significantly
with increasing age. Frequency and duration of tapping (Tap)
also declined with age, and declined slightly faster in the
presence of predator cues (Fig. 2). The most telling indicator
of senescence in males is the significant increase in grooming
activity with age, whether or not predator cues were present
(Fig. 4). Like many spiders, wolf spiders cease molting at
maturity (Foelix 2011). Physical traits, such as the tufts of
foreleg bristles male S. ocreata use in signaling to females,
would be subject to wear as males age and thus an increase in
maintenance behaviors like grooming is to be expected. Any
shift in time allocation to grooming, though, must be balanced
by shifts in other behaviors. If males maintain consistent
courtship effort as they age, as it appears they do (Fig. 1), then
this allocation shift may explain the decline in less critical
display behaviors like leg raise or tapping (Figs. 2, 3).

ACKNOWLEDGMENTS

We would like to thank Ryan Bell and Samantha Herrmann
for their support and guidance as this project progressed. We
are also grateful to the many undergraduate students who
helped rear and maintain spiders for this work, especially M.
Campbell, B. Paniccia, I. Ackers, and B. Zajd. This work was
supported, in part, by two OSU Dean of Arts and Sciences,
Departmental Research Grants (BN), an OSU Undergraduate
Student Government Academic Enrichment Grant (BN), and
an Ohio State Newark Scholarly Activities Grant (JAR).
Voucher specimens are maintained in the collections of the
corresponding author (JAR) and the Denver Museum of
Nature and Science.

NICKLEY ET AL.— PREDATOR INFLUENCE ON COURTSHIP SENESCENCE

181

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2016. Journal of Arachnology 44:182-193

Spatial patterns and environmental determinants of community composition of web-buiMing spiders in
understory across edges between rubber plantations and forests

Booppa Petcharad', Tadashi Miyashita^, George A. Gale^, Sunthorn Sotthibandhu' and Sara Bumrungsri*: 'Department of
Biology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla, 901 12, Thailand. E-mail: zigzagargiope@
yahoo.com; "Laboratory of Biodiversity Science, Graduate School of Agricultural and Life Science, The University of
Tokyo, Japan; ^Conservation Biology Program, School of Bioresources and Technology, King Mongkut’s University of
Technology Thonburi, Thailand

Abstract. Rubber plantations in Southeast Asia have expanded greatly in recent decades, thereby increasing the amount
of edges bounding natural forests. In this study, we focused on the effects of rubber plantation-forest edges on species
diversity and abundance of web-building spiders. We also aimed to reveal environmental determinants that influence such
patterns. We visually searched and collected spiders within 85 quadrats from October to January (heavy rain period), and
160 quadrats from May to September (light rain period). The quadrats were placed in five sites representing rubber
plantations, rubber plantation-forest edge, and forest interior up to 150 m from the edge. We examined understory
characteristics, microclimate, and potential prey within each quadrat. Certain species were abundant in rubber plantations,
others were abundant at the edge or within the forest, and others showed no pattern. Species richness was not related to the
edge whereas species diversity and total abundance of the spiders was higher in the rubber plantation and decreased at the
rubber plantation-forest edge and into the forest interior. Temperature range and average temperature appear to drive the
distribution patterns of species diversity and total abundance. Characteristics of understory, namely dry twigs and
seedlings also tended to affect such patterns. Temperature probably affected the spiders’ ability to maintain favorable body
temperatures whereas dry twigs and seedlings probably provide reliable web support and suitable refuges.

Keywords: Alpha diversity, Araneae, edge effect, peninsular Thailand, temperature

Recent deforestation in Southeast Asia has been rapid due
to the large-scale expansion of rubber plantations (Li et al.
2007). Replacing natural forests by rubber plantations can
reduce biodiversity through habitat fragmentation as well as
forest degradation (Zhai et al. 2012). An increase of edge
habitats and effects of edges are among the phenomena caused
by the fragmentation; they may affect the distributions and the
interactions of organisms in the ecosystem (Ries et al. 2004).
Despite the situation mentioned above resulting in an increase
of rubber plantations-forest edges, studies on the effect of this
edge type on biodiversity are sparse.

Spiders are reliable bioindicators of environmental change
in tropical ecosystems (Malumbres-Olarte et al. 2013). Web
builders are sit-and-wait spiders that directly use webs for
capturing prey; they stay within the small range of their webs,
so have small home ranges (Miyashita et al. 1998). They are
highly sensitive to environmental changes (Lessard et al.
2010). Accordingly, the web builders appear to be suitable
animal models to assess edge effects, especially in small-scale
ecosystems.

A number of studies have shown edge effects on arthropod
abundance and richness (Bogyo et al. 2015; Lacasella &
Zapparoli 2015). Their patterns along edge gradients varied
depending on taxa or vegetation type (Albrecht et al. 2010;
Rykken et al. 201 1). Some studies reported no detectable edge
effects on the abundance of several groups of arthropods
(Jabin et al. 2004). On spider diversity, most studies have
shown positive edge effects (Galle & Feher 2006; Rodrigues et
al. 2014) while a few studies have shown negative edge effects
(Rodrigues et al. 2014) or no edge effects (Pearce et al. 2005).
Also, a few studies have shown intermediate effects on spider
diversity whereby at a plantation/pasture edge, spiders were

more abundant relative to the forest plantation, but less
abundant relative to the grass pasture (Downie et al. 1996).
Specific studies on distribution patterns of web-building spider
assemblages along edge gradients and environmental variables
influencing these patterns are described by Baldissera et al.
(2004, 2008). The edge between Araucaria forest and Pinus
plantation did not significantly affect richness and abundance
of web-building spiders (Baldissera et al. 2008), while the
richness and abundance were positively affected by the edge
between pasture and Araucaria forest. The latter pattern was
positively influenced by vegetation species richness (Baldissera
et al. 2004).

In this study we focused on the effects of rubber plantation-
forest edge on web-building spiders. Our first objective was to
investigate changes in species diversity, richness, and abun¬
dance of the spiders from rubber plantations across edges
toward forest interiors. The edge effects can occur both at
community and species levels of spiders. At a community level,
we expected that the spider diversity, richness, and total
abundance would be highest in rubber plantations, where the
understory habitat has relatively high complexity and density,
and that it would decrease at the edge toward the forest which
had a relatively sparse understory in our study area. At the
species level, we expected that abundance of different species
would vary in response to edge because of differences in
microhabitat preference. Although it is broadly known that
environmental conditions influence spiders (Entling et al.
2007), the key environmental variables are not well under¬
stood, especially in the inter-habitat transition zones. Conse¬
quently, our second objective was to analyze environmental
variables determining the patterns of spiders along the edge
gradients. Based on previous research mentioned above, we

182

PETCHARAD ET AL — FACTORS DRIVING EDGE EFFECTS IN SPIDERS

183

!

A Sampling location
• Village location
/\/Main road

Evergreen forest
I I Rubber plantation
Forest plantation

mu Annual cropland

research station

S] Others

Figure 1. — A map of the study area, including the Khuan Khao Wang Forest Park (the dark grey component), and the rubber plantation zone
(the white component). The black triangular symbols indicate the sampling locations where belt transects were set to extend from the rubber
plantation into the forest.

predicted that vegetation complexity and density of understo¬
ry would be the primary determinants.

METHODS

Study area. — The study was carried out in Khuan Khao
Wang Forest Park (area = 3.26 km^), Hat Yai District,
Songkhla Province, southern Thailand (6°59"N, 100°18"E, 200
m a. s. 1.) (Fig. 1). This forest park is a secondary forest
remnant composed of semi-evergreen lowland trees, and has
been naturally reforested for about 25 years since the
termination of the logging concession. Logging began in
1970 and was terminated a few years later. Then, in 1995, the
forest was assigned protected area status. The dominant trees
in the forest park were Bairmgtonia spp., Diospyros spp.,
Dipterocarpus alatus Roxb. Ex G. Don, Eugenia spp., Fagraea
fragrans Roxb., Intsia spp., Lithocarpus spp., Morinda spp.,
Pterocarpus spp., and Shorea spp. This protected area has a
hill (200 m a. s. 1.) and a few seasonal streams, and is
surrounded by rubber plantations, forest plantations, a small
area of cropland, a few palm plantations, and houses. Outside

the boundaries of the forest park, the monoculture rubber
plantations are dominant. The forest plantations have
Dipterocapus alatus Roxb. ex G. Don, Intsia palemhanica
Miq., Hopea odorata Roxb., Shorea roxburghii G. Don,
Azadiraclita excels (Jack) Jacobs, and Casuarina equisetifolia
J.R. & G. Forst. Most of the rubber plantations are mature
(7-25 years old) with a canopy height of approximately 14 m;
the rubber trees within each plantation are about the same age
and height. Generally, the rubber trees are planted at 3 m
intervals within each row, and 7 m spacing between the rows.
The understory vegetation consists of grasses, sedges, herbs,
ferns, vines, woody seedlings, and lianas, which are signif¬
icantly denser in the rubber plantations than in the forests.
Generally, the woody seedlings are dominant in the forest
understory, while grasses are almost absent. In contrast,
various species of grasses and herbs are the dominant
vegetation in the rubber plantations. Human disturbances,
including mowing and latex tapping, take place regularly in
the rubber plantations. Traditionally, farmers slash or mow
the understory in their plantations once a year, and they
routinely walk the tracks along the rubber tree rows in order

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JOURNAL OF ARACHNOLOGY

Figure 2. — The arrangement of 15 X 2 m sampling plots, crossing
the forest boundary and extending to the forest interior and the
rubber plantation. The understory, sapling, and tree densities were
assessed by sampling these plots.

to tap the latex. The mean annual precipitation during 2003-
2012 was 1890.3 ± 122.4 mm (mean ± SE). Generally, there
are two seasons in the study area; dry and wet. Based on Mohr
(1944), the dry season is from February to mid-April (mean
monthly precipitation across 2003-2012 = 58.3 ± 15.2 mm).
The wet season can be divided into two periods: the period of
light rain from May to September (mean monthly precipita¬
tion across 2003-2012 = 86.1 ± 7.7 mm), and the period of
heavy rain from October to January (mean monthly precip¬
itation across 2003-2012 = 309.9 ± 34.6 mm) (Rattaphum
meteorological station, unpublished data).

Edge determination. — The vegetation characteristics were
assessed along paths from the rubber plantation into the
forest, in order to determine the position and width of the edge
zone between the rubber plantation and the forest. We selected
rubber plantations of at least 15 years of age that had not had
herbicides or insecticides applied for the last 10 years (based
on interviews of rubber farmers), and had not had understory
mowing during the last 6 months. We identified the line across
which the vegetative contrast was strongest, approaching the
forest from the rubber plantation (Cadenasso et al. 2003). We
established belt transects of 15 m width, extending 20 m into
the rubber plantation and 50 m into the forest from the forest
boundary (the contrast line), spaced 30 m apart. We outlined
15 X 2 m plots in the rubber plantation and in the forest, on
both sides of the forest boundary and then at every 10 m (Fig.
2), and counted saplings and trees (woody plants > 1.5 m tall)
in the plots. We further outlined 1 X 1 m subplots at the center,
as well as in the upper right and lower left corners of each plot,
and assessed the understory in these subplots (see “Assessment
of environmental variables” for details).

Study design. — We applied an interrupted belt transect
sampling method on the rubber plantation across the edge
toward the forest interior. Each transect was at least 50 m
away from the outer bounds of the rubber plantation and the

•tI

o

g

I

(T

§

Figure 3. — The arrangement of 3 X 2 m quadrats for collecting
spiders and assessing environmental variables at each site along belt
transects spaced 20 m apart during the first session of data collection.
A site was located at the edge, and others at 50 m from the edge into
the rubber plantation, and 50, 100, and 150 m from the edge into the
forest. During the second session of data collection, the same five sites
along the transects were used, but this time 1 X 1 m quadrats were
spaced 10 m apart along the transects.

forest (horizontal distance shown in Fig. 3). We conducted the
study in two sessions. The first session, from October 2008 to
January 2009 (in the period of heavy rain) was to examine
whether the edges affect the distribution of spiders. We laid 17
belt transects (3 m wide) spaced 20 m apart, and placed 3X2
m quadrats to collect spiders at five sites along each transect.
The sampling sites on the transects were; at the edge, 50 m
from the edge into the rubber plantation (RP); and 50 m
(F050), 100 m (FlOO), and 150 m (FI 50) from the edge into the
forest (Fig. 3). The second session, from June to September
2012 (during the light rain period) was to confirm an existence
of edge effects and assess environmental determinants of
spider distribution along the edge gradients. Because the heavy
rains obstructed spider collection, we conducted data collect¬
ing of the second session in the light rains instead of the heavy
rains. Spiders and data on environmental variables likely to
affect their distribution patterns (see “Spider sampling and
identification” and “Assessment of environmental variables”
for details) were collected. We laid 32 belt transects (1 m wide)
spaced 10 m apart, and placed 1 X 1 m quadrats at every 50 m
for five sites along each transect, in a similar arrangement as
for the first session but at a different place to avoid

PETCHARAD ET AL.— FACTORS DRIVING EDGE EFFECTS IN SPIDERS

!85

pseudoreplication (Fig. 3). We downsized the sampling
quadrats in the second session, to be able to complete both
spider and environmental variable samplings of each transect
within the same day. In the rubber plantation, we placed
sampling quadrats only between the rows of rubber trees and
away from tracks, in order to avoid disturbance by walking
farmers.

Spider sampling and identification. — Within each quadrat,
we found spiders during the daytime, on days without rain,
from the ground up to 1.5 m height visually surveying all
understories, saplings, trees, stones, and dry leaves/twigs/
branches. We searched for spiders for 25 min. in the 3 X 2 m
quadrats, and for 10 min. in the 1 X 1 m quadrats, to collect as
many as possible. The time taken to collect spiders was
excluded from the sampling time. Along each transect we
randomized the order of quadrats for collecting spiders, on
every collection day, to avoid temporal confounding effects
related to the time of a day. Kleptoparasitic spiders were not
included in this sampling. We identified mature spiders mainly
on the basis of morphological characteristics, to the extent
possible. For certain spiders, we used DNA analyses for
identification, focusing on the mitochondrial cytochrome
oxidase subunit I sampled from specimens preserved in 75%
ethanol. All the procedures for DNA extraction, polymerase
chain reaction, and sequencing, followed Tanikawa (2012),
except for the DNA extraction kit. We used a FavorPrep
Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech
Corp, Ping-Tung, Taiwan). We applied the nomenclature after
Platnick (2014). Specimens were stored in 75% ethanol in vials,
and deposited in the Princess Maha Chakri Sirindhorn
Natural History Museum at Prince of Songkla University,
Hat Yai, Thailand.

Assessment of environmental variables. We collected data
on vegetation structure for edge determination. Although
vegetation structure is well known to influence web-building
spiders, microclimate (Sattler et al. 2010) and potential prey
(Halaj et al. 2000) have been also suggested. Accordingly, to
analyze environmental determinants of distribution patterns
of the spiders along the edge gradients, we measured
vegetation structure, microclimate and potential prey avail¬
ability for evaluating determinants of spider distribution
patterns (in the second session).

Vegetation structure: For edge determination, we counted
the number of stems or trunks of trees and saplings in each
plot. We quantified the density of understory vegetation by
counting leaves of grasses/sedges/ferns, all stems of vines and
lianas, and primary stems of herbs/seedlings in each subplot.

For determinants of spider distribution patterns, we
assessed densities of grasses, sedges, herbs, ferns, vines, lianas,
seedlings, saplings, and trees by counting their buttresses,
trunks, branches, stems, twigs, rachises, leaves, or inflores¬
cences within each quadrat. We then obtained a measure of
vegetation complexity from the combination of all plant
categories above (McCoy & Bell 1991). We measured the
cover percentage of canopy by sighting with a cardboard tube
with a crosshair through the canopy. This was repeated at the
center and in every corner of each quadrat (simple point
intercept method: James & Shugart 1970). We estimated the
cover percentage of litter on the ground, and measured the
litter depth in all four corners and at the center of each

quadrat. We counted the numbers of stones on the ground and
also counted arboreal dead leaves/twigs/branches in the
quadrats up to a height of 1.5 m.

Microclimate: We programmed data loggers, HOBO U12
Temp/RH/Light/External Data Logger - U12 - 012 (Onset
Corporation, Bourne, MA), to record temperature, relative
humidity, and light intensity at 30 min intervals, and placed
them in transparent plastic rain shelters at 1 m height in every
site along each transect, for a continuous period of 48 h. In
each site, we randomly selected two from five points, four
corners and at the center, within each quadrat. We also
randomized the order of such two points for measuring
microclimate in a quadrat and placed a data logger for 24 h at
point one and moved to point two for continuing measure¬
ment another 24 h. From the resulting data, we calculated the
daily ranges (maximum - minimum) and average values for
each of the microclimate variables (Vandergast & Gillespie
2004).

Prey availability: For insect sampling, we applied sticky
traps made from 15 X 15 cm transparent plastic pads coated
with sticky glue. Within each quadrat along the transects, we
placed the traps above the ground at 0, 0.5, 1.0, and 1.5 m
heights, for 72 h. Insects captured by these traps were
identified to order level following Borror et al. (1989). The
trapped insects were counted, and their body lengths were
measured. Dry biomass of each insect was estimated using the
formula fF= 0.0305L^ where W is the dry mass in mg, and
L is the length in mm (Lumsden & Bennett 2005).

Statistical analysis. — We applied the Shannon-Wiener
diversity index to provide a measure of relative diversity of
the spiders (Magurran & McGill 201 1). To standardize species
richness of spiders across sampling plots, we estimated rarefied
species richness by using a function from the library “vegan”
in R (Oksanen 2015). To designate dominant species, we
calculated the proportion of individuals of each species
divided by total number of individuals. We defined dominant
species as those making up > 3% of individuals in the sample
following Spiller & Schoener (1998). To compare the
differences in spider diversity, species richness, and abundance
of species in total and each dominant species between sites, we
used one-way ANOVA, where “site” was used as a fixed
factor. We checked the normality of spider data, using the
Wilk-Shapiro test, and tested homogeneity of variance using
Bartlett’s test. The dependent variables were transformed by
natural logarithms in cases where the data lacked normality or
homogeneity of variance. We used Kruskal-Wallis tests when
normality was not met. For post hoc multiple comparison
tests, we applied Tukey’s test following one-way ANOVA and
Mann-Whitney U-test following Kruskal-Wallis tests. Be¬
cause we used the Mann-Whitney U-test, which is a pairwise
comparison for simultaneous inference, we adjusted the
significance level by using the Dunn-Sidak procedure, in order
to reduce the possibility of Type I errors (Quinn & Keough
2002). For dominant species, since their occurrences are not
independent and we repeatedly applied the test on different
species, we used Bonferroni correction to reduce the possibility
of Type II errors (Cabin & Mitchell 2000). For spider diversity
and total abundance that demonstrate patterns along edge
gradients, we evaluated key environmental variables influenc¬
ing the patterns. For abundance of the dominant species that

186

JOURNAL OF ARACHNOLOGY

also demonstrated patterns along the edge gradients, we could
not evaluate key environmental variables influencing their
patterns because of too many zeros in the response variable
data set.

We applied a Gaussian generalized linear model (GLM)
with an identity link to evaluate the relationship between the
environmental variables and spider diversity. For the spider
abundance of all species combined, we applied zero-inflated
models (Zuur et al. 2009), i.e., the ZIP or the ZINB models
using the “pscl” library in R (Jackman 2012). This approach
was appropriate because our data had an excessive number of
zeros. We used the MuMIn package in R (Barton 2012) to
construct a set of alternative full models. We applied the
Akaike Information Criterion (AIC) for model selection and
presented only the best models (Burnham & Anderson 2002).
To evaluate whether there are effects of spatial autocorrelation
in parameters, we assessed spatial autocorrelation of the final
model with correlograms using a spline function in the ncf
package in R (Bjornstad 2015). There was no significant
spatial autocorrelation. We used each best model to predict
the values of spider diversity and total abundance, as functions
of the environmental variables, to assess the effect sizes of
these variables (Martin et al. 2005). We computed the
percentage of the effect size of each key environmental
variable on spider diversity and the total abundance following
Pilosof et al. (2012), dividing the predicted minimum value by
the predicted maximum value of spider diversity and total
abundance, and multiplying the result by 100. We performed
all the analyses in R v.3.1.0 (R Core Team 2013).

RESULTS

Vegetation change and edge determination. — Understory
density was higher in the rubber plantations than in the
forests, decreasing sharply within 10 m of the forest boundary
(from RPIO to FOO, Fig. 4A). The tree density was lower in the
rubber plantation than in the forest, and had a steep increase
at the transition zone to the forest (from RPOO to FOO) (Fig.
4B). Plant density changed conspicuously from 12 m within
the rubber plantation to 2 m within the forest, measured from
their boundary, and this defined an edge zone of approxi¬
mately 14 ni width (Figs. 2, 4).

Distribution of environmental variables. — The temperature
range and seedling density differed significantly between the
sites (Kruskal-Wallis tests, temperature range: H4 = 54.3, P <
0.001; seedling density: H4 = 24.3, P < 0.001, Fig. 5). The
temperature range was wider at the RP than at the edge and in
the forest. Likewise, seedling density in the RP was
significantly greater than at the edge and in the forest. The
average temperature and dry twig density did not differ
significantly between the sites (Kruskal-Wallis tests, average
temperature: H4 = 9.6, P = 0.05; dry twig density: H4 = 8.7, P —
0.07, Fig. 5).

Distribution pattern of web-building spiders. — During the
first session (heavy rains), a total of 1753 spiders were collected
including 917 (52.3%) juveniles and 836 (47.7%) adults. Adults
belonged to 67 species of 14 families. Nine species were
considered dominant, and these nine species accounted for
74% of total abundance. During the second session (light
rains), a total of 908 spiders were collected, including 611
(67.3%) juveniles and 297 (32.7%) adults. Adults belonged to

Figure 4. — Plots of the distribution of understory (A) and the tree
density (B) along the transect extending from rubber plantation (RP)
into forest (F). RPOO, RPIO, and RP20 are at distances 0, 10, and 20
m from the forest boundary to the rubber plantation, while FOO to
F50 are at distances 0 to 50 m into the forest from the boundary.
Points are means. Whiskers show SE.

50 species of 12 families. Nine species were considered
dominant, accounting for 71% of total abundance.

During the first session (heavy rains), significant differences
between rubber plantation and forest sites were found in
Crassignatha sp2, Araneidae gen. sp3, and Mysmenidae gen.
sp3 (Table 1). The abundance of Crassignatha sp2 was
significantly higher in the FI 50 than in RP (Fig. 6A). The
abundance of Araneidae gen. sp3 was significantly higher at
the edge than at RP and at FI 50 (Fig. 6B). The abundance of
Mysmenidae gen. sp3 was significantly higher at RP than at
the edge, F050, and FI 50 (Fig. 6C). The abundance of other
dominant species, namely, Araneidae cf. Nemoscolus sp.,
Leucauge argentina (Hasselt, 1882), Linyphiidae gen. spl,
Mysmenidae gen. spl, Octonoba spl, Zonia dibaiyin Miller,
Griswold & Yin, 2009, did not differ significantly between
rubber plantation and forest sites.

During the second session (light rains), we found significant
differences between the sites in Araneidae cf. Nemoscolus sp.,
Mysmenidae gen. spl, and Theridiidae gen. spl (Table 1). The
abundance of Araneidae cf. Nemoscolus sp. was significantly
higher in the FI 50 than in RP (Fig. 6D). The abundance of
Mysmenidae gen. spl was significantly higher at the edge than
at FlOO (Fig. 6E). The abundance of Theridiidae gen. spl was
significantly higher at the rubber plantation than in the forest
(Fig. 6F). The abundance of other dominant species, namely,
Araneidae gen. sp3, Belisana kbaosok Huber, 2005, Lycosidae
gen. sp., Linyphiidae gen. spl, Symphytognathidae gen. spl.

PETCHARAD ET AL.— FACTORS DRIVING EDGE EFFECTS IN SPIDERS

187

Plot location relative to plantation-forest edge

Plot location relative t© plantation-forest edge

Plot location relative to plantation-forest edge Plot location relative to plantation-forest edge

Figure 5. — The comparison of key environmental factors, temperature range (A), average temperature (B), seedling density (C), dry twig density
(D), across the edge from rubber plantation into the forest. Bars are means. Whiskers are SE. Different letters indicate significant differences.

Table 1. — A list of dominant spider species, total abundance, and
results of Kruskal-Wallis test (df = 4) comparing the abundance of each
species between sites from rubber plantation and forest. Bold letters
indicate significant differences between sites following a post hoc test.

Total

abundance

Kruskal-Wallis test

Species

H

P

Heavy rain period

Araneidae cf. Nenioscolus sp.

67

5.4

0.25

Araneidae gen. sp3

33

19.9

< 0.001

Crassignathci sp2

93

21.9

< 0.001

Leucauge cirgentimi (Hasselt, 1882)

38

11.9

< 0.05

Linyphiidae gen. spl

74

2.4

0.66

Mysmenidae gen. spl

229

3.6

0.46

Mysmenidae gen. sp3

21

31.2

< 0.001

Octonoba spl

23

6.2

0.19

Zonia dibaiyin Miller, Griswold
& Yin, 2009

39

14.7

< 0.01

Light rain period

Araneidae cf. Nemoscolus sp.

32

16.6

< 0.001

Araneidae gen. sp3

9

0.8

0.94

Belisana khaosok Huber, 2005

10

6.1

0.19

Lycosidae gen. sp.

12

8.3

0.08

Linyphiidae gen. spl

16

4.7

0.31

Mysmenidae gen. spl

54

15.3

< 0.005

Theridiidae gen. spl

35

27.3

< 0.001

Symphytognathidae gen. spl

10

9.5

0.05

Symphytognathidae gen. sp2

34

9.5

0.05

Symphytognathidae gen. sp2, did not differ significantly
between the sites (Table 1).

During the first session (heavy rains), the diversity of spiders
differed significantly between the sites (Kruskal-Wallis test,
H4=14.9 , P < 0.01). It was significantly higher in the RP than
at F050 (Fig. 7A). Species richness and total abundance of
spiders did not differ significantly between the sites (one-way
ANOVA, richness: F4, 95= 1.0, P = 0.41; abundance: F4, 95 =
1.1, P — 0.36, Fig. 7B, C). During the second session (light
rains), the diversity and total abundance of spiders differed
significantly between the sites (diversity: one-way ANOVA,
F4, 95 = 3.9, P < 0.01; abundance: Kruskal-Wallis test, H4 =
16.4, P < 0.01). The diversity of spiders was significantly
higher at RP than at FI 00 (Fig. 7D). The abundance was
significantly higher both at RP and at EDGE than at FI 00
(Fig. 7F). Species richness of spiders did not differ significantly
between the sites (Kruskal-Wallis test, H4 = 4.0, P = 0.40, Fig.
7E).

Variables influencing the distribution pattern of web-building
spiders. — The average temperature and the temperature range
were significant variables affecting total abundance and
diversity of spiders (Table 2). Not only temperatures but also
seedlings and dry twigs affected the total abundance and the
diversity. The models suggest that a wider temperature range
contributed to the increase in total abundance and diversity of
spiders, while increasing the average temperature reduced the

188

JOURNAL OF ARACHNOLOGY

Heavy rain

Light rain

Plot location relative to plantation-forest edge

Plot location relative to plantation-forest edge

Plot location relative to plantation-forest edge

Plot location relative to plantation-forest edge

Figure 6. — Median values with range of abundance for the dominant species of spiders from rubber plantation into forest. Dashes are
medians. Whiskers show ranges. Different letters indicate significant differences.

total abundance and the diversity. The numbers of seedlings
and dry twigs positively influenced the total abundance and
the diversity (Table 2). The zero-inflated negative-binomial
model that we applied for total abundance of spiders did not
show significant results (dry twig: j3 = — 0.274, Z = -0.963, P =
0.336). The fitted GLM explained 19.3% of deviance in the
diversity (Table 2). Species diversity was increased by 69%
(from 1.15 to 1.67), 46% (from 1.24 to 2.71), and 47% (from

1.22 to 2.60) by temperature range, seedling abundance, and
number of dry twigs, respectively. The diversity declined by
62% (from 1.68 to 1.05) with average temperature (Fig. 8).
Temperature range, seedling abundance, and dry twigs
increased the total abundance 22% (from 0.85 to 3.91), 9%
(from 1.28 to 14.10), and 8% (from 1.15 to 14.15), respectively.
The total abundance declined by 20% (from 3.47 to 0.68) with
average temperature (Fig. 9).

FETCH ARAD ET AL.— FACTORS DRIVING EDGE EFFECTS IN SPIDERS 189

Heavy rain Light rain

Plot location relative to plantation-forest edge Plot location relative to plantation-forest edge

Plot location relative to plantation-forest edge Piot location relative to plantation-forest edge

Piot iocation reiative to plantation-forest edge Plot location reiative to plantation-forest edge

Figure 7. — The species diversity, the species richness, and the total abundance of web-building spiders from rubber plantation into forest.
Mean values with SE are shown for the species diversity (D) during light rain period, and for the species richness (B) and the total abundance (C)
during heavy rain period; dashes are means; whiskers are SE, Median values with range are shown for the species diversity (A) during the heavy
rain period, and for the species richness (E) and the total abundance (F) during the light rain period; dashes are medians; whiskers indicate the
range. Different letters indicate significant differences.

DISCUSSION

Effect of edge on the distribution pattern of web-building

spiders. — Certain species of web-building spiders indicated the
existence of the edge effect. As in previous studies (Baldissera
et al. 2004; Vandergast & Gillespie 2004), the pattern of edge
responses in abundance of spiders varied among different
species. Edge influenced spider distribution in periods of both
heavy and light rain, despite the fact that different taxa
occurred in each period.

Crassignatha sp2, Mysmenidae gen. sp3, and Theridiidae
gen. spl, which were found only in a single period and
responded to the edge, are probably sensitive to environmental
change. The effects of edge on Araneidae gen. sp3, Araneidae
cf. Nemoscolus sp., and Mysmenidae gen. spl, which were
found in both sampling periods varied. Araneidae gen. sp3
was influenced by edge in the period of heavy rain but not in
the period of light rain. Araneidae cf. Nemoscolus sp. and
Mysmenidae gen. spl were influenced by edge in the light rain

190

JOURNAL OF ARACHNOLOGY

Table 2. — Summary of GLM testing the effect of four environ¬
mental variables on the diversity and total abundance of spiders. Bold
values are significant at F < 0.05.

Type of
model

Response variable

Explanatory variables

Estimate

P

Count model

Diversity

Average temperature

-0.120

<0.001

Temperature range

0.080

<0.001

Seedlings

<0.001

0.020

Dry twigs

<0.001

0.040

Count model

Total abundance

Average temperature

-0.393

<0.001

Temperature range

0.253

<0.001

Seedlings

0.001

0.007

Dry twigs

0.002

0.018

period while neither species was influenced by edge in the
heavy rain period. We postulated that these three species are
intermediate in sensitivity to environmental change along the
edge gradients. Linyphiidae gen. spl was found in both the
periods of heavy and light rain and showed no edge effect,
suggesting that it is insensitive to environmental change along
the edge gradients.

This is the first report of an edge effect on the diversity of
web builders. The distribution pattern of spider diversity
showed an intermediate stage between the positive and
negative effects of the edge between rubber plantation and
forest. This was consistent across seasons, even though the
magnitude of the effect varied seasonally. The positive effect
of the edge between rubber plantation and forest was observed
in spider abundance in the light rain period. This pattern is in
accord with the patterns in abundance of web-building spider
assemblage described by Baldissera et al. (2004) and Vander-
gast & Gillespie (2004). No edge effects on spider abundance
during heavy rains and species richness in both seasonal
periods as revealed in the present study are similar to the

results of Baldissera et al. (2008) but different from Baldissera
et al. (2004).

Variables influencing the distribution pattern of web-building
spiders. — The influence of the temperature range and the
average temperature on the diversity and abundance of web¬
building spiders indicate that both these environmental
variables mainly drive spider distribution patterns. Although
Chaladze et al. (2014) and Kwon et al. (2014) showed influence
of temperature on spiders, those studies did not examine small
variations in temperature parameters between sites as in the
present study. The response of web-building spiders to small
variations in average temperature (26.2-27.3 °C) along the
edge gradients observed here suggests the significant impor¬
tance of temperature for determining spider distribution. In
the present study, even though there is a small temperature
range (5. 7-8. 8 °C) along the edge gradients, this is the most
important variable positively driving distribution patterns of
spider diversity and total abundance. In contrast to our
results, Coyle (1981) reported that a wider range of
temperatures decreased the diversity and the abundance of
web-builders in clear-felled temperate areas. In the present
study, the maximum temperature range was 22.5-37.0 °C, and
the daily range was typically 8.8 °C in the rubber plantations,
which may not be too extreme for spiders. It is possible that
the canopy of the rubber plantations alleviates the effect of
maximum temperature compared with the clear-felled areas.
Based on our results, we postulate that web-building spiders
prefer a wider range of temperature within the range of
suitable temperatures.

A wider temperature range would support greater diversity
and abundance. Different species may require different
temperature optima for various essential activities, i.e., web
building, prey capture, egg hatching, molting, development
(Prestwich 1977; Li & Jackson 1996). Also, different species of
spiders need appropriate ranges of ambient temperature to
reach and maintain their favorable body temperatures
(Krakauer 1972). The longer the activity, the more prey can

s

c

(9

E

I

E

■a

Q.

W

Dry twig density

Figure 8. — Plots of the impacts of average temperature (A), temperature range (B), seedlings (C), and dry twigs (D), on the total abundance of
web-building spiders of the top models. The solid lines are mean predicted values and the dashed lines indicate 95% confidence intervals.

PETCHARAD ET AL.— FACTORS DRIVING EDGE EFFECTS IN SPIDERS

191

Average temperature

Temperature range

Figure 9. — Plots of the impacts of average temperature (A), temperature range (B), seedlings (C), and dry twigs (D), on the species diversity of
web-building spiders of the top models. The solid lines are mean predicted values, and the dashed lines indicate 95% confidence intervals.

be captured and the more food is consumed; this contributes
to early production of offspring and increased fecundity
(Logan et al. 2006). Additionally, temperature affects silk
properties (Yang et al. 2005). Certain spider species need
specific temperatures to produce the best quality web, which is
associated with the efficiency of prey capture (Barghusen et al.
1997). Previous research suggests that temperature is a
significant factor driving distribution patterns of web-building
spiders at a local scale (Finch et al. 2008), and our study
suggests temperature may also be important on a very fine,
microsite scale.

Only density of seedlings and dry twigs positively influenced
the distribution patterns of spiders. The positive association
with particular characteristics of the understory and spider
diversity and total spider abundance is similar to Grill et al.
(2005) and Blamires et al. (2007). Notably, the influence of
twig density on the patterns also agrees with Gillespie (1987).
The present study specifically indicates influence of seedling
density on these patterns for the first time. Dry twigs and
seedlings here could provide reliable architectural supports for
various sizes and types of webs of most spiders (Miyashita et
al. 2004) and proper refuges for spiders against their predators
such as lizards (Hoffmaster 1982).

Edge effect penetration. — The edge effect between the
rubber plantation and the forest was found up to 50 m into
the forest during the heavy rain period, and up to 100 m
during the light rain period. Thus, the penetration of the edge
effect in the light rain period was deeper into the forest than in
the heavy rain period. A key variable driving the patterns
appeared to be the temperature; this could explain the edge
effect being stronger during the dry season (Pohlman et al.
2009). Obvious changes in diversity and total abundance of
web-building spiders across very short edge gradients in our

study as compared with those found in beetle communities
suggested that web-building spider assemblages could also be
an efficient bioindicator of edge effects, especially on a small
spatial scale (Ewers & Didham 2008).

Conservation implications. — Certain dominant species were
abundant in the forest while others were abundant in the edge
or rubber plantations. These patterns suggest that every
position along the edge gradients is crucial for harboring
particular species of web-building spiders. High diversity and
total abundance of web-building spiders in rubber plantations
compared to forest in the present study are the result of edge
effects, and further study on such patterns in rubber
plantation farther from forest is recommended. Since
increased seedling and dry twig density in rubber plantations
supports higher spider diversity, less disturbance to the
understory could reduce losses of spider diversity (Beukuma
et al. 2007). Generally, for rubber plantations, most farmers
frequently clear the understory with herbicides or mechanical
cutting. However, a few farmers, who practice agroforest
rubber plantation, leave the understory undisturbed and plant
more forest tree seedlings. It would be particularly informative
to assess spider diversity between these different practices of
rubber plantations.

ACKNOWLEDGMENTS

This research was mainly funded by the Development and
Promotion of Science and Technology Talented Project
(DPST), under the Institute for the Promotion of Teaching
Science and Technology (IPST), Thailand. It was also
financially supported by the Graduate School of Prince of
Songkla University. We are very grateful to Yutaka Osada,
Graduate School of Agricultural and Life Sciences, The

192

JOURNAL OF ARACHNOLOGY

University of Tokyo, Japan, for his valuable statistical advice,
critical reading of the manuscript, and invaluable comments.
We are deeply indebted to Dr. Akio Tanikawa, Graduate
School of Agricultural and Life Sciences, The University of
Tokyo, Japan, for help in spider identification, and for being a
great teacher of taxonomy to the first author. We thank
Ratana Tongyoi, SOUTHGIST, Prince of Songkla University,
for providing detailed maps of study area. We thank the head
of Khuan Khao Wang National Park and the owners of the
rubber plantations for their permissions and support of data
collection. We thank the staff of Khuan Khao Wang Forest
Park for their support of the forest survey in the beginning of
this research. For English editing of our first draft manuscript,
we would like to express several thanks to Assoc. Prof. Dr.
Seppo Karrilla, Faculty of Science and Industrial Technology,
Research and Development Office (RDO), Prince of Songkla
University, Thailand. For English editing of our revised
manuscript, we are genuinely grateful to Prof. Paul Racey,
University of Aberdeen, United Kingdom, and Prof. Doug
Armstrong, Massey University, New Zealand.

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Manuscript received 2 April 2015, revised 29 March 2016.

2016. Journal of Arachnology 44:194-198

First record of a representative of Ballarrinae (Opiliones: Neopilionidae), Americovibone remota sp. nov.,

from New Zealand

Christopher K. Taylor: Department of Environment and Agriculture, Curtin University, GPO Box U1987, Perth, WA
6845, Australia; School of Animal Biology, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009,
Australia. E-mail: Chris.Taylor@curtin.edu.au

Abstract. Americovibone remota sp. nov. is described as the first New Zealand representative of the Ballarrinae, a
Gondwanan-distributed group of harvestmen (Arachnida: Opiliones: Palpatores), from a female collected at Dart Hut in
Mount Aspiring National Park. Though closely allied by external and ovipositor morphology to Americovibone
kmfrcmcoae Hunt & Cokendolpher, 1991 of southern South America, A. remota lacks the reflexed pedipalpal tibia
previously regarded as characteristic of the Ballarrinae. The genus Americovibone is restricted to austral Nothofagus forests
which have a similar trans-Pacific distribution.

Keywords: Arachnida, Palpatores, taxonomy, biogeography, Gondwana

http://zoobank.org/References/EEE95A2C-9951-4EF3-B7D7-82EA70A09671

The Ballarrinae are a highly distinctive but little-studied
group of long-legged harvestmen (Opiliones: Palpatores)
found in continents of the Southern Hemisphere. They are
immediately recognizable from their distinctive pedipalps,
which have an elongate patella, reduced tibia and lack a tarsal
claw (Hunt & Cokendolpher 1991). They are also noteworthy
for including some of the smallest of all Opiliones, with body
lengths less than 1.5 mm in some species (Hunt & Cokendol¬
pher 1991).

When first described by Hunt & Cokendolpher (1991), the
Ballarrinae exhibited a near-classic Gondwanan distribution,
with species found in southern parts of each of South America,
Africa and Australia. However, they have hitherto appeared
to be curiously absent from New Zealand. Other Gondwanan-
distributed groups of Opiliones, such as Pettalidae (Boyer &
Giribet 2009), Triaenonychidae (Forster 1954) and Enantio-
buninae (Taylor 2011; Fernandez et al. 2014), are diverse in
the region and make up the greater part of the local Opiliones
fauna. Recently, while sorting through material on loan from
the New Zealand Arthropod Collection (NZAC), I discovered
a specimen of Ballarrinae in a collection from the Dart Hut in
New Zealand’s Mount Aspiring National Park. Though only a
single female specimen was available, this represents a
significant development in our understanding of the New
Zealand harvestman fauna.

The New Zealand specimen is very similar to the southern
Chilean species Americovibone lanfrancoae Hunt &. Cokendol¬
pher, 1991 and is here described as a second species of the
same genus. This implies a closer relationship of the New
Zealand Ballarrinae to South America than to the Australian
genera Plesioballarra Hunt & Cokendolpher, 1991, Arrallaba
Hunt & Cokendolpher, 1991 and Ballarra Hunt & Cokendol¬
pher, 1991.

The specimen was sourced from the New Zealand Arthro¬
pod Collection (NZAC), Landcare Research, Auckland. It
was examined using a Nikon SMZ1500 stereo microscope, and
photographs and measurements were taken using the NIS-
Elements D 4.00.03 program. The ovipositor was removed and
partially cleared using 50% lactic acid, and the ovipositor, a

separated chelicera, and the original specimen were examined
using a Leica DM2500 compound microscope. The ovipositor
and separated chelicera were retained in a microvial with the
original specimen. Coloration is described as preserved in
alcohol. Measurements are reported in millimeters (mm).

Family Neopilionidae Lawrence, 1931
Subfamily Ballarrinae Hunt & Cokendolpher, 1991
Genus Americovibone Hunt & Cokendolpher, 1991

Americovibone Hunt & Cokendolpher, 1991: 165.

Type species. — Americovibone lanfrancoae Hunt & Coken¬
dolpher, 1991, by original designation.

Remarks. — Americovibone remota sp. nov. is consistent with
the description of Americovibone provided by Hunt &
Cokendolpher (1991). Important diagnostic features of this
genus include: chelicera with ventral spur at base of segment I;
pedipalp with patella longer than tibia and tarsus, femur and
patella of female pedipalp pseudosegmented; leg claws with
lateral teeth; ovipositor corpus with more than two segments,
two spermathecae present. Americovibone remota also has a
spermathecal morphology very similar to those of A.
lanfrancoae.

Americovibone remota sp. nov.

(Fig. 1)

http://zoobank.org/NomenclaturalActs/

F6D622B7-A474-4553-BE09-683D85C12E2D

Type material. — Holotype female. NEW ZEALAND: Dart
Hut, Mount Aspiring National Park, 44°32'S 168°33'E, 920 m,
13-14 February 1980, J. S. Dugdale, pan trap in bush
(NZAC).

Etymology. — From the Latin remotus, remote, in reference
to both the remoteness of the type locality in New Zealand,
and the species’ remoteness from its closest ally in South
America.

Diagnosis. — Americovibone remota differs from A. lanfran¬
coae in having the pedipalpal tibia not reflexed on the patella.

194

Figure 1. — Americovibone remotci sp. nov., holotype female. A. Body, dorsal view; B. Body, lateral view; C. Right pedipalp, lateral view, setae
omitted; D. Ovipositor, showing position of spermathecae as dotted lines; E. Photograph of spermatheca; F. Line diagram of spermatheca.

with the dorsal angle between the two segments greater than
180°. As in other Phalangioidea, the patella-tibia junction of
the pedipalp in Ballarrinae is able to flex laterally but not
dorsoventrally (Wolff et ah, in press), so the difference in

palpal disposition between the two species is not an artefact of
preservation. The two species may also differ in color pattern,
with A. lanfrancoae illustrated by Hunt & Cokendolpher
(1991) as having the propodeum medially brown, and the

196

JOURNAL OF ARACHNOLOGY

dorsum of its opisthosoma more extensively pale with the
brown transverse striping broken medially. However, this
should be treated with caution due to the reported poor
condition of the A. lanfrancoae type specimens. Comparison
of body size between A. remota and A. lanfrancoae is also
impeded by the condition of the single known female specimen
of the latter, in which the main body is distorted and the legs
missing (Hunt & Cokendolpher 1991). However, differences
between cheliceral and pedipalpal measurements of the two
species are minimal except that the pedipalpal femur and
tarsus are relatively longer in A. lanfrancoae (1.97 and 0.98
mm, respectively; patella 1.4X length of tarsus in A.
lanfrancoae vs 1.6X in A. remota).

Description (female holotype). — Prosoma length 0.29, pro¬
soma width 0.73, body length 1.18. Dorsum unarmed,
without prominent setae. Ozopores small, round, not raised
on lobes. Propodeum cream-colored; ocularium black.
Mesopeltidium, metapeltidium and dorsum of opisthosoma
mostly mottled brown, with cream-colored transverse stripes
and cream-colored lateral margins on opisthosoma. Venter
cream-colored, without prominent setae; coxapophysis II
angled slightly rearwards. Chelicerae: Segment I 0.25,
segment II 0.46. Unarmed. Base of segment I with ventral
spur; spinose scales absent. Fingers relatively slender, bent
mesad in frontal view; teeth all small. Pedipcdps (Fig. IC):
Femur 1.89, patella 1.34, tibia 0.59, tarsus 0.84. Femur more
than 1.5 times main body length, with ten pseudosegments.
Patella more than two times length of tibia, with eight
pseudosegments whose boundaries are concentrated towards
midlength; patella with small swelling retrodistally. Tibia not
reflexed relative to patella; dorsal angle between patella and
tibia slightly more than 180°. Tarsus evenly concave on
dorsal margin. Sparse plumose setae along entire length of
pedipalp, becoming denser distally on tarsus; plumes
restricted to one side of each seta, reaching about halfway
down length of each seta on femur to tibia, extending further
down each seta on tarsus. Few non-plumose setae present at
apex of tarsus. Tarsal claw and microtrichia absent. Legs:
Leg I femur 1.57, patella 0.33, tibia 1.38; leg II femur 2.96,
patella 0.34, tibia 2.92; leg III femur 1.58, patella 0.31, tibia
1.41; leg IV femur 2.40, patella 0.36, tibia 2.32. All segments
unarmed. Femora of all legs with, respectively, 6, 12, 4-5 and
7-8 pseudosegments; tibia II with 12 pseudosegments, tibia
IV with 3 pseudosegments, tibiae I and III not pseudoseg-
mented. Metatarsi elongate but not pseudosegmented. Tarsal
claws of legs I, III and IV with sharp bend near base; dentate
lateral carina present on either side; tarsal claw of leg II
weaker, without lateral carina. Ovipositor (Fig. ID-F); Furca
three-segmented, distal segment of furca elongate (about four
times as long as wide). Two spermathecae present in second
and third segments; spermathecae consisting of short loop
with lateral extension on each side.

Remarks. — As the new species described herein is more
similar to A. lanfrancoae than any other species of Ballarrinae,
it is provisionally assigned to the same genus pending the
discovery of male specimens. It might be questioned whether it
is appropriate to describe a new species of Opiliones from a
single female specimen, considering the pre-eminent position
of male genitalic characters in Opiliones taxonomy (see e.g.,
Maci'as-Ordonez et al. 2010; Perez-Gonzalez 2011; Pinto-da-

Rocha et al. 2012), the presence in many groups of Opiliones
of significant sexual dimorphism, and the potential difficulty
of distinguishing females of closely related species (see e.g.,
Taylor 2004). However, this particular example represents the
first record in New Zealand of a significant group of Opiliones
whose presence there has not hitherto been recognized. Many
parts of the south-western South Island of New Zealand are of
limited accessibility, and so have not been extensively
collected. The type locality of A. remota. Dart Hut, is
positioned in the eastern part of Mount Aspiring National
Park at the junction of Snowy Creek and the Dart River (Fig.
2). Dart Hut is two days’ walk from the nearest road, with the
entire Rees-Dart track taking four or five days (Department of
Conservation 2013).

Significant sexual dimorphism has not yet been recorded
from Ballarrinae, though males are generally smaller than
females (Hunt & Cokendolpher 1991). The main external
differences between A. lanfrancoae males and females are that
male pedipalps lack the pseudosegments found in females, and
the male pedipalpal tarsus is dorsally convex rather than
concave. Hunt & Cokendolpher (1991) did not identify any
significant differences between the sexes in coloration,
ornamentation or cheliceral development.

As noted above, the close similarity of A. remota to A.
lanfrancoae suggests a closer relationship of New Zealand
Ballarrinae to South American than to Australian species.
Though less common than the converse, other examples of
this pattern of biogeographic relationships include the plant
genus Aristotelia (Elaeocarpaceae) and certain members of
the midge subfamily Podonominae (Diptera: Chironomidae)
(Crisci et al. 1991). However, it is also noteworthy that Dart
Hut is positioned in a beech (Nothofagus) forest (map in
Sommerville et al. 1982), which is also the known habitat for
the South American species (Hunt & Cokendolpher 1991).
Nothofagus has only a relictual distribution in mainland
Australia, being found in Tasmania, southern Victoria and
across the New South Wales-Queensland border (Knapp et
al. 2005). Where habitat has been recorded, Australian
Ballarrinae are mostly known from Eucalyptus woodlands,
though Ballarra cantrelli Hunt & Cokendolpher, 1991 and
Plesiohallarra crinis Hunt & Cokendolpher, 1991 are possibly
within the range of Nothofagus moorei (Hunt & Cokendol¬
pher 1991). No Ballarrinae have as yet been described from
Tasmania.

It is also possible that local extinctions have complicated the
biogeography of Ballarrinae. The endemic New Zealand bat
genus Mystacina is more closely related to South American
taxa in the Noctilionoidea than to any living Australian bats
(Teeling et al. 2005). Nevertheless, the fossil record supports
an Australian origin for Mystacina, with its probable sister
genus Icarops being present in the Miocene of northern
Australia (Hand et al. 1998).

Though the Ballarrinae were initially united on the basis of
their distinctive pedipalpal morphology (Hunt & Cokendol¬
pher 1991), a recent molecular analysis of Palpatores has
questioned the monophyly of ballarrines (Groh & Giribet
2015). In this study, the South African Vibone vetusta Kauri,
1961 failed to form a clade with the Australian Ballarra
longipalpus Hunt 8l Cokendolpher, 1991. Conversely, B.
longipalpus and the South American A. lanfrancoae did form

TAYLOR— NEW ZEALAND BALLARRINAE

197

Figure 2. — Map of Southern Hemisphere, showing distribution of Ballarrinae and Nothofagus. Symbols indicating known localities of main
Ballarrinae subgroups (Hunt & Cokendolpher 1991): = Americo\’ih(me\ diamond^ Vibone\ triangle = Australian clade {Ballarra, Aircillaha

and Plesiohallarra). Distribution of Nothofagus shown in grey (based on Knapp et cil. 2005, modified for scale). Inset: southern South Island,
New Zealand, with black square indicating type locality of Americovibone remota.

a clade when included in a morphological analysis of
Neopilionidae by Taylor (2013). However, neither of these
analyses included more than two species of Ballarrinae nor
had the Ballarrinae as their main focus. The Australian
Ballarrinae are united by their distinctive male genital
morphology, with a barbed process on the left ventral side
of the penis (Hunt & Cokendolpher 1991). This process is
absent in A. lanfrancoae. The male of V. vetiista is
unfortunately unknown but it is noteworthy that the
ovipositor morphology of this species, as described by Kauri
(1961), is unique in the Phalangioidea, with the main body of
the ovipositor largely unsegmented.

The absence of a dorsal reflexion between the patella and
tibia of the pedipalp in A. remota, previously regarded as a
defining feature of the Ballarrinae, gives credence to the
possibility that the unusual ballarrine palpal morphology may
have evolved independently. Glandular setae on harvestmen
pedipalps produce sticky secretions that are used in prey
capture (Wolff et al. 2014), and it is possible that the
‘ballarrine’ pedipalp represents a convergent adaption to the

active predation of fast-moving prey such as springtails.
Further investigation into the monophyly of this group is
required.

ACKNOWLEDGMENTS

I would like to thank Grace Hall of Landcare Research for
the loan of specimens from the New Zealand Arthropod
Collection. Research for this paper was conducted using
facilities at Curtin University, Western Australia, and the
University of Western Australia. Comments and suggestions
for the original manuscript were provided by Mark Harvey
and two anonymous reviewers. The maps used in Fig. 2 were
modified from freely available figures at Wikimedia Commons
(https://commons.wikimedia.org).

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Manuscript received 14 September 2015, revised 15 February 2016.

2016. Journal of Arachnology 44:199-209

Mechanical properties of male genitalia in Leiobumim harvestmen (Opiliones: Sclerosomatidae)

Mercedes Burns’'^ and Jeffrey W. Shultz^- 'Department of Entomology and BEES Graduate Program, University of
Maryland, College Park, MD 20742; “Present address: Department of Biology, San Diego State University, San Diego,
CA 92182. Email: mercedes.burns@gmail.com

Abstract. The morphology of arthropod intromittent organs evolves rapidly and is often species specific, phenomena
widely attributed to sexual selection. Similar patterns in biomechanical properties may also exist, but practical challenges
in manipulating small structures and measuring minute forces has impeded experimental biomechanical analysis. Here we
describe a device that displaces a small structure while measuring its resistance, and use it to examine the biomechanics of
penile flexure in the eastern North American harvestman genus Leiolmnum C.L. Koch, 1839. Several Leiobunum lineages
have lost primitive penis-associated nuptial-gift sacs and have gained apparent female pregenital barriers, a co¬
evolutionary pattern consistent with shifts from precopulatory enticement to more-antagonistic strategies. We tested for an
association between losses of nuptial-gift sacs and increases in penile flexural resistance using five sacculate and five non-
sacculate species. We measured three mechanical variables — resistance force, elastic efficiency and viscoelastic relaxation
time — under lateral, dorsal, and ventral flexion. Our functional assumptions about sacculate and non-sacculate penes
anticipated two biomechanically-defined species clusters, but three were found: a diverse sacculate group, a monophyletic
non-sacculate group and an unanticipated mixed group. This work demonstrates that experimental genital biomechanics in
arthropods is possible, and we discuss the functional implications of our results.

Keywords: Reproduction, viscoelasticity, elastic efficiency, phylogenetic comparative methods

Explaining the remarkable diversity of reproductive struc¬
tures in arthropods and other animals is a perennial goal of
evolutionary biologists (Day & Young 2004; Leonard &
Cordoba-Aguilar 2010). Attention has centered on the often
species-specific and sometimes exaggerated or complex traits
of males (Hosken & Stockley 2004), although several recent
authors have highlighted the importance of genital variation in
females as well (Brennan et al. 2007; Sanchez et al. 2011;
Tanabe & Sota 2013; Ah-King et al. 2014). The persistent bias
toward research on male structures, especially intromittent
organs, likely reflects their proven value for delimiting species
(Edwards & Knowles 2014), their relatively rapid rate of
evolution (Cayetano et al. 2011; Cassidy et al. 2014; Masly &
Kamimura 2014), and the numerous evolutionary factors that
have been invoked to explain their diversity (Leonard &
Cordoba-Aguilar 2010), including female preference (Kokko
et al. 2003), sperm competition (Parker et al. 2013), and
cryptic female choice (Eberhard 1996; Albo et al. 2013). An
understanding of the contributions that different selection
mechanisms have made in shaping genitalic diversity should
benefit from detailed information about the mechanical
properties of genitalia (Cayetano et al. 2011), but existing
information is largely based on inferences drawn from static
anatomy or associated behavior rather than from experimen¬
tal measurement of biomechanical variables (Bonduriansky &
Day 2003; Marquez & Knowles 2007). Consequently, despite
active interest in the roles of female enticement and coercion
as male mating strategies, there is little information about the
intrinsic ability of male intromittent organs to respond
mechanically to female movement or to overcome female
resistance during antagonistic interactions (but see Brennan et
al. 2010). Here we focus on the mechanical properties of penes
in the eastern North American species of harvestmen from the
genus Leiobunum C.L. Koch, 1839 (Fig. 1), a clade for which
reproductive diversity is increasingly well documented (Burns

et al. 2012, 2013; Fowler-Finn et al. 2014; Burns & Shultz
2015).

Harvestmen are unusual among arachnids in having a true
penis and in mating face to face (Machado et al. 2015; Fig. 1).
The reproductive structures in both sexes are enclosed within a
pregenital chamber that occupies the ventral part of the
abdomen and opens anteriorly just posterior to the mouth.
This chamber is enclosed ventrally by a large sclerite, the
genital operculum, which articulates with the abdomen
posteriorly via a transverse hinge to open and close like a
trapdoor. The penis is essentially a cuticular tube that usually
has a subterminal joint that divides it into a long proximal
shaft and short distal glans, which has a thin terminal stylus
that bears the small primary genital opening. The glans-shaft
joint is operated by a bi- or multi-pinnate muscle that arises
from the walls of the shaft and inserts via a long tendon on the
ventral surface of the joint. The penis is externalized anteriorly
by the combined effects of protractor muscles and hydraulic
eversion of the flexible walls of the pregenital chamber.

Two basic types of penes have been distinguished based on
the presence or absence of a subterminal pair of cuticular sacs
(Fig. lA). The sacs carry a male-generated nuptial gift that
may be accessed orally by the female early in mating (Fig. 1 D),
a behavior that was often confused with copulation by early
naturalists due to the proximity of the mouth and pregenital
opening. Penile sacs were lost several times in Leiobunum
(Burns et al. 2013); losses typically accompanied by the
evolution of a sclerotized pregenital barrier in females. These
correlated transformations suggest that female enticement via
nuptial gifts was important in the primitive premating strategy
in Leiobunum but was replaced multiple times by other
mechanisms, including antagonistic interactions between the
penis and the opening to the female pregenital chamber (Burns
et al. 2013). Recent work indicates that the relative maximum
forces produced by the penis protractor muscle and by the
closer of the female genital operculum coevolved and are

199

200

JOURNAL OF ARACHNOLOGY

L calcar^^Z

L euserratipalpe'illizzzz

L nigropalpi 5~~

L. politum cr ~

L bracchiolum i

L vittatum
L uxorium
L aldrichi c

L ventricosum^
L verrucosum<^

1 mm

Figure 1. — Male reproductive morphology and phylogeny of Leiobimwn. A. Penes (to same scale) from 10 Leiohmmm species depicted on a
pruned maximum clade credibility tree (Burns et al. 2013). We hypothesized that these discrete classes should be highly correlated with genital
function, such that mechanical force traits might discriminate them. B-E. Mating in Leiohimum venucosum (legs removed for clarity). B. Male
encounters receptive female, male palps preparing to grasp female, penis extruded. C. Male clasps female with palps posterior to leg coxa II,
delivers initial nuptial gift to female from penile sacs. D. Female feeds from male glands, penis lodged near female pregenital opening. E.
Intromission associated with reorientation of bodies.

BURNS & SHULTZ— GENITAL BIOMECHANICS OF OPILIONES

201

higher in non-sacculate species, suggesting that greater
mechanical forces are produced and resisted in non-sacculate
forms (Burns & Shultz 2015).

We hypothesized that the mechanical properties of penes in
Leiobunum have changed from those that accommodate
female preferences to those that can transmit or resist
mechanical forces when interacting with the female’s pregen¬
ital opening. Specifically, we expect penes used in forceful
precopulatory interactions to resist higher bending forces than
those used principally for enticing females with nuptial gifts.
We also predicted changes in two parameters associated with
cuticular viscoelasticity. In structures composed of ideally
elastic materials, the energy used in deforming a structure is
stored as elastic potential energy and recovered as kinetic
energy as the penis regains its resting state, regardless of the
duration or rate of loading or unloading (Vincent 2012).
However, in viscoelastic materials, some energy is lost to heat
during deformation, with the amount being time dependent.
Thus, we also predicted that the amount and rate of energy
loss would be higher in penes that are adapted to accommo¬
date female nuptial-gift feeding and lower in penes adapted for
applying large or prolonged forces to the female

We tested our hypotheses by bending penes from 10
Leiobunum species while measuring both flexural resistance
and flexural displacement. Measurements were obtained from
a phylogenetically diverse sample of five sacculate species and
representatives from two non-sacculate clades, the calcar and
vittatum groups (Fig. lA). We measured three biomechanical
variables — maximum resistance force for a flexural displace¬
ment of 5% of beam length, the efficiency of elastic energy
storage, and the rate of viscoelastic relaxation in static
bending. Phylogenetic multivariate analyses of our data
recovered two species clusters (the sacculate group and
monophyletic calcar clade) that were consistent with our
biomechanical predictions but also a third cluster that was not
anticipated. Ultimately, this work demonstrates that mechan¬
ical properties of reproductive structures can be measured and
that data derived from these measurements can be used to test
hypotheses about arthropod mating systems.

METHODS

Animals. — We examined 60 male specimens representing ten
Leiobunum species (Fig. lA), constituting a subset of species
examined in Burns & Shultz (2015). Five species have penes
with subterminal gift-bearing sacs and females that lack
pregenital barriers (i.e., L. ventricosum (Wood, 1868), L.
verrucosum (Wood, 1868), L. aldrichi Weed, 1893, L. politum
(Weed, 1889), L. bracchiolum McGhee, 1977), which we term
“sacculate species”. Five species lack gift-bearing sacs and
females have sclerotized pregenital barriers (i.e., the vittatum
group: L. uxorium Crosby & Bishop, 1924, L. vittatum (Say,
1821) and the calcar group: L. nigropalpi (Wood, 1868), L.
euserratipalpe Ingianni, McGhee & Shultz, 2011, L. calcar
(Wood, 1868)), which we term “non-sacculate species”.
Specimens were collected and maintained up to a week in
laboratory terraria with food (pulverized fish food) and water
provided ad libitum in cotton-stoppered vials. See Table 1 for
additional species information.

Apparatus. — We assembled a device to measure forces
associated with penile flexural resistance and flexural displace¬

ment (deflection) in fresh harvestman penes mounted as a
cantilever (Fig. 2). We attached a force transducer (Aurora
Scientific, Inc.: Model 404A: range, 0-100 mN; sensitivity; 10.0
mN; resolution, 2000 nN) to a vertically mounted, computer-
controlled translation stage (Thor Labs: OptoDC Servo
Motor Driver #001). A displacement transducer (Microstrain:
SG-DVRT-4: max. linear stroke, 24 mm; resolution, 6 pm)
recorded linear movement of the force transducer. The
translation stage was programmed to move at 1 mm/s. A
stiff, hooked, non-magnetic wire was attached to the input
tube of the force transducer and used in deflecting the penis.

Protocol. — Adult specimens were sacrificed by placing them
in a freezer at — 20°C for 10 minutes, after which the penis was
removed and affixed at its proximal end to a round glass cover
slip using ethyl cyanoacrylate gel (Super Glue®). A drop of
accelerant (Turbo Set I, Palm Labs Adhesives, Inc.) was
applied to the glue bead to fix the penis as a full-moment
cantilever (Fig. 2B). The cover slip with attached penis was
submerged in a clear-sided, open-top polyacrylic box filled
with room-temperature Ringer’s solution. The cover slip was
affixed to the side of the box using 1/8 inch x 1/8 inch (.3175
cm X 3175 cm) neodymium magnets to facilitate repositioning
(Fig. 2A).

The hooked wire from the force transducer was brought
into contact with the penis at a point one third of free-penis
length from the distal end. This length was selected to avoid
applying force to the sac region of penes from sacculate species
(Fig. lA). The translation stage was programmed to bend the
penis with a deflection of 5% of the beam length. Three
consecutive hysteresis loops (Fig. 3A) were obtained from
each penis, with the penis displaced and returned to its resting
position at 1 mm/s. Hysteresis loops were obtained for dorsal,
ventral and horizontal deflection from the resting position, as
each of these flexural directions may be imposed by the female
pregenital chamber upon the penis during precopulation. We
also deflected each penis to 5% of beam length and held it for
at least 180 s while measuring the viscoelastic relaxation of the
restoring force (Fig. 3B). Displacement and force data were
sampled every 50 ms using Easylogger Dual Version 1.0
software (EasySync Ltd.).

Mechanical variables. — We calculated three mechanical
variables each from dorsal, ventral and lateral flexures — force
at maximum experimental displacement (or cF,,,^,.^ when
corrected for body size), elastic efficiency R, and relaxation
time to 90% (T^o) — resulting in a total of nine variables.

Mean values for all variables were established for each
specimen from three replicates, and mean species values were
calculated from specimen means.

F,„ax is the force required to achieve a vertical deflection of
the penis equal to 5% of beam length. Consequently, F,„ax can
be viewed a measure of stiffness at a geometrically similar
flexural displacement across specimens. To adjust the magni¬
tude of F,„ax for body size, we derived a method of size
correction from the standard equation for cantilevered beams
fixed at one end (Vogel 2003):

FZ)’’ = 3£/L“^

(1)

and its proportional representation:

[FD“'] = EIL-^

(2)

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Table 1. — Taxon sampling for molecular phylogenetic reconstruction and mechanical force trait evaluation. Accession numbers are for the
GenBank genetic sequence repository; numbers GQ870643-GQ870668 and GQ872152-GQ872185 are derived from Hedin et al. (2010). Column
5: penile nuptial gift sac presence, grouping variable used in testing for trait mean differences. Column 6: numbers of male specimens analyzed for
mechanical force traits.

Species

GenBank accession numbers

Molecular
specimen locality

Mechanical data
specimen locality

Penile
nuptial
gift sac
presence

Number

of

specimens

Leiobimum

alclrichi

GQ870650, JQ432342, JQ432284,
GQ872154, GQ870649, JQ432343,
JQ432285, GQ872153, JQ432344,
JQ432286, JQ432238

USA: MI: Calhoun Co.

USA: MD: Frederick Co.

Present

2

L. hracchiohnn

JQ432330, JQ432272, JQ432230

USA: NC: Guilford Co.

USA: MD: Montgomery Co.

Present

2

L. calcar

GQ870653, JQ4323I6, JQ432258,
GQ872157, JQ432317, JQ432259,
JQ432223, JQ432319, JQ432261,
GQ870655, JQ432320, JQ432262,
GQ872158, JQ432318, JQ432260

USA: MD: Frederick Co.

USA: TN: Carter Co.

Absent

9

L. euserratipalpe

JQ432321, JQ432263, GQ870656,
JQ432322, JQ432264, GQ872160

USA: MD: Montgomery Co.

USA: MD: Frederick Co.,

USA: MD: Montgomery Co.

Absent

10

L. nigropalpi

JQ432323, JQ432265, JQ432224,
JQ432324, JQ432266, JQ432225,
JQ432325, JQ432267, JQ432226

USA: MD: Frederick Co.

USA: MD: Montgomery Co.,
USA: TN: Washington Co.,
USA: VA: Fairfax Co.

Absent

6

L. poliluin

JQ432326, JQ432268, JQ432227,
JQ432327, JQ432269, JQ432228,
jQ432328, JQ432270, JQ432229,
JQ432329, JQ43227I

USA: AR: Lawrence Co.

USA: MD: Montgomery Co.

Present

6

L. uxoriwn

JQ432339, JQ432281, JQ432235,
JQ432338, JQ432280, JQ432236

USA: VA: Smythe Co.

USA: MD: Montgomery Co.

Absent

8

L. veutricosum

JQ432348, JQ432290, JQ432349,
JQ432291, JQ432242, JQ432350,
JQ432292, JQ432243

USA: TN: Blount Co.

USA: TN: Washington Co.

Present

5

L. verrucosum

JQ432351, JQ432293, JQ432244,
JQ432347, JQ432289, JQ432241

USA: TN: Cumberland Co.

USA: MD: Montgomery Co.,
USA: TN: Washington Co.

Present

3

L. vittation

JQ432333, JQ432275, JQ432232,
GQ870651, JQ432334, JQ432276,
GQ872155, JQ432335, JQ432277,
JQ432233, JQ432336, JQ432278,
JQ432234,GQ870652, JQ432337,
JQ432279, GQ872156

USA: TN: Davidson Co.

USA: MD: Montgomery Co.

Absent

4

where F is the imposed bending force (in Newtons), D is
vertical displacement of the beam at the point where F is
applied (in meters), E is the elastic modulus of the material, / is
the second moment of area of the beam, and L is the distance
(in meters) between the base of the beam and the point where
F is applied. E was assumed to be the same in all penes and
was treated as a constant. / is a measure of architectural
stiffness and reflects the amount and distribution of material
around the flexural axis of a beam. It varies in proportion to
d^, where rf is a characteristic length of an isometric system.
We used the width of the propeltidium of the carapace
between coxae I and II {d) as the characteristic length; the
propeltidium is a single sclerite and is largely unaffected by
nutritional or reproductive status of an adult specimen.

To determine the size correction for F, we solved Equation 2
for F by converting all other parameters to d" by dimensional
analysis:

[Fi] = DIL-^ = d^d^d~^ =d^ (3)

Thus, we size corrected by dividing its measured value by
the square of carapace width to obtain cF„,ax-

We obtained elastic efficiency R by dividing the area under
the unloading portion of the hysteresis loop (i.e.,
mechanical energy out) by the area under the corresponding
loading portion of the loop Wi (i.e., mechanical energy in)
(Fig. 3A, C, D). Given our assumptions of isometry and
constant E (see above), no size correction for R was required.

Tgo was the time (in seconds) required for the force of
flexural resistance to undergo viscoelastic relaxation to 90%
Fmax- We did not attempt to correct Tgg for size given the
absence of a time dimension in Eq. 1. In cases where 90% F,„ax
was not reached after 180 s, 180 s was used as the relaxation
time. cF,„ax and Tgo were log-transformed to minimize
heteroscedasticity between dorsal, ventral and lateral bending
and between sacculate and non-sacculate groups.

Data analysis. — We used phylogenetic comparative methods
in statistical analysis to control for covariance due to shared
evolutionary history. We established a phylogenetic frame¬
work by pruning a maximum clade credibility tree from a
previous Bayesian-likelihood analysis of leiobunine phylogeny

BURNS & SHULTZ— GENITAL BIOMECHANICS OF OPILIONES

203

Figure 2. — Experimental apparatus. A. Forces associated with flexural displacement of harvestman penes were measured using a force
transducer mounted vertically on a computer-driven translation stage, with vertical movement recorded by a displacement transducer. Penes
were glued to a glass coverslip in cantilevered position. The mounted penis was placed in a bath of Ringer's solution and the coverslip was
secured to the side of the bath with neodymium magnets (nm). A non-magnetic wire bent at 90° was attached to the input tube of the force
transducer, brought into contact with the penis and used to bend the penis in three directions: dorsal, ventral and lateral. B. Lateral view of penis
oriented for dorsal flexion. L, beam length, D, vertical displacement of beam (max. 0.05L). C. Photo of apparatus.

(Burns et al. 2013) to include only the 10 species examined here
(Fig. 1 A). The geiger package (Harmon et al. 2008), written in
the statistical programming language R (R Development Core
Team 2008), was used to evaluate evolutionary models for
each variable. The models included Brownian motion,
directional evolution (Brownian motion with a trend), Pagel’s
lambda (phylogenetic signal), kappa (punctuated equilibrium),
and delta (time-dependent rates or comparable to early burst
evolutionary model) (Pagel 1997, 1999). Evolutionary models
were evaluated using the corrected Akaike information
criterion (AICc), adjusted by the number of estimated
parameters for each model. Model probability was determined
by AICc weights (Burnham & Anderson 2004).

We performed phylogenetic principal components analyses
using the R-based package phy tools (Revell 2012) to explore
covariation among mechanical variables. The predictions
presented in the introduction anticipate positive covariation
among variables that should be reflected in similarities in
variable loadings and the placement of sacculate and non-
sacculate species into separate clusters. Differences in me¬
chanical variables between sacculate and non-sacculate groups
were investigated further using phylogenetic multiple analysis
of variance (pMANOVA) (Garland et al. 1993) as implement¬
ed in the geiger package. The Wilk’s lambda test statistic and
significance level were calculated for the data and for one
million Brownian-motion simulations based on the evolution¬
ary variance/covariance matrix estimated from the data across

the phylogeny. Thus, model significance indicated by the
standard MANOVA is supported by the commonality of the
actual-data test statistic compared to a null distribution. We
conducted Shapiro-Wilk’s and Levene’s tests (using the R
package asbio; Aho 2014) on each variable to assess normality
and homoscedasticity.

RESULTS

Models of evolution. — We used the fitContinuous function
in the R package geiger to determine the best fit based on
AICc for five potential models of evolution for each
mechanical variable (Table 2). Most variables were best
modeled by Brownian motion. This result was reinforced by
maximum likelihood estimates of Pagel’s lambda, which
equaled or approached 1.0 for several traits across the three
bending directions, including dorsal logTgg, dorsal and lateral
logcF,„^,v, and ventral and lateral R. A lambda value of 1 .0 is
considered equivalent to a Brownian motion evolutionary
model (Boettiger et al. 2012) and indicates that covariance can
be largely attributed to shared evolutionary history (i.e.,
length of shared branches.) Two mechanical variables had
lower AICc scores for non-Brownian models; lateral XogTgo
was best modeled by the kappa branch transformation (k =
6.6E-214, AICc = 252.56) and lateral R was best modeled by
the lambda branch transformation (L = 0.715, AlCc = 4.06). In
both cases, the Brownian model had the next highest AIC

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Figure 3. — Diagrammatic plots illustrating biomechanical variables and ventral sample data. A. Hysteresis loop showing parameters used to
calculate elastic efficiency R. The work generated v/hen a penis is loaded in flexion is proportional to the area under the loading curve (Wi). The
v/ork generated by the penis against the force transducer during re-extension (Wo) reflects the elastic energy stored in the penis. Thus, WJWi
equals R. B. Plot illustrating determination of Tgg, the time required for the maximum force of flexural resistance F,„c,x to relax to 90% F,„ax- We
anticipated that a penis specialized for coercive interaction with females should have a higher F,„^x, Tgo and R than penes adapted to
accommodate female preferences. C. Hysteresis loop for ventral flexion in Leiobunum politum specimen (sacculate). D. Hysteresis loop for ventral
flexion in a L. euserratipalpe specimen (non-sacculate, calcar species-group).

weight (lateral Tpo^O.!?; lateral .R = 0.31), indicating that the
alternative mode! did not provide significant improvement
over Brownian motion. However, it may be meaningful that
the traits best modeled by the more-complex functions were
both derived from lateral bending.

Exploring covariation in niec.hanical variables using principal
components analysis. — We performed a phylogenetic principal
components analysis using a multivariate lambda model of
evolution to account for phylogenetic covariance due to
species relatedness (k — 6.9E-5, LogL A, = 36.1). Variable
loadings are given in Table 3 and plotted alongside species
scores in Fig. 4. Principal components ! and 2 accounted for
about 80% of total variance. Several mechanical variables for
dorsal and ventral bending loaded highly on PCI, particularly
dorsal and ventral logr9o ^nd dorsal R. Variables associated
with lateral bending tended to load more heavily on PC2.

Principal component 1 separated the L. calcar group from
all other species, indicating that its members had compara¬
tively high dorsal and ventral logTso- Principal component 2
separated four sacculate species (L. verrucosum, L. aldricM, L.

politum, L. bracchiolum) into one cluster and the non-sacculate
vittatum group (L. vittatum, L. uxorium) plus the sacculate L.
ventricosum in another. The ventricosumivittatum group was
characterized by comparatively high lateral and ventral R,
long lateral logTpo, and short dorsal and ventral logTgo-

Mechanical comparisons between sacculate and non-sacculate
penes. — Results from group-mean comparisons using phylo¬
genetic MANOVA are summarized in Fig. 5. No significant
difference between sacculate and non-sacculate groups was
found for R (Wilk’s A. = 0.33, F3,6 = 4.04, model P = 0.069,
phylogenetic P = 0.68) or logcF,,,^,^ (Wilk’s A. = 0.7, F3,6 =
0.856, model R = 0.512, phylogenetic F = 0.19) for any of the
three flexural direction. High phylogenetic P-values for these
models indicate that similar group means were achieved in
most simulations, where phylogenetic branch lengths are
randomly rescaled to allow greater potential change along
longer segments.

There was a significant difference between sacculate and
non-sacculate species in logTgo (Wilk’s A, = 0.26, Fa^e = 5.77,
mode! P < 0.05) (Fig. 5D), with non-sacculate penes taking

BURNS & SHULTZ— GENITAL BIOMECHANICS OF OPILIONES

205

Table 2. — Evolutionary model selection for body size and mechanical force traits. Akaike information criterion corrected for small sample size
(AICCwt) standardized weights mechanical reproductive traits. Models included Brownian motion (random walk), Directional (Brownian motion
with a trend), kappa (punctuated equilibrium), lambda (phylogenetic signal), and delta (time-dependence) (Pagel 1999, 1997). Unstandardized
weights were calculated with the equation AICcwt = (Burnham & Anderson 2004). Preferred model (greatest AlCc^t) is

indicated with asterisk (*).

Model

Mechanical variable

Brownian

(AICcw.)

Directional

(AICCw,)

kappa

(AICcwt)

lambda

(AICcwt)

delta

(AICCwt)

Elastic efficiency (R)

Dorsal

*0.4098

0.0719

0.1628

0.1553

0.2002

Ventral

*0.5729

0.0768

0.1173

0.1141

0.1189

Lateral

0.3069

0.0528

0.1432

*0.3596

0.1373

90%-relaxation time (Tgo)

Dorsal

0.6216

0.1962

0.0564

0.0564

0.0694

Ventral

0.5843

0.0851

0.0536

0.1017

0.1752

Lateral

0.2695

0.0453

*0.4407

0.1377

0.1067

Maximum experimental displacement force (F,„oJ

Dorsal

*0.6211

0.0866

0.0882

0.0563

0.1476

Ventral

*0.4059

0.0720

0.1293

0.1742

0.2185

Lateral

0.6525

0.0823

0.0839

0.0592

0.1221

significantly more time to reach 90% of However, this
result was not robust to evolutionary data replication
(phylogenetic P = 0.7815), indicating that a significant group
difference based on sac presence would not be found under the
majority of logTpo simulations with identical evolutionary
conditions. Tests of data normality and heteroscedasticity
indicated that all variables were normally distributed, but
there were unequal variances between sacculate and non-
sacculate species for many variables, primarily those measured
during dorsal flexion (dorsal log(:F„,„v; Fi g = 16.7, P <0.01;
logTet): Fi g = 8.61, P <0.05). Heteroscedasticity within the
non-sacculate category is consistent with results from the
principal components analysis (Fig. 4), where the vittatum
group tended to cluster with the sacculate species, and not
with the non-sacculate calcar group.

Although phylogenetic simulation did not identify a
significant difference in \ogT<)o between sacculate and non-
sacculate species, we performed three follow-up phylogenetic
univariate tests comparing means of dorsal, ventral, and

Table 3. — Trait loadings of phylogenetic principal component
analyses, eigenvalues, and percent variance explained by first two
principal components (PC).

Mechanical variable

PC 1

PC 2

Elastic efficiency (R)

Dorsal

-0.783

0.254

Ventral

-0.661

-0.655

Lateral

-0.476

-0.811

90% relaxation time {Tgo)

Dorsal

-0.977

-0.018

Ventral

-0.741

-0.008

Lateral

-0.566

-0.775

Max. resistance force(F„,aJ

Dorsal

-0.726

0.579

Ventral

-0.829

0.452

Lateral

-0.596

0.481

Eigenvalues

4.67

2.52

% Variance

51.89

28.05

lateral XogTgo. We found significantly longer relaxation times
for dorsal (Fi g = 5.16, P <0.05) and lateral (F| g = 19.21, P
<0.001) bending in non-sacculate species, and a similar,
though non-significant, trend for higher ventral logTpo (Fi g =
0.639, P — 0.78). These results demonstrate significant
differentiation of elastic responses in penile cuticle between
sacculate and non-sacculate species.

Following the result from the phylogenetic principal
components analysis, which separated non-sacculate species
into vittatwnlventricosum and calcar groups, we repeated the
phylogenetic MANOVA with three group-mean comparisons,
separating trait values by sacculate, vittatumiventricosum, and
calcar group membership. This model yielded significant
differences in all three variables (R: Wilk’s 1 = 0.05, Fs.io =
5.76, model P < 0.01; logcF),,^^: Wilk’s A, = 0.1 16, Fg lo = 3.23,
model P < 0.05), although in phylogenetic simulation only
\ogTgo was found to be significantly different between groups
(Wilk’s I = 0.0079, Fgjo = 17.107, model P <0.0001,
phylogenetic P <0.01). A phylogenetic ANOVA with Holm-
Bonferroni posthoc test to compare bending directions for
each of the three variables identified significantly higher dorsal
logTpo (Fj j = 20.83, P <0.05) in the calcar group as compared
to the two other groups, as well as a hierarchy of significant
differences in lateral logT^o (Fu = 43.52, P <0.01) between
the sacculate, vittatwnlventricosum, and calcar groups (Fig. 5).
Using this method, we additionally found significant differ¬
ences between the sacculate and calcar groups in ventral R
(F) 7 = 14.06, P <0.05) and between the calcar and vittatum
groups in dorsal logcF,,,^^ (Fi^y = 18.05, P <0.05) and ventral
logcF„,„v (Fi,7= 15.18, P <0.05). Values of R and logcF„,„.,. for
the vittatum + L. ventricosum and sacculate groups were
statistically indistinguishable.

DISCUSSION

Previous work on the evolution of reproductive structures
and mating systems in Leiobunum and related taxa (Burns &
Shultz 2015) identified the coevolution of relative maximum

206

JOURNAL OF ARACHNOLOGY

Figure 4. — Phylogenetic principal components analysis of mechanical force data. The pPCA, applied to dorsal, ventral, and lateral measures of
maximum flexural resistance (log cF,„a^), elastic efficiency (R) and time to 90% relaxation of F„,ax (logTgo). Principal components 1 and 2 together
account for 80% of total data variance. Trait loadings are indicated by arrows, color coded by flexural orientation. Non-sacculate species indicated
by white shapes — squares for the calcar group, diamonds for the vittatum group — and sacculate species indicated by black circles.

force produced by the penis protractor muscle and by the
closure of the female genital operculum. These relative forces
are higher in non-sacculate species, which suggests that greater
mechanical forces are produced and resisted in non-sacculate
forms. Following this line of evidence, we tested our
expectation that presence and absence of penile nuptial gift
sacs in Leiohiamm correspond to differences in the flexural
biomechanics of the penis shaft. We predicted that penes in
sacculate and non-sacculate species differ in the magnitudes of
three biomechanical variables — maximum resistance to exper¬
imental penile flexure (F„,ax), efficient storage of elastic energy
(i?) for use in restoring the flexed penis to the resting state, and
persistence of the restoring force during static flexion (Tgo) —

with the non-sacculate species having higher values. We
obtained these values for each of three biologically relevant
bending directions, and used modern phylogenetic compara¬
tive methods to find covariation between nuptial gift sac
presence and biomechanical specialization. Our success in
doing so demonstrates that biomechanical data can be
obtained from arthropod genitalia and may be useful in
resolving functionally distinct groups.

Results from phylogenetic MANOVA showed no significant
differences between sacculate and non-sacculate species
groups, except perhaps in Tgo. These findings are consistent
with those obtained from multivariate analysis based on
measurements and functional inferences from static reproduc-

BURNS & SHULTZ— GENITAL BIOMECHANICS OF OPILIONES

207

Figure 5. — Phylogenetic ANOVA results for logrpo. Bar graph
results summarize phylogenetic ANOVA tests comparing the logio-
transfonned viscoelastic relaxation times of penes displaced dorsally,
ventrally, and laterally for sacculate, vittatumiventricosiim and calcar
clusters. Bars are group means plus standard error for sacculate
(black), vitlatumjventricoswn (hatched lines) and ca/cr/r-group (white)
species for each of three bending aspects. Asterisks indicate significance
level of between-group comparisons (‘*’ P, <0.05; P, <0.01).

tive morphology (Burns & Shultz 2015). Specifically, canonical
correlation, bivariate correlation and principal components
analyses placed leiobunine species along a continuum of
“antagonistic specialization” in which sacculate species
dominated one end and non-sacculate species dominated the
other, with a broad region of overlap that precluded
classification into distinct sacculate/enticement and non-
sacculate/coercion groups. In contrast, results from the
current study differ in that pPCA (Fig. 4) appeared to recover
three species clusters rather than a single cluster or continuum.
PCI may represent the antagonistic potential associated with
dorsal and ventral penile flexure, with higher values toward
the left side of the plot. This axis separates the non-sacculate
calcar group, with high antagonistic potential, from all other
species, including the non-sacculate vittatum species-group.
Furthermore, PC2 appears to correspond to the antagonistic
potential associated with lateral flexure and separates the
vittatum species-group plus the sacculate L. veiitricosum from
the remaining sacculate taxa.

More specifically, PCA results from our current study
indicate that penes in the calcar group offer greater resistance
to dorsal and ventral flexure relative to body size icF,„ax) than
those of the other species examined. Further, relatively more
elastic energy is stored in the flexed penis for use in flexural
resistance during re-extension (R), and the restoring force
persists for a longer time (Tgo). This result is consistent with
initial predictions, but was not found in the vittatum group,
and the results were thus inconsistent with predictions about
non-sacculate penes generally. The species outside the calcar
group were separated primarily by factors associated with
lateral flexure, with sacculate species (except L. ventricosum)
having greater resistance to lateral flexion relative to body size
than the vittatumjventricosum cluster. The relatively greater
flexural compliance of the vittatumjventricosum cluster may be
attributed to the greater relative length (L) of penes and the
low second moment of area (/ ) of the penis shaft in the
vittatum group, which is narrow and circular in cross section
rather than wide and dorsoventrally compressed as in
sacculate species (see Eq. 1; Fig. 1). In contrast, lateral

bending in the penes of the vittatumjventricosum cluster
showed viscoelastic properties higher than those of most
sacculate species (i.e., with higher energy storage and longer
relaxation times).

Given the rather low sample size and high experimental
variance in some of our data, it is premature to conclude that
the three clusters recovered by PCA correspond to functional
groups or mating strategies. Furthermore, we modeled
displacement assuming penes were the equivalent of a beam,
although unlike standard beams, penes of these species are not
consistent in width across length. While we did not vary the
application site of forces, we could expect incremental changes
in second moment of area (/) as the site of displacement (L) is
repositioned. However, the combination of mechanical
properties within each cluster, together with other morpho¬
logical and behavioral observations, may offer new insights
and testable hypotheses. Specifically, we propose that the
calcar group still exemplifies an antagonistic mating system in
which male coercion and forced penetration have played an
important role in shaping genitalic morphology and biome¬
chanics. This conclusion is consistent with the robust
pedipalps in males that are used to clasp the female during
mating and a sclerotized latch mechanism at the pregenital
opening of females (Ingianni et al. 2011). The cluster of four
sacculate species may represent a mating system dominated by
male enticement of females. Members of this group have short
but dorsoventrally flexible penes with gift-bearing sacs. The
penes are poorly designed for imposing significant mechanical
forces and, in fact, are mounted on a fluid-filled turret
(haematodocha) during mating (Fig. 1) that would tend to
accommodate rather than resist female movements. In
addition, females of the species group have no special
elaboration of the pregenital opening that might resist forceful
penetration, although larger body size alone may be a
sufficient defense. Finally, we speculate that the genitalic
features of the mixed vittatumjventricosum cluster could reflect
specializations that allow females to coerce prolonged delivery
of nuptial gifts from males by entrapping or imposing possible
damage to the penis (as in Kuriwada & Kasuya 2011). Such a
system would combine elements of enticement by the male and
antagonism by the female and might thus account for the
intermediacy between enticement and antagonism revealed in
our previous study (Burns & Shultz 2015).

A few other lines of evidence are consistent with female
coercion of males via penis entrapment. Specifically, recent
behavioral analyses of mating in L. vittatum from Wisconsin
indicate that females may regulate the duration of mating
contact, in part, by resisting male attempts at separation
(Fowler-Finn et a!. 2014) and that males sometimes show signs
of physiological exhaustion after mating (K. Fowler-Finn,
pers. comm.). Interestingly, male L. vittatum from Massachu¬
setts have been found with no other damage than a broken
penis (Fig. 6A-C), which is consistent with persistent penile
entrapment by the female. Although we previously character¬
ized the sclerotized sterno-opercular mechanism in female L.
vittatum as a pregenital barrier (Burns et al. 2013; Burns &
Shultz 2015), the pregenital mechanism could also be used for
entrapping the penis. In contrast, our own video-based
observations of mating in L. ventricosum show no obvious
signs of female coercion during mating. However, Leiobunum

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JOURNAL OF ARACHNOLOGY

Figure 6. — Male Leiobumim vittatum found with broken penes, suggesting mechanical damage incurred during mating. A. Mating L. vittatiim
(female left, male right). Male clasps female coxa II (as in Fig. 1C) with damaged penis extended. Female palpates male haematodocha. Lateral
and dorsal drawings of L. vittatum penis are inset. B. Ventrolateral view of male L. vittatum with broken penis. C. Ventral view of same male. In
both examples, male penis is broken at the upturned glans portion. All pictures courtesy of Joe Warfel/Eighth Eye Photography, Massachusetts,
USA.

holtae McGhee, 1977 (not included in the present study), a
derived species that appears to have evolved from L.
ventricosum-Mke. ancestors (Burns et al. 2012), is morpholog¬
ically similar to L. vittatum in having a long, thin non-
sacculate penis. In addition, the female pregenital opening has
a large, horizontally-arranged opercular plate that can be
pressed against a large sternite dorsally. Again, this mecha¬
nism could be interpreted as either a barrier (which seems
substantially overdesigned given the apparent compliance of
the penis) or a penis trap. It is therefore possible that the
condition in L. holtae evolved from a less extreme form of
female coercion that may be practiced by L. ventricosiim.
Testing this speculative hypothesis will require new, intensive
studies of mating behavior and functional morphology.

ACKNOWLEDGMENTS

We are grateful to Mary E.L. Burns and the attendees of the
2013 American Arachnological Society held at East Tennessee

State University in Johnson City, TN, for their assistance in
specimen collection and to Joe Warfel for photographs of L.
vittatum. We additionally thank Dan Gruner, David Haw¬
thorne, Sara Bergbreiter, Priscila Chaverri, and four anony¬
mous reviewers for discussion of the manuscript. MB was
supported by the University of Maryland and GRF, DDIG,
and PRFB grants from the National Science Foundation. JWS
was supported by the Maryland Agricultural Experiment
Station. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of
the manuscript.

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2016. Journal of Arachnology 44:210-217

Mating behavior of the solitary neotropical harvestman Pachyloides thovellii (Arachnida: Opiliones)

Estefania Stanley', Gabriel Francescoli" and Carlos A. Toscano-Gadea': 'Laboratorio de Etologia, Ecologia y Evolucion,

Institute de Investigaciones Biologicas Clemente Estable, Avenida Italia 3318, C.P. 11.600. Montevideo, Uruguay. E-
mail: estefaniastanley@gmail.com; “ Seccion Etologia, Facultad de Ciencias, UdelaR, Igua 4225 esquina Mataojo, C.P.
11.400. Montevideo, Uruguay

Abstract. In order to study how sexual selection takes place during mating, it is necessary to have a clear knowledge of
the interactions that occur throughout mating and which morphological and behavioral traits are involved. Available
information about harvestman reproductive biology is mainly restricted to anecdotal field observations, most of them
lacking a detailed description and quantification of mating behavior. In this paper, we study the reproductive behavior of
the gonyleptid Pachyloides thorellii Holmberg, 1878 (Pachylinae) and provide quantitative and descriptive information
about its sexual behavior. We observed 15 matings, measured females and males, and analysed our behavioral data in the
context of individuals’ sizes. We observed conspicuous pre-copulatory, copulatory and post-copulatory courtship. We also
found that females have several strategies to reject males’ mating attempts. Like most gonyleptids, males and females of P.
thorellii mate in face-to-face position; however, we observed that both male and female clasp their chelicerae mutually.

This behavior has not previously been reported for the suborder Laniatores. The information obtained through this study
establishes the basis for further studies on this species’ reproductive biology and supports the suitability of this species as a
model to explore the importance of male copulatory courtship for female choice and sperm use.

Keywords: Sexual behavior, pre-copulatory courtship, chelicerae clasp, copulatory courtship

Opiliones is the third most diverse order within arachnids
and it is divided into four suborders: Cyphophthalmi, Eupnoi,
Dyspnoi, and Laniatores (Pinto-da-Rocha & Giribet 2007). In
general, harvestmen are omnivorous, nocturnal creatures,
showing high morphological and behavioral diversity (Savory
1938; Coddington et al. 1990; Adis & Harvey 2000). In
Cyphophthalmi, reproduction can be achieved asexually
through parthenogenesis or sexually through a spermatophore
transference (Tsurusaki 1986; Machado & Macias-Ordonez
2007). However, the most widespread sperm transfer mecha¬
nism in the order is direct copulation. Males possess an
eversible penis that is introduced into the females’ operculum
to achieve sperm transfer (Machado & Macias-Ordonez 2007).

Although studies regarding harvestman reproductive be¬
havior have significantly increased in the last decade, there are
still many gaps in our knowledge (Machado et al. 2015). Most
studies on harvestman sexual behavior are field studies on
Neotropical species of the suborder Laniatores, particularly of
Gonyleptidae, with some kind of parental care. Sexual
interactions in harvestmen mainly follow the scheme presented
by Machado et al. (2015) where the mating process is divided
into three stages: Pre-copulatory, Copulatory and Post-
copulatory. In each stage, different sources of selection may
shape both the morphology and the behaviors observed in
different species (Fowler-Finn et al. 2014). Therefore, having a
detailed description of the behaviors observed during these
stages is the first step towards understanding sexual selection
in each species.

During the Pre-copulatory stage, individuals generally
evaluate their partner through courtship and decide whether
to continue with further mating (Andersson 1994; Machado et
al. 2015). In harvestmen, this stage is brief and courtship
involves touching their partner’s body (by males, females or
both) using legs I and II. There is a small number of species for
which courtship has been described; in Chavesincola inex-
pectahilis Soares & Soares, 1946 and Pseudopucrolia sp.

(Gonyleptidae), the male taps the female’s genital opening
with legs II and gently touches her dorsum with legs I
(Nazareth & Machado 2009, 2010), while in Zygopachylus
albomarginis Chamberlin, 1925 (Manaosbiidae) it is the female
that initially taps the male carapace and legs, and if the male
returns the taps then copulation takes place (Mora 1990).

The Copulatory stage involves a closer evaluation of the
partner, intromission, and sperm transfer. Stimulation
through copulatory courtship is generally the most extended
way in which such evaluation occurs (Eberhard 1996;
Machado et al. 2015). Copulatory courtship is generally
performed by males and consists of touching or grazing the
legs and/or dorsum of the female. Males of Discocyrtus
pectinifemur Mello-Leitao, 1937 and C. inexpectabilis (Gony¬
leptidae) tap females’ bodies with legs II and I, respectively,
during intromission. In other gonyleptid species such as
Acutisoma longipes (Roewer, 1913), males intensively tap the
dorsum and hind legs of females (Machado & Macias-Ordonez
2007; Nazareth & Machado 2009).

Finally, mating is followed by a mate guarding period (Post-
copulatory stage). In this stage, the male remains with the
female and continues to court and/or stimulate her in order to
reduce sperm competition and increase reproductive success
(Simmons 2001; Machado et al. 2015). In some harvestman
species, males remain with the female, touching her from time
to time with legs I and II, until she lays one or more eggs. This
period can range from a few minutes (Z. albomarginis (Mora
1990); Iporongaia pustulosa Mello-Leitao, 1935 (Requena &
Machado 2014)) to several days {A. longipes (Machado &
Olivera 1998); C. inexpectabilis (Nazareth & Machado 2009)).

The goal of the present study was to describe the mating
behavior of the gonyleptid Pachyloides thorellii Holmberg,
1878. This is the first mating description for a solitary species
lacking any kind of post-oviposition parental care. We first
provide information about the behaviors that conform to the
Pre-copulatory, Copulatory and Post-copulatory stages with a

210

STANLEY ET AL.— MATING BEHAVIOR IN SOLITARY HARVESTMEN

211

Figure 1. — Measure taken for males (a) and females (b) of P. tlwrellii. dsl: dorsal scute length.

flow diagram. Second, we examine relative sizes of the sexes in
the context of the observed behaviors to evaluate whether
body size affects mating duration. And finally we provide, for
the first time, detailed photographs of the genitalia of this

species.

METHODS

Study organisms. — Pachyloides tlwrellii inhabits cryptozoic
environments in southern Uruguay, characterized by the
presence of leaf litter and pieces of tree bark. Males and
females have similar sizes and, contrary to what is observed in
other species of the family, they do not seem to be sexually
dimorphic (Pinto-da-Rocha & Giribet 2007; Giuliani 2008;
Willemart et al. 2008). Females start ovipositing approximate¬
ly a month after copulation and perform several ovipositions
in which each egg is placed in isolation under a rock or inside a
tree bark fissure (Stanley 2011).

Collection and maintenance in captivity. — We collected adult
individuals of P. thorellii at Marindia (Canelones, Uruguay;
34°46'S, 55°49'W), during January 2008 and 2009. The
individuals were taken to the Laboratorio de Etologia,
Ecologia y Evolucion (I.I.B.C.E., Montevideo, Uruguay) and
held individually in Petri dishes of 9 cm diameter and 1.5 cm
height, with sand as substrate and wet cotton wool as a water
supply. They were fed ad libitum once a week with apple and
cucumber pieces, cat food, and pieces of Tenebrio molitor
(Coleoptera) larvae. We maintained individuals under natural
photoperiod. The average temperature during breeding was
26.3 °C (± 1.8 SD, range = 17.5-37).

Behavioral observations. — The experiments were performed
in March 2008 and 2009, with a room temperature of 24 °C (±
1.2 SD, range = 20-30). Females were placed in Petri dishes of

14.5 cm diameter and 2.5 cm height (encounter arena), with
sand as substrate and wet cotton wool to maintain humidity,
24-48 h before the experiments, for acclimation and stress
reduction. Males were placed inside the arena immediately
before the beginning of each encounter. Males were carefully
picked up with forceps by one of their legs IV, to prevent the
release of chemical substances that could affect behavior.
Then they were gently placed approximately at 10 cm from the
female. Each trial lasted 30 minutes after the introduction of
the male or until the end of mating. If mating was not
observed, the same couple was tested again 24 h later.

All the observations took place under red light (placed 50
cm from the arena). We recorded each encounter with a Sony
Handycam video camera (DCR-SR40 Nightshot; Sony Corp.,
Tokyo, Japan) and took notes of all interactions. We analyzed
the video recordings with J Watcher computer program
(Version 0.9, Blumstein et al. 2000), to determine the
frequency and duration of each behavior. We used the
frequency of transition from one behavior to the other and
expressed them in percentages to construct the flow diagram.

Morphological features. — After the trials, individuals were
fixed and preserved in ethanol 95%. Both males and females
were photographed with a digital camera (Nikon Coolpix
5100) mounted on a stereoscopic microscope (Nikon SMZ-IO;
Nikon Corp., Tokyo, Japan). We took three separate pictures
per individual and using ImageTool software (Version 3.0;
Wilcox et al. 1995) software, we measured the length of dorsal
scute (Fig. 1). We analyzed the average of the measures taken
from the three pictures. Following Willemart et al. (2008), we
used dorsal scute length as a size reference to calculate an
index of size difference for each couple (dorsal scute length of
male divided by dorsal scute length of female). This index was
related to mating duration in a linear regression, transforming

212

JOURNAL OF ARACHNOLOGY

Table 1. — Description of F. thorellii mating behavior units observed. Rejection units were only performed by females.

Behavior

Category

Description

Touch with leg II

Pre-copulatory

Mutual touches with the tarsus of the second pair of legs. The individuals stand still on the
substrate touching dorsum, sides and/or first three pair of legs of the partner.

Touch with leg I and II

Pre-copulatory

Male intensively taps female’s dorsum with the tarsus of the first pair of legs, while Touches
with leg II continues.

Rush

Pre-copulatory

Male extends its pedipalps and quickly approaches the female.

Male over female

Copulatory

Male climbs over female’s dorsum and slides over it while he extends its pedipalps and
grazes female’s dorsum. Touch with leg I and 11 continues and this behavior ends when
the male locates himself in front of her in a face-to-face position.

Grabbing

Copulatory

Once in face-to-face position the male uses the claw of his pedipalps to grab the coxae of the
female’s first pair of legs. Both grab each other’s chelicerae (Fig. 5). Male continues Touch
with leg I and II.

Elevation

Copulatory

Using his fourth pair of legs as support, the male elevates the anterior part of his body
together v/ith the anterior part of the female’s body forming a 90° angle between them.

Copulatory courtship

Copulatory

Male puts the tarsus of both legs I in the dorsum of the female and slides them towards the
sides of her body. He maintains his second pair of legs in the air alternating between right
and left to touch the female on the sides and dorsum.

Pulls

Copulatory

Female pulls backwards from the male using her third and fourth pair of legs as support.

Leg II movements

Copulatory

Female moves the second pair of legs slowly.

Lowers body

Copulatory

Female bends her legs lowering her body.

Separation

Post-copulatory

Male retracts penis and releases female’s pedipalp and chelicerae as the female releases
male’s chelicerae.

Operculum Cleaning

Post-copulatory

Female scraps the operculum with the claws of her pedipalps and takes them to her mouth.
She repeats this several times.

Leg Cleaning

Post-copulatory

Individuals slide their legs through their chelicerae.

End

Post-copulatory

One or both individuals move far away from the other.

Rejection units

Run away

Pre-copulatory

Female quickly moves away from the male when he touches her.

Bending

Pre-copulatory

Female retracts legs I, II and pedipalps towards her body while elevating the abdomen and
lowering the cephalothorax to the substrate.

Kicking

Pre-copulatory

The female rapidly extends leg IV when male approaches.

each variable into logarithmic values. Voucher specimens were
deposited in the Coleccion Entomologica de la Facultad de
Ciencias, Montevideo, Uruguay.

Scanning electron microscope (Jeol JSM 5900LV) images
were used to visualize the structures present on the penis and
the ovipositor of P. thorelin individuals. Samples were critical
point dried and sputter coated with gold, and scanned at the
Servicio de Microscopi'a Electronica de Barrido y Micro-
analisis, Facultad de Ciencias, Montevideo, Uruguay.

Statistical analysis. — All statistical analysis was performed
using PAST (Version 1.18, Hammer et al. 2003). We selected P
= 0.05 as the limit for statistical significance. We tested the
behavioral and morphological data for normality and
homogeneity of variances using a Shapiro-Wiik test and
Levene test, respectively. If variables showed normality and
homogeneity of variances, we used the parametric Student’s /-
test; if any of these conditions was not met we used the non-
parametric Mann-Wliitney 17-test. We compared dorsal scute
length between males and females to determine whether there
was any size difference. Then we performed a multiple
regression test using size differences within couples as the
independent variable and the duration of the different stages
defined during mating as the dependent variable. Finally, we
performed a multiple logistic regression using presence and
absence of rejection as the categorical variable and mating
duration and size differences within couples as continuous
variables.

RESULTS

Sexual behavior. — The average duration of the analyzed
mating sequences was 690 seconds (± 198 SD, range = 486-
1182 s, n = 15). We defined 14 behaviors (see Table 1 for
description) and displayed the transitions from one behavior
to the other in a flow diagram (Fig. 2). The mating process was
divided into the three stages proposed by Machado et al.
(2015): Pre-copulatory, Copulatory and Post-copulatory.

Pre-copulatory behavior. — Interactions between male and
female began when one or both individuals waved their second
pair of legs simultaneously or alternately while they remained
still or walked around the arena. Contact was initiated by the
male in 87% (« = 13) of the cases, by directing his second pair
of legs and walking towards the female. In the remaining cases
(13%, n = 2), females initiated contact in a similar way as the
majority of males had. When they were close to each other,
both male and female touched each other’s dorsum and legs
with the tarsi of their first and second pair of legs. The Pre-
copulatory Behavior stage showed a mean duration of 18 s (±
12 SD, range = 2-48 s). This stage began with the behavior
Touch with leg I and I! and ended with the behavior Grabbing.
The male touched the female with leg I and rapidly climbed
over her dorsum {Rush). Once over the female, the male
touched the female dorsum both with legs I and pedipalps and
the touches with leg II accelerated. He immediately slid over
the female until reaching a face-to-face position {Male over

STANLEY ET AL.— MATING BEHAVIOR IN SOLITARY HARVESTMEN

213

TOUCH WITH

LEG II

RUSH

TOUCH WITH LEG!
AND II

LEG II

I

MOVEMENTS I

PULLS * — -

LOWERS BODY •

i SEPARATION 5^

TOUCH WITH
LEG II

A

Q!

LEG CLEANING

I TOUCH WITH I
I LEG II I

i

I

LEG CLEANING |

* OPERCULUM CLEANING '

■ Mana aaeaa

I END

Precopulatory

Behavior

Copulatory

Behavior

Postcopulatory

Behavior

71-100 %

57-70 % -¥■ 30-56 %

10-29% ---> 0-9%

Figure 2. — Flow diagram of P. tliorellii's mating. Squares with continuous lines contain behavioral units that were performed only by males.
Squares with dashed lines contain behavioral units that were performed only by females, and squares with dotted lines represent behavioral units
that were performed by both individuals. Arrow thickness represents different frequencies of transition between units and their value is expressed
in percentages.

female). During this behavior, the female was able to reject or
offer certain resistance to the male’s grabbing attempts.
Finally, once in front of the female, the male grabbed the
base of her first pair of legs with the claw of his pedipalp and
then they both grabbed each other’s chelicerae {Grabbing)
(Fig. 3).

Thirty-three percent (« = 5) of the females accepted male
courtship and mated without resistance; of the remaining 67%
{n — 10), 60% {n — 6) resisted male attempts to mate but
accepted later in the same trial, and 40% {n = 4) rejected males
but accepted them 24 h later without resistance. The behaviors

observed during female resistance and rejections were Run
away. Bending and Kicking (see definitions in Table 1). Neither
size difference nor mating duration were correlated with the
presence and absence of rejection (logistic regression: Size
difference: x" = 2.1, P = 0.15; Mating duration: x‘ = 0.67, P —
0.46).

Copulatory behavior. — The Copulatory Behavior stage had
a mean duration of 654 s (± 152 SD, range = 403-954 s), and
started with the behavior Elevation. After grabbing the female,
the male elevated the front part of his body together with the
female, reaching copulatory position {Elevation; see Table 1

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JOURNAL OF ARACHNOLOGY

Figure 3. — Ventral detail of mutual cheliceral grabbing during copulation in P. tlwrellii. Black arrows show the sites where male chelicerae
grabbed female chelicerae and the white arrow shows female site of grabbing, r Ch: Right chelicerae; 1 Ch: Left chelicerae; p: penis.

for further detail). Simultaneously, the male raised his third
pair of legs until he got them on top of the female’s second
pair of legs. In this position, while performing Copiilatory
courtship, the male inserted his penis into the female’s
gonopore and did not withdraw it until the mating ended.
At this point, we observed a decrease in the intensity of the
male touches with legs I, which then remained constant until
the couple separated. Females remained almost immobile
during most of this stage, except at the beginning and near the

Figure 4. — SEM image of female ovipositor of P. tliorellii. White
arrow shows ovipositor opening.

end of the stage when they tried to pull away from the males’
grasp. Males maintained their grasp and continued the
copulatory courtship. Before Separation, females slowly
started moving legs II {Leg II movements) and lowered their
bodies by flexing their fourth pair of legs, which obliged males
to withdraw the penis and release the female chelicerae and
legs (/? =11). Males sometimes finished mating by freeing the
female in absence of any of the mentioned female displays (« =

4).

Post-copulatory behavior. — The Post-copulatory Behavior
stage started immediately after the couple separated and had a
mean duration of 63 s (± 75 SD, range = 12-258 s). During
that stage, both male and female stayed close to each other
(approximately 1 cm away), touching each other’s dorsum and
legs with legs II. At the same time, each of them performed
Leg cleaning, and during this stage all females were observed
carrying out Operculum cleaning.

We did not observe a statistically significant relationship
between the size ratio of the members of each couple and
mating duration or duration of any of the stages (multiple
regression: r = 0.63, P — 0.23).

Genital apparatus. — The female’s ovipositor has four lobes,
each carrying three long setae that point towards the center of
the ovipositor, covering the entrance (Fig. 4). The male’s penis
has several ornamentations on its pars distalis (Fig. 5). We
observed on both sides, two groups of three sensilla, one at the
distal end and the other on the base (Figure 5a). Between those
groups of sensilla, there is a spiny area that covers the edge of
the distal part of the penis (Figure 5b). In the glans, we observe
both the ventral process and the stylus (Figure 5). In a closer

STANLEY ET AL.— MATING BEHAVIOR IN SOLITARY HARVESTMEN

215

Figure 5. — SEM image of male penis of P. thorellii. a, b: detail of
sensilla and spines in the pars distalis; c: ventral process; S: Stylus.

look, the ventral process shows several processes on its sides
(Figure 5c).

DISCUSSION

We found that P. thorellii mating behavior is one of the
longest found so far in the suborder Laniatores, clearly
showing the three stages — Pre-copulatory, Copulatory and
Post-copulatory — proposed by Machado et al. (2015). Males
touched females with legs I and II from the beginning to the
end of the interactions and females were able to resist and/or
reject males. We observed that females cooperate with the
male during copulation, through cheliceral holding and are
able to end mating by lowering their bodies, forcing males to
withdraw the penis and release their chelicerae. We found no
relationship between the size ratio of each couple and either
the probability of rejection or the duration of any stage or the
whole mating.

As observed in other harvestman species, individuals of P.
thorellii seem to identify conspecifics and differentiate males
from females after touching them (Willemart et al. 2006;
Fowler-Finn et al. 2014). Normally, individuals use their
second pair of legs to orient themselves to nearby objects;
these legs are not used for locomotion and they are constantly
moving in a similar way to insect antennae (Machado et al.
2007). As it was observed, prior to contact, individuals direct
their second pair of legs towards their conspecific, approach
them, and finally contact takes place. Pre-copulatory court¬
ship in P. thorellii is similar to that reported for other
Laniatores (see Table 12.1 in Machado & Maci'as-Ordonez
2007). Particularly in Gonyleptidae, courtship is short and
initiates when one individual touches the other. Once the male
detects the female, he tries to grab her and mate. However,
females can accept mating or resist it. Resistance behaviors in
P. thorellii resemble those observed in other members of the
family (Gnaspini 1995; Elpino-Campos et al. 2001; Machado
& Macias-Ordonez 2007; Willemart et al. 2008; Nazareth &
Machado 2009, 2010; Requena & Machado 2014). There was
no relationship between rejected males and size ratio within
couple members, and rejected males reinitiated courtship

several times by touching the female with legs I and II; this
behavior may have the function of increasing the probability
of female mating acceptance as suggested by Willemart et al.
(2006). These facts together with the pre-copulatory behaviors
reported by Machado & Macias-Ordonez (2007) in other
species, suggest that Laniatores may rely more on courtship
than on coercive behaviors as observed in Eupnoi (Machado
& Macias-Ordonez 2007). The time individuals remain in pre-
copulatory courtship represents a window for evaluation of
the potential partner and the length of this stage may be
correlated with the capacity of the female to control sperm
afterwards. It would be necessary to compare courtship
duration and the frequency and duration of the behaviors
observed during courtship with the number of offspring
obtained from virgin females to assess the function of such
behaviors.

As mentioned before, the copulatory behavior in P. thorellii
is one of the longest found so far for the suborder (Matthiesen
1983; Gnaspini 1995; Machado & Oliveira 1998; Elpino-
Campos et al. 2001; Machado & Maci'as-Ordonez 2007;
Nazareth & Machado 2009, 2010; Buzatto et al. 2011) and
within species of other suborders (Eupnoi: Macias-Ordonez
1997, 2000; Willemart et al. 2006; Dyspnoi: Pabst 1953;
Martens 1969), and was characterized by tactile courtship
(touches with legs I and II) like many other harvestmen
(Machado & Macias-Orddnez 2007). Copulatory position
(face-to-face and forming a 90°angle) is similar to what is
observed in other harvestmen; the mutual chelicerae holding
has not been reported for the suborder Laniatores. Until now
it has only been mentioned that females of Zygopachylus
alhomarginis extend their chelicerae and pedipalps and grab
males by their cephalothorax to bring them closer, but there
has been no mention of male cheliceral holding (Mora 1990).
Male cheliceral holding was reported for two species of
Trogulus Latreille, 1802 (Dyspnoi), in which a male grabs a
female’s body with legs I and II and her chelicerae with his
chelicerae (Pabst 1953). In females, cheliceral holding was
observed in a few species of the genus Ischyropsalis C.L. Koch,
1839 (Dyspnoi) (see Table 12.1 in Machado & Macias-
Ordonez 2007). Females grab the base of male’s chelicerae
with her chelicerae, bringing them close to her mouth and
maintaining that position until mating ends (Martens 1969).
The fact that females actively participate in holding and
maintaining mating position suggests they have a greater
control of mating duration. In fact, it was observed in these
species and both P. thorellii and other gonyleptids that females
are able to end mating (Pabst 1953; Martens 1969; Nazareth &
Machado 2009). P. thorellii females lower their bodies, forcing
males to withdraw the penis and release the chelicerae. The
fact that the male and female hold each other’s chelicerae
could enable a more firm and stable position during mating
and such stability could explain the longer matings observed.
Males could use part of the mating time for several purposes:
to remove sperm from previous matings (Thomas & Zeh 1984;
Eberhard 1996; Birkhead & Moller 1998), to transfer
accessory substances (nutritious or inhibitory of future
matings) (Parker 1970; Simmons 2001) and/or to stimulate
females for longer periods (Eberhard 1998). We found that the
penis in P. thorellii has spines, sensilla and other projections,
such as the ventral process, that could promote penetration of

216

JOURNAL OF ARACHNOLOGY

the penis, remove sperm, and/or stimulate the ovipositor
during mating (Macias-Ordonez et al. 2010). However, the
function of these ornaments in this and other harvestman
species is still unknown.

After mating, males remain near the female touching her
with legs I and II; this fact could indicate the presence of Post-
copulatory courtship. A similar behavior was observed in
other Gonyleptidae species: in Chavesincola inexpectabilis,
females oviposit immediately after mating (Nazareth &
Machado 2009) and in Goniosoma spelaeiim (Mello-Leitao,
1933) and A. longipes, males stay close and approach to
reinseminate the female (Gnaspini 1995; Machado & Olivera
1998). In P. thorellii, females oviposit between 30 and 40 days
after mating (Stanley & Toscano-Gadea, unpublished data).
Males could stay with them for long periods during which they
could reinseminate them and protect the female from other
males. Even though in this study we separated the couple after
they moved away from each other, Stanley (2012) observed
that the same couple was able to mate up to six times with a
separation of 24-48 h between matings. She also observed that
males may fight with one another immediately after matings.
These facts could imply that both post-copulatory courtship
and mate guarding could be occurring in P. thorellii.

Females perform Operculum cleaning during most of the
Post-copulatory stage. Due to the fact that this behavior is
observed immediately after mating it is possible that females
are removing and eating sperm (Pinto-da-Rocha & Giribet
2007; Macias-Ordohez et al. 2010). Females of the fly
Prochyliza xanthosoma prefer males that transfer great
amount of sperm, because after mating they expel part of
the ejaculate and feed from it (Bonduriansky & Rowe 2003;
Bonduriansky et al. 2005). If females of P. thorellii are in fact
expelling sperm, this would be one more feature in favor of
female control over sperm in this species. Future studies
should identify and quantify the substance that the female
takes to her mouth and determine if the observed behavior is
related with sperm dumping or with other substances with
nutritional value being transferred to females as nuptial gifts
(Eberhard 1998; Arqnvist & Nilsson 2000; Bonduriansky &
Rowe 2003; Elgar et al. 2003; Bonduriansky et al. 2005, Peretti
& Eberhard 2009).

P. thorellii seems to be a promising model in which to study
the mechanisms responsible for sexual selection. This work
provides the framework required for future sexual behavior
research in the species. Studies involving courtship influence
on mating duration and sperm use in female reproductive
tract, as well as the causes promoting male fights, are already
taking place.

ACKNOWLEDGMENTS

We are grateful to Fernando G. Costa for his help in
capturing the individuals and for useful discussions of the
original idea and results. We also thank Fernando Perez-Miles
and Miguel Simo from Facultad de Ciencias, for letting us use
their laboratory equipment for measuring the individuals. We
also would like to thank Anita Aisenberg and J. Henderson
for improving the first version of this study and revising the
English. And finally we are grateful to two anonymous
reviewers that constructively criticized and significantly
improved this article.

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Manuscript received 22 April 2015, revised 27 January 2016.

2016. Journal of Arachnology 44:218-226

The smallest known solifuge: Vempironiella aguilari^ new genus and species of sun-spider (Solifugae:

Mummuciidae) from the coastal desert of Peru

Ricardo Botero-Trujillo: Division Aracnologia, Museo Argentine de Ciencias Naturales “Bernardino Rivadavia”,

Avenida Angel Gallardo 470, CP: 1405DJR, C.A.B.A., Buenos Aires, Argentina. E-mails: rbt@macn.gov.ar &
pachyurus@yahoo.com

Abstract. A new genus and species in the South American sun-spider family Mummuciidae, Vempironiella aguilari gen.
nov., sp. nov., is herein described from a series of specimens from the coastal desert of Punta Hermosa, Peru. Vempironiella
can be readily distinguished from all other known mummuciid genera, by the absence of the cheliceral movable finger MM
tooth and the presence of a diastema between the RFA and RFP teeth on the fixed finger. With this description, the
number of valid species of mummuciids is 19, three of which have been described from Peru. Males of V. aguilari measure
3.90-5.85 mm in total body length making it the smallest solifuge species known to date. The cheliceral morphology of V.
aguilari is discussed and some hypotheses on the function of morphology are provided.

Keywords: Solifuges, Punta Hermosa, Peruvian coastal desert.

The South American sun-spider family Mummuciidae
Roewer, 1934 encompasses small to moderate-sized species
of solifuges. Traditionally, eight genera have been included in
the family, namely Mummucia Simon, 1879, Gaiicha Mello-
Leitao, 1924, Metacleobis Roewer, 1934, Mianinucina Roewer,
1934, Mimmnicipes Roewer, 1934, Gauchella Mello-Leitao,
1937, Cordohulgida Mello-Leitao, 1938 and Uspallata Mello-
Leitao, 1938 (Harvey 2003; Bird et al. 2015). Although the
catalogue of Harvey (2003) listed ten genera in the family, two
of them, i.e., Miimmuciona Roewer, 1934 and Sedna Muma,
1971, had been transferred to Ammotrechidae by Maury
(1982, 1987). Until recently, 20 species were recognized for
Mummuciidae (Bird et al. 2015); however, two were discov¬
ered to belong to Ammotrechidae (Botero-Trujillo & luri
2015).

Mummuciid species have been described mostly from Brazil
and Argentina, with six and four species respectively, followed
by Paraguay, Chile and Peru, each with two species, and
Bolivia and Ecuador, with a single species each (Maury 1998;
Xavier &. Rocha 2001; Martins et al. 2004; Rocha & Carvalho
2006; Carvalho et al. 2010; Gonzalez-Reyes & Corronca 2013;
Botero-Trujillo & luri 2015). These numbers are not accurate
estimators of species diversity, however, and enormous areas
across the geographical distribution of the family remain
unsampled (e.g., Maury 1998: fig. 4). As summarized by
Harvey (2003), a few species have been allegedly recorded for
more than one country [e.g., Mummucia variegata (Gervais,
1849)]. Whilst some of those correspond to rather old records
[e.g., Simon’s mention of M. variegata for Peru (Simon 1879;
152)], determining the actual geographic range of species
requires additional dedicated fieldwork and comprehensive
efforts to delimit species.

Thus far the recognition of mummuciid genera is a
challenging task, for these are poorly defined (Maury 1998;
Botero-Trujillo 2014), rendering the validity of some ques¬
tionable. Because of this, it is often easier to identify new
species than it is to place them into a genus. As a consequence,
some authors of newly-named species have opted for placing
them into the type genus, Mummucia, as a conservative
approach without taxonomic support (Xavier & Rocha 2001;

Martins et al. 2004; Rocha & Carvalho 2006; Carvalho et al.
2010). Two genera with more than one species, Mummucia and
Mummucina, have neither been revised nor had their
monophyly yet demonstrated. Meanwhile, the other six genera
remain monotypic. Due to this taxonomic confusion, only the
study of the type species of the different genera can shed light
on where a new species should be placed.

In the present contribution, Vempironiella gen. nov. is
created to accommodate a remarkable new species, Vempir¬
oniella aguilari sp. nov., from the coastal desert of the district
of Punta Hermosa, Peru. After direct comparison with the
type species of the eight former genera of Mummuciidae, the
new species proved to exhibit a unique morphology that does
not fit into any of the currently recognized genera, all of which
are more similar to one another than any is to the new genus.
Vempironiella aguilari is the smallest known solifuge, with
males measuring 3.90-5.85 mm in total body length, with the
second smallest being the southern African melanoblossiid
Lawrencega minuta Wharton, 1981 whose males measure 5-8
mm (Bird et al. 2015).

Vempironiella aguilari represents only the third mummuciid
described from Peru, along with Mummucina exlineae Mello-
Leitao, 1943 and Mummucina masculina Lawrence, 1954, and
brings the known diversity of the family to 19 species.

METHODS

Terminology used for referring to cheliceral teeth and other
cheliceral structures follows Bird et al. (2015). The ierm fixed
finger retrofondal diastema (frfd) is here introduced to refer to
a toothless diastema present between the RFP and RFA teeth.
Abbreviations rlf)^ are here used to identify a set of four
individual principal retrolateral finger setae, as defined by Bird
et al. (2015: 173). These r If setae, which are common to all
mummuciid species and are present in at least some other
families (e.g., Daesiidae Kraepelin, 1899; see Bird et al. 2015:
pi. 145), differ in position across mummuciid taxa (i.e., with
respect to particular teeth) and bear some relevant taxonomic
usefulness. Identification of individual teeth used the criteria
of Bird et al. (2015: 83) for primary homology assessment of

218

BOTERO-TRUJILLO— THE SMALLEST KNOWN SOLIFUGE

dentition. Leg segmentation terminology follows Shultz
(1989). In line with Bird & Wharton (2015), the terms basi-
and telotarsus are used for the pedipalp segments traditionally
refered to as metatarsus and tarsus. The term ‘spiniform setae’
(equivalent to spine-like setae) refers to rigid, socketed
macrosetae and is preferred over ‘spines’ (broadly used before
by various authors), following recent works on solifuges (e.g.,
Botero-Trujillo 2014; Bird & Wharton 2015; Botero-Trujillo &
luri 2015). The formula used to describe the pattern of
spiniform setae on telotarsi of legs follows luri et al. (2014),
where a dash line (-) stands for incomplete segmentation and a
slash (/) for complete segmentation. Pedipalp setae terminol¬
ogy follows Cushing & Casto (2012).

The ‘row of rigid hairs along the posterior margin of the
post-spiracular sternite IP (4‘*’ post-genital sternite) is the
same structure referred to as ‘specialized setae’ by Botero-
Trujillo (2014) and as ‘comb of rigid hairs’ by Botero-Trujillo
& luri (2015). Maury (1984) referred to it as “ctenidia in the
form of a comb of rigid hairs”. Here the term ‘ctenidia’ is used
only for the long, single-tipped (non-bifid) and flexible seta-
like structures that, in the new species, are present on the 3"^^*
and 4'*^ post-genital sternites. Unlike the rigid hairs which are
arranged in a row, ctenidia are irregularly distributed in the
sternites (Figs. 22, 23).

The “variation” section deals with observations performed
on the cheliceral dental pattern formula and teeth (FSD,
FSM) counts (no other significant variation was observed);
dental pattern formula follows that proposed by Bird et al.
(2015: 67).

Specimens were examined with Leica M165 C and Leica
S8AP0 stereomicroscopes. Photographs were taken with a
Leica DFC 290 digital camera mounted on the Leica M165 C
stereomicroscope and the extended focal range images
composed with Helicon Focus 6.2.2 Pro software (http://
www.heliconsoft.com/heliconsoft-products/helicon-focus/). Il¬
lustrations of the chelicerae were prepared with CorelDRAW
12 by superimposing vectors on previously obtained micro¬
graphs. Images were edited with Adobe Photoshop CS3 (10.0).
Measurements, in millimeters, were obtained using an ocular
micrometer fitted to a Leitz Wetzlar stereomicroscope.

Some chelicerae were manipulated, after dissection, to allow
full display of the dentition. Fine forceps were carefully placed
between the finger mucra, as close as possible to the bases of
FD and MSM teeth. The tips of the forceps were gradually
separated by carefully inserting between them the tip of
another set of forceps, while controlling the first forceps such
that it opened only as desired, i.e., to prevent an abrupt
opening that could damage the fingers. Chelicerae were
opened enough to expose all the teeth, or until the muscle
keeping the movable finger closed had detached. For scanning
electron microscope (SEM) preparations, specimens were
dissected, cleaned with a fine-bristle paintbrush followed by
ultrasonication, dehydrated via 80% - 87% - 96% - 100%
ethanol series, fixed to aluminum stubs, and gold-palladium
coated in a VG Scientific SC 7620 mini sputter-coater. SEM
micrographs were taken under high vacuum with a Philips FEI
XL30 TMP.

Material examined. — Specimens used in the present work
belong to the following institutions: American Museum of
Natural History, New York, U.S.A. (AMNH); Museo

219

Argentino de Ciencias Naturales “Bernardino Rivadavia”,
Buenos Aires, Argentina (MACN); Museo de Historia
Natural, Universidad Nacional Mayor de San Marcos, Lima,
Peru (MUSM); Museu de Ciencias Naturais, Funda^ao
Zoobotanica do Rio Grande do Sul, Porto Alegre, Brazil
(MCN); Museu Nacional do Rio de Janeiro, Rio de Janeiro,
Brazil (MNRJ); Museum National d’Histoire Naturelle, Paris,
France (MNHN); Senckenberg Forschungsinstitut und Natur-
Museum, Frankfurt, Germany (SMF).

Specimens of 17 of the other 18 species currently placed in
Mummuciidae (all but Mummucia dubia Badcock, 1932) were
examined, including type specimens of most of them. A list of
material examined belonging to the type species of all other
genera is provided below.

Cordohulgida bnichi Mello-Leitao, 1938: female holotype
(MNRJ): Labels verbatim: “Cordobuigida bnichi M. L. / Aha
Gracia / Bruch leg. j 58160". “520 a-D / Leg.: Dr. C. Bruch /
Aha Gracia (Cord.) / 14.xii.l934" . ARGENTINA: Cordoba,
Alta Gracia, La Granja, under rocks, i.l939, C. Bruch, 2
juveniles (MACN-Ar); Cordoba, Alta Gracia, La Granja,
i.l938, C. Bruch, 1 male, 1 female, 1 juvenile (MACN-Ar).

Gaucha fasciata Mello-Leitao, 1924: male holotype (MNRJ,
currently at MCN): Label verbatim: “Gaucha fasciata M. L. /
Porto Alegre / Gliesch j 42682" . “Laboratorio de Zoologia /
Solifugosj Solpugidae / Gaucha fasciata / M. Lei t do". 1 male, 2
female paratypes (MNRJ; currently at MCN): Label verbatim:
“Laboratorio de Zoologia / Solifugosj Solpugidae / Gaucha
fasciata / M. Leitdo". BRAZIL: Rio Grande do Sul, Porto
Alegre, Jardim Botanico, granito, 46 m elev., 30‘’03'13.11" S
51°10'35.18" W, 19.xi.2012'^ 3 males, 1 female (MCN-Sol-020);
03.xii.2012, 2 males, 2 juveniles (MCN-Sol-021); xii.2014, R.
Ott & R. Botero Trujillo, 1 male (96% ethanol, MCN).

Gaiichella stoeckeli (Roewer, 1934): 2 males, 1 female
syntypes (SMF): Label verbatim: “Araclm. Coll. Roewer -
Lfd. No. 2984 j Solifuga: / No. 73 / Gaucha stoeckeli n. sp. /
2(3, 19 j Bolivia, La Paz / Typus / Roewer det. 1933".

Metacleobis fulvipes Roewer, 1934: male holotype (SMF):
Label verbatim: “Araclm. Coll. Roewer - Lfd. No. 4556 j
Solifuga: j No. 365 j Metacleobis fulvipes j 16 j u. g. u. sp. j
Brasil: Mat to Grosso, Cuyabo j Typus / Roewer det. 1933" .
“4756".

Mummucia variegata (Gervais, 1849): 3 female syntypes
(MNHN): Labels verbatim: “17849 j Mummucia varegata [sic]
/ Chili j Gervais / Vid. Kraep." “59.” CHILE: V Region,
Valparaiso, Puente Las Bayicas, 24 km E of Algarrobo,
09. xi. 1988, E. Maury, 15 males, 1 female, 2 juveniles (MACN-
Ar).

Mummucina titschacki Roewer, 1934: ECUADOR: Chim¬
borazo, Road 35th, 3 km N of Riobamba, 1 km before San
Andres, 100 m from “Cantera (quarry) San Andres”, 3000 m
elev., 01°35'57" S 78°41'50" W, manual capture and pitfall
traps (12:00 to 15:00 hs), 22-23. iii. 20 14; R. Botero Trujillo, 31
males, 3 females, 4 juveniles (MACN-Ar).

Mummucipes paraguayensis Roewer, 1 934: 2 males, 1 female
syntypes (SMF): Label verbatim: “Araclm. Coll. Roewer - Lfd.
No. 4753 / Solifuga: j No. 362 / Mummucipes paraguayensis /
26,19 i n. g. n. sp. j Paraguay: Asuncion / Typus / Roewer det.
1933". “4753".

Uspallata pulchra Mello-Leitao, 1938: ARGENTINA:
Mendoza, Las Heras, 10 km N of Uspallata, 2014 m elev.,

220

JOURNAL OF ARACHNOLOGY

Figures 1~4. — Vempiroiiiella aguikiri gen. nov., sp. nov. 1-2. Male holotype (MACN-Ar-35453); 1. Habitus, dorsal view; 2. Prosoma, dorsal
view. 3-4. Adult female paratype (MACN-Ar-35454); 3. Habitus, dorsal view; 4. Prosoma, dorsal view. Scale bars: 1 mm (Figs. 1, 3); 0.3 mm
(Fig. 2); 0.5 mm (Fig. 4).

32°32'30.8" S 69°18'22.2" W, manual capture, 22.i.2014; H.A.
luri, R. Botero Trujillo, A. A. Ojanguren Affilastro, 1 female
(96% ethanol, MACN-Ar).

NOTE: Mello-Leitao (1938) only reported one type
specimen for C. bnichi which was thus far considered lost
(Kury & Nogueira 1999). One specimen, accompanied by a
label in Mello-Leitao’s handwriting and with collection data
matching that reported in the original description, was
recently found in the collection of the MACN. Although the
specimen is not accompanied by any label identifying it as a
type, the morphology and wear pattern of its chelicerae (which
is very particular) allowed the author to determine that it is,
without a doubt, the same specimen illustrated by Mello-
Leitao (1938: figs. 72, 73). Therefore, this specimen is
considered to be the holotype of C. hriichi.

TAXONOMY

Family Mummuciidae Roewer, 1934
Vempiroiiiella gen. nov.

Type species. — Veiupironiella aguikiri sp. nov.

Etymology. — The generic name is an arbitrary combination
of letters that resembles the word “vampire” inspired by the
shape of the cheliceral teeth which are reminiscent of the fangs
of vampires. Feminine in gender.

Diagnosis. — A member of the family Mummuciidae because
of having a three-dark-band pattern on the opisthosomal
dorsal surface (Figs. 1, 3), a row of rigid hairs along the
posterior margin of post-spiracular sternite II (4**’ post-genital
sternite), lacking spiniform setae on pedipalps (Fig. 16), and
the male flagellum of the composite type, retrolaterally
compressed with ipsilateral opening, and immovably attached
to the cheliceral fixed finger (Figs. 12, 13) (Maury 1984; Bird et
al. 2015; Botero-Trujillo & luri 2015). The new genus differs

from all other genera in the family in various aspects, mostly
of its cheliceral morphology: i) Fixed finger with retrofondal
diastema (frfd) between the RFA tooth and the RFP tooth
(intermediate retrofondal teeth absent) (Figs. 5, 6). ii)
Movable finger with MP and MSM teeth only, MM tooth
absent (Figs. 7, 11). Hi) Movable finger MSM tooth markedly
pronounced and columnar (Figs. 7-11). iv) Movable finger of
female aculeus-like, with very long and slender mucron, and
teeth located in a noticeably basal position on the finger (Figs.
5, 7). v) Movable finger of female with mucron cylindrical,
retrolateral carina obsolete (represented by shallow granules
on the base of finger and edge carina on the apex), and gnathal
edge carina identified only by a sclerotized line along the
mucron dorsal margin (Figs. 5, 7). vi) Opisthosomal lateral
pleural membranes, sub-dorsal dark bands with white marks
surrounding the insertion socket of some setae, instead of
similar but black marks on the sub-ventral whitish bands of
the membrane.

Comparisons. — All other eight genera currently recognized
in the family, most importantly their type species, differ
substantially from the above description by: i) Cheliceral fixed
finger retrofondal teeth series is uninterrupted, without
diastema, ii) Movable finger with MP, MSM and MM teeth
present. Hi) Movable finger MSM tooth small to moderately
pronounced and sub-triangular, iv) Movable finger mucron of
female moderately long and more robust than that of
VempiroiHella, with teeth located in a sub-medial position on
the finger, v) Movable finger of female with retrolateral carina
moderately to densely granular and gnathal edge carina
identified by pronounced angle formed by adjacent surfaces,
which gives the appearance of a cutting edge along the
mucron. vi) Opisthosomal lateral pleural membranes, sub-
ventral whitish bands with black marks, and not the other way
around, except for Mummucina in strict o sensu (i.e., M.

BOTERO-TRUJILLO— THE SMALLEST KNOWN SOLIFUGE

221

Figures 5-6. — Vempironiella agidlari gen. nov., sp. nov. Schematic representation of the cheliceral morphology in retrolateral aspect. 5.
Female and juvenile morphology; 6; Male morphology. Abbreviations; RFP, RFA, FP, FM, FD, particular fixed finger teeth for reference; MP,
MSM, movable finger teeth; r{fi_4, set of four principal retrolateral finger setae; Fgl, flagellum; sa, setose areas; MRLC, movable finger
retrolateral edge carina; FRLC, fixed finger retrolateral edge carina; frfd, fixed finger retrofondal diastema. Scale bars: 0.25 mm (Fig. 5); 0.1 mm
(Fig. 6).

titschacki) which shares the pattern described above for
Vempironiella.

Vempironiella aguilari sp. nov.

Figures 1-23; Table 1

Mummucia variegata (misidentification): Aguilar 1977: 91 [as
Mumnnicia variegata (?)”].

Type material. — Holotype male: PERU: Lima, Lima, Punta
Hermosa, “40 km S of Lima”, 03. xi. 1974, P. Aguilar (MACN-
Ar-35453). Paratypes: PERU: same data of holotype, 12
males, 2 females, 2 juveniles (MACN-Ar-35454), 1 male, 1
juvenile (AMNH), 1 male, 1 juvenile (MUSM). All specimens
preserved in 80% ethanol.

Etymology. — The species is named after the prominent
Peruvian Biologist, Dr. Pedro G. Aguilar Fernandez (1926-
2013). Doctor Aguilar Fernandez was the collector of the type
material and, in one of his 1977’s publications, presented some
information about the natural history of this species.

Diagnosis. — As for genus.

Description of male. — Meristic data in Table 1.

Color: (Figs. 1, 2). On 80% ethanol-preserved specimens.
General coloration yellow with iridescent white areas.
Propeltidium with yellow central area, longer than wide, and
two yellow areas on posterior margin, all forming an arrow¬
like design that is surrounded by white pigment; ocular
tubercle yellowish-brown, except for the border of the eyes
which is black. Chelicerae manus yellow, ornamented with
longitudinal white bands which fuse together on the distalmost
region of the setose area; fingers yellow, translucent. Meso-,
metapeltidium, and dorsal surface of opisthosoma with a
three-dark-band design typical of the family: tergites with
median, longitudinal light brown band, and paired lateral
white bands; lateral pleural membranes with sub-dorsal dark-
brown and sub-ventral white bands; dark bands of opistho-

somal pleural membrane with white marks surrounding the
insertion socket of some setae; sternites immaculately irides¬
cent white. Ventral aspect of prosoma, legs and pedipalps
uniformly yellow, with hint of iridescence; sternum lighter
than coxae. Malleoli yellow, translucent.

Prosoma: (Fig. 2). Propeltidium wider than long; with
bifurcated setae of variable size, the longest setae arranged in a
bilaterally symmetrical distribution on propeltidium; anterior
margin procurved, with ocular tubercle elevated; complete and
shallow median longitudinal furrow present; anterolateral
propeltidial lobes separated from the propeltidium principal
shield by incomplete lateral groove. Meso- and metapeltidium
wider than long, with bifurcated setae of variable size. Coxae
densely covered with bifurcated setae; some of which are
longer and exhibit a bilateral symmetrical distribution, and
one or two other long single-tipped setae present at least on
coxae HI. Sternum glabrous.

Clielicera-dentition and processes: (Figs. 6, 11-15). Fixed
finger with median teeth series comprising all primary teeth,
i.e., FP, FM, FD, markedly pronounced and columnar;
secondary teeth arranged in two (FSM and FSD) categories,
similar to principal teeth but slightly shorter; retrofondal teeth
series comprising RFA, RFP and RFSP teeth only, interrupt¬
ed by retrofondal diastema (frfd) between the RFA and RFP
teeth; RFA and RFP larger than RFSP, both similar to teeth
of median series; profondal teeth series with three teeth (PFSP,
PFP, PFM); PFM tooth visible in retrolateral aspect through
the frfd. Movable finger with median teeth series comprising
only two teeth, markedly pronounced and erect MP, and
pronounced and columnar MSM, arranged as MP>>MSM;
teeth placed in a sub-basal, rather than medial, position on the
finger. Movable finger without any trace of MM tooth and
without subproximal (MSP) or subterminal (MST) teeth;
retrolateral carina incomplete and obsolete, consisting of one
or two low granules basal to MP tooth, and keel-like section

JOURNAL OF ARACHNOLOGY

222

Figures 7-10. — Vempiroiiiella agidkiri gen. nov., sp. nov. SEM images. Juvenile (presumably subadult) and adult female paratypes (MACN-
Ar-35454). 7. Juvenile, right chelicera, retrolateral aspect; 8. Adult female, left chelicera, retrolateral aspect; 9. Juvenile, right chelicera, prolateral
aspect; 10. Juvenile, right chelicera, apex of movable finger mucron, retrolateral aspect (gnathal edge carina indicated by white arrows;
retrolateral edge carina indicated by black arrow). Scale bars: 0.25 mm (Figs. 7-9); 50pm (Fig. 10).

(i.e., retrolateral edge carina) evident only on the apical region
of the mucron. Closure of FP tooth distal to MP. Fixed finger
with prodorsal carina complete (along the entire length of the
asetose area), starting approximately at level of the attachment
point of the flagellum and of RFA tooth, predominantly
straight, without angular dorsal crest; proventral carina long,
starting approximately at level of FM tooth and present in the
entire mucron area; mucron long and slender, ventral margin
gently curved, without subterminal flange (STF), apex (FT
tooth) ventrally curved. Movable finger mucron very long and
slender, with obsolete gnathal edge carina, identified by subtle
angle formed by adjacent surfaces.

Chelicera-setose areas and strididatory plate: (Figs. 6, 1 1-
15). Retrolateral and dorsal surfaces with abundant bifurcated
retrolateral manus {rim) and retrolateral finger (;7/) setae, of
different sizes; some of these setae are arranged in a bilaterally
symmetrical pattern, including four evident principal retro¬
lateral finger (jyrincipal rlf) setae, i.e., /7//_/, with distribution in
the fixed finger as shown in Fig. 6. Prolateral surface with
array of setal types, as follows: proventral distal {pvd) setae
consisting of (apparently) two rows of plumose setae, the
ventral reaching the level of the fondal interdigital articular
membrane {fiam) and the dorsal reaching the prolateral
interdigital condyle (pic)', proventral subdistal setae made up

of few thick and blunt setae {pvsd comb) at level of the

stridulatory apparatus, and a few others, thinner, in more
distal position {pvsd)\ carpet-like field of sparse barbed and
bristle-like promedial {pm) setae, covering the distalmost
quarter of manus. Stridulatory plate slightly longer than high,
occupying most of manus, dorso-apically with a six-ridged
stridulatory apparatus (variability in ridge number was not
measured). Prolateral setose area of movable finger with setal
insertions reaching the level of MP tooth; movable finger
prodorsal (mpd) setal series consisting of plumose setae
arranged in one staggered row or two rows, followed by
sparse setae of different length and thickness corresponding to
the movable finger promedial (mpm) and proventral (mpv)
setal series, the distalmost setae of each of which is longer.

Flagellum: (Figs. 6, 11-14). A thin, translucent, membra¬
nous structure immovably attached prodorsally to the fixed
finger; ipsilateral opening present. General aspect drop-like,
moderately inflated and narrowing anteriorly; ventral margin
sinuous. Visible (prolateral) surface almost smooth, with very
sparse minute spicules, barely identifiable along regions of
prodorsal margin; apex without visible spicules; apex of the
flagellum reaching about midway between the apex of the
mucron and FD tooth.

BOTERO-TRUJILLO— THE SMALLEST KNOWN SOLIFUGE

223

Figures !1-15. — Vempironiella aguilari gen. nov., sp. nov. SEM images. Male paratypes (MACN-Ar-35454). 11. Right chelicera, retrolateral
aspect (broken FD tooth indicated by arrow); 12. Right chelicera, prolateral aspect (broken FD tooth indicated); 13. Right chelicera, flagellum,
prolateral aspect; 14. Left chelicera, fixed finger, proventral aspect (retrofondal diastema indicated by arrow); 15. Ibid., retroventral aspect. Scale
bars: 0.1 mm (Figs. 11, 13, 14); 0.25 mm (Fig. 12); 50 pm (Fig. 15).

Pedipalp: (Figs. 16-18). Segments robust, all coated with
bifurcated setae {sensu Cushing & Casto 2012) of different
sizes; femur, basitarsus, and especially tibia with ventral set of
very long setae, some of them longer than tibia; clubbed setae
{sensu Cushing & Casto 2012) only present on basi- and
telotarsus; spiniform setae absent. Randomly distributed slit
sensilla present at least on tibia, basi- and telotarsus.

Leg I: (Fig. 1). Similar to pedipalp with respect to the types,
density and distribution of setae; with neither claws nor
spiniform setae. Slit sensilla, if present, could not be identified.

Walking legs: (Fig. 1). Covered with abundant small- to
medium-sized bifurcated setae, and a few longer setae. Legs II
and III: tibia and basitarsus with array of pro- and
retroventral rows of spiniform setae; on basitarsus apparently
a row of three proventral, row of three retroventral, and one
distal subventral spiniform setae, in a 2.2.3 pattern; telotarsus
bi-segmented with pro- and retroventral rows of spiniform
setae, each apparently with five and three, respectively, in a
1.1. 2/2. 2 pattern. Leg IV: Tibia with row of three/four
spiniform setae on proventral surface and single distal
spiniform seta on retroventral surface; basitarsus apparently
with row of four provental and one distal retroventral
spiniform setae, in a 1.1. 1.2 pattern; telotarsus bi-segmented
with incomplete (ventral) segmentation on first (basal)
tarsomere, with pro- and retroventral rows of six spiniform
setae each, in a 2. 2. 2-2/2. 2 pattern.

Opisthosoma: (Figs. 1, 20-23). Tergites with abundant
bifurcated setae of variable size. Sternites with several
bifurcated setae. Ctenidia present on 3'^'^ and 4”’ post-genital
sternites (post-spiracular sternites I and II); ctenidia filiform
and setae-like, similar in thickness to the bifid setae, but
distinguishable because ctenidia are longer, single-tipped (non¬
bifid), and flexible; ctenidia similar in the two sternites. Post-
spiracular sternite II with row of rigid hairs along posterior
margin. Two pairs of microsetae, of the same type reported by
luri et al. (2014) and Botero-Trujillo (2014), present in the
posterior half of the genital plate, and 2"*^ post-genital
sternites (spiracular sternites); one of these microsetae is also
present on each side of post-spiracular sternite I (these could
not be seen in other sternites due to dense setation).

Female. — Meristic data in Table 1. Figs. 3-5, 7-10. Similar
to male but larger and more robust; propeltidium wider.
Ctenidia present in the same sternites and similar to those of
male. Chelicera without the sexual specializations of males.
Fixed and movable fingers very sharp, with sharp teeth. Fixed
finger dorsal surface more elevated than manus, evidently
curved on lateral aspect and without dorsal crest; fixed finger
highest elevation at level of mucron. Movable finger mucron
aculeus-like, with teeth located in a noticeably basal position
on the finger; mucron cylindrical; vestigial retrolateral carina
present on basal third of finger (where granulose) and on the
apex (i.e., retrolateral edge carina); gnathal edge carina

224

JOURNAL OF ARACHNOLOGY

Figures 16-23. — Vempiroiiiella cigiiilari gen. nov., sp. nov. SEM images. Male paratypes (MACN-Ar-35454). 16. Right pedipalp, prolateral
aspect; 17. Ibid., detail of telotarsus; 18. Tip of clubbed seta on pedipalp telotarsus; 19. Leg IV malleoli; 20. Genital plate; 21. Pair of microsetae
on genital plate; 22. Post-spiracular sternite I (arrows indicate some ctenidia); 23. Post-spiracular sternite II (arrows indicate some ctenidia). Scale
bars: 0.5 mm (Fig. 16); 0.1 mm (Figs. 17, 19, 20, 22-23); 5 pm (Fig. 18); 10 pm (Fig. 21).

obsolete, not elevated and identified only by a sclerotized line
along the mucron dorsal margin.

Variation. — Dental pattern formula: In females and juve¬
niles: FD-(l-2)-FM-(i-2)-FP-(lRFA, IRFP, IRFSP); in
males: FD-(i-2)-FM-(l)-FP-(l RFA, IRFP, IRFSP).

Number of teeth on the FSD secondary teeth category:
Males: n (chelicerae) = 24; 21 with one, 3 with two FSD;
females: n = 4; 4 with two FSD.

Number of teeth on the FSM secondary teeth category:
Males: n (chelicerae) = 24; 24 with one FSM; females: n = 4; 2
with one, 2 with two FSM.

Notes. — Resulting from a year-round (1974-1975) ecologi¬
cal study of the arthropod fauna of the Tillandsial of Punta
Hermosa, Aguilar (1977: 91) reported V. agiiilari [as ''Mum-
miicia variegata (?)”] as the most abundant arachnid species.
Aguilar (1977) did not mention if the specimens were to be
deposited in a collection; however, he specified that some
arachnid samples from his study had been sent to Dr. M. E.
Galiano, formerly at the MACN where the material herein

referred was found. The information contained in the label
with the specimens accurately indicates that these are from
Aguilar’s survey of the spring of 1974 (September to
November). According to Aguilar (1977: fig. 4), around 40
specimens of only that solifuge species were captured in that
season, while other, about 190 specimens were captured
during the rest of the year (mostly in summer). So far, only
the specimens here referred are known to be deposited in a
formal collection, the rest remain unlocated.

Even though V. aguilari appeared to be, back then, fairly
abundant throughout the year, a two-day survey to the type
locality conducted by the author in early March 2014, aimed
at collecting additional material of this species, was unsuc¬
cessful. Whether the population density might have decreased,
or which variables might be related to the species not having
been found, cannot be determined at this time.

Habitat. — The coastal-desert area where V. aguilari was
collected is characterized by the presence of the xerophyte
Tillandsia latifolia (Bromeliaceae) (Aguilar 1977).

BOTERO-TRUJILLO— THE SMALLEST KNOWN SOLIFUGE

225

Table 1. — Meristic data for Vempironiella aguilari gen. nov., sp.
nov. Measurements in millimeters for male and female. L = length; W
= width; H = height. 'Measured along medial axis, from the
propeltidium anterior margin to the opisthosoma posterior margin.
“Measured in dorsal view at widest point. '’Measured in retrolateral
view parallel to longitudinal axis of chelicera, from the fixed finger
apex to anterolateral propeltidial lobe anterior margin. "’Measured in
retrolateral view, along vertical axis at widest part of manus. ^Sum of
individual segment lengths. ^Measurement excludes claws. * Range
for males (n = 15). ** Measurement unavailable (legs IV absent).

Male holotype
(MACN-Ar-35453)

Female paratype
( adult )

(MACN-Ar-35454)

Total body L:

With chelicerae:

5.72 (holotype)

[3.90 - 5.85] *

8.11

w/o chelicerae;'

4.66 (holotype)

[3.19-4.79] *

5.72

Propeltidium:

L:

0.90

1.57

W:2

1.00

2.10

Chelicera:

L:'

1.23

2.83

W:2

0.43

0.97

H:"*

0.37

0.97

Pedipalp total L;^

3.37

5.33

Femur L:

1.17

1.83

Tibia L:

1.00

1.67

Tibia W:^

0.23

0.42

Basitarsus -t-

1.20

1.83

telotarsus L:

Leg I total L:^

2.70

4.11

Patella L;

0.90

1.17

Tibia L:

0.82

1.30

Basitarsus L:

0.58

0.97

Telotarsus L:

0.40

0.67

Leg IV total L

4.57

**

(w/o claws):^

Patella L:

1.50

**

Tibia L:

1.37

**

Basitarsus L:

1.03

**

Telotarsus L: ^

0.67

**

DISCUSSION

The cheliceral morphology of Vempironiella aguilari is
challenging to interpret, especially that of the movable finger.
Bird et al. (2015) consider in corollary 1 of their structural
criterion of homology that secondary teeth are more likely to
be absent than primary teeth. On the other hand, corollary 2
argues that teeth are more prone to be absent the more distal
its position is on the finger (except within secondary teeth
categories where the opposite can be true).

The chelicerae of V. aguilari bear only two teeth on the
movable finger, the proximal of which is larger than the distal.
In interpreting this dentition pattern in the light of the
corollaries of Bird et al. (2015), it could be argued that it is the
MSM tooth which is absent, the smallest tooth on the movable
finger of this species corresponding to MM. Three things
suggest, however, that this is not the case and that it is the
MM tooth which is indeed absent. First, in all known
mummuciid species, the MM tooth closes just slightly

proximal to its serial homolog on the fixed finger, FM;
therefore if MM is presumed to be present in V. aguilari, then
its closure with respect to FM would deviate from that pattern
considering that the two teeth would be well distant when the
fingers are closed. In addition, the two teeth on the movable
finger of V. aguilari are placed in a clearly basal position on
the finger, while the finger mucron is very long. If compared
with other species in the family, it is reasonable to consider
that it was the absence of MM tooth, instead of the MSM,
which makes the mucron of this species that long as compared
to the whole finger length. The absence of MM tooth would
also more easily explain why the teeth are placed in a basal
instead of median position on the finger, the latter being more
widely distributed across mummuciid taxa. Likewise, the
anterior-most tooth on the movable finger of V. aguilari is
considerably smaller than MP, as it most frequently happens
with MSM and MP teeth, respectively, throughout the order
(Bird et al. 2015). Although the former tooth is indeed much
more developed compared to the MSM of other species in
family Mummuciidae, it is similar in size to the secondary
teeth of the fixed finger, and therefore the hypothesis that it is
the MSM tooth remains feasible.

The frfd in the chelicerae of V. aguilari involves the absence
of retrofondal teeth (including RFM). This diastema, which is
present in adults of both sexes as well as in juveniles, is to our
knowledge not shared with any other described solifuge. The
frfd is unlikely to be homologous to the fondal notch of many
male eremobatids, the later of which is situated immediately
proximal to the FP tooth, whereas the frfd is proximal to
RFA. The frfd is not either considered homologous to the
medial notch of some other families (e.g., Solpugidae), since
such diastema is situated between FM and FSM and does not
involve the lack of teeth (Bird et al. 2015).

The shape of the chelicerae of solifuges has been proposed
to be associated with dietary preferences and burrowing
abilities (Van der Meijden et al. 2012; Bird et al. 2015). For
instance, species with multidentate chelicerae are presumed to
be especially successful at hunting small, fast-running prey, at
the cost of lower force as compared to species with robust
chelicerae (Bird et al. 2015). The chelicerae of V. aguilari are
neither multidentate nor especially robust, and these might
also be associated with feeding and burrowing adaptations.
The long and delicate shape of the fingers of V. aguilari
suggests that these solifuges are not especially adapted for
burrowing or that they burrow in soft substrates (e.g., loose-
sand removal instead of hard-substrate excavation). In
contrast, it is possible that the long, sharp aculeus-like
movable finger can serve as a “killing weapon” that easily
penetrates soft-bodied animals or articular membranes. In
addition, the large size of the MP tooth might grant these
animals the ability to break small, hard-shelled preys (e.g.,
ants), or to prevent them from escaping, for instance, by
crushing them or keeping them trapped against the frfd. These
hypotheses, however, have not yet been confirmed with live
animals and direct observations will be necessary.

The cheliceral morphology of V. aguilari is remarkable and
very different from that of most species in the family.
Interestingly, there is some resemblance between the chelicerae
of this species and that of M. mauryi (see Xavier & Rocha
2001). In both, the chelicerae are slender with finger tips sharp.

226

JOURNAL OF ARACHNOLOGY

the movable finger mucron is long and delicate, and the fixed
finger highest elevation in female is at level of the mucron.
These resemblances probably result from independent adap¬
tations in two distant taxa. As for the two other Peruvian
species, M. exlineae and M. masculina, neither is assignable to
the new genus and their systematic position remains to be
determined.

ACKNOWLEDGMENTS

The author is thankful to Mariajose Deza (and her family)
and Diana Silva Davila for their assistance during his 2014
trip to Punta Hermosa and visit to the MUSM collection,
respectively. Andres A. Ojanguren Affilastro and Hernan
Augusto luri have contributed to several discussions regarding
the author’s ongoing project on Mummuciidae. Thanks to
Tharina L. Bird for some useful opinions about the cheliceral
teeth of the new species. Additional thanks are due to Martin
J. Ramirez, Daniel N. Proud, AAOA and HAI for performing
reviews on an earlier version of the manuscript, and to the
anonymous referees of the submitted version, all of whom
provided valuable feedback. Thanks also to the curators of
institutional collections who loaned (or otherwise facilitated
access to) the specimens used during the present study and
listed in the text: Peter Jager (SMF), Mark Judson (MNHN),
Adriano B. Kury (MNRJ) and Ricardo Ott (MCN). The
author is supported by a Doctoral Fellowship associated with
PICT 2011-01007 from the “Fondo para la Investigacion
Cientifica y Tecnologica-FONCyT”, Argentina. Financial
support was received from PICT 2010-1764 to AAOA and
PICT 2001-1007 and PIP 2012-0943 to MJR.

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Bird, T.L. & R.A. Wharton. 2015. Description of a new solifuge
Mehmoblossia aiisie sp. n. (Solifugae, Melanoblossiidae) with notes
on the setiform flagellar complex of Melanoblossiinae Roewer,
1934. African Invertebrates 56:515-525.

Bird, T.L., R. Wharton & L. Prendini. 2015. Cheliceral morphology
in Solifugae (Arachnida): primary homology, terminology and
character survey. Bulletin of the American Museum of Natural
History 394:1-355.

Botero-Trujillo, R. 2014. Redescription of the sun-spider Mummucina
litschacki Roewer, 1934 (Solifugae, Mummuciidae) with notes on
the taxonomy of the genus. Zootaxa 3884:319-332.
Botero-Trujillo, R. & H.A. luri. 2015. Chileotrecha romero (Kraus,
1966) comb. nov. and Pseudocleobis patagonicus (Roewer, 1934)
comb. nov. transferral from Mummuciidae to Ammotrechidae
(Arachnida, Solifugae). Zootaxa 3990:437^43.

Carvalho, L.S., D.F. Candiani, A.B. Bonaldo, L. Suesdek & P.R.R

Silva. 2010. A new species of the sun-spider genus Mimmmcia
(Arachnida: Solifugae: Mummucidae) from Piaui, northeastern
Brazil. Zootaxa 2690:19-31.

Cushing, P.E. & P. Casto. 2012. Preliminary survey of the setal and
sensory structures on the pedipalps of camel spiders (Arachnida:
Solifugae). Journal of Arachnology 40:123-127.

Gonzalez- Reyes, A.X. & J.A. Corronca. 2013. A new Solifugae
species of Mummucina Roewer, 1934 (Solifugae, Mummuciidae)
from the northwest of Argentina. Zootaxa 3737:538-544.

Harvey, M.S. 2003. Catalogue of the Smaller Arachnid Orders of the
World: Amblypygi, Uropygi, Schizomida, Palpigradi, Ricinulei
and Solifugae. CSIRO Publishing, Collingwood, Victoria, Austra¬
lia.

luri, H.A., M.A. Iglesias & A. A. Ojanguren-Affilastro. 2014. A new
species of Chileotrecha Maury, 1987 (Solifugae: Ammotrechidae)
from Argentina with notes on the genus. Zootaxa 3827:20-30.

Kury, A.B. & A.L.C. Nogueira. 1999. Annotated check list of type
specimens of Arachnida in the Museu Nacional-Rio de Janeiro. I.
Scorpiones, Pseudoscorpiones and Solifugae. Publicagoes Avulsas
do Museu Nacional 77:1-19.

Martins, E.G., V. Bonato, G. Machado, R. Pinto-da-Rocha & L.S.
Rocha. 2004. Description and ecology of a new species of sun
spider (Arachnida: Solifugae) from the Brazilian Cerrado. Journal
of Natural History 38:2361-2375.

Maury, E.A. 1982. SohTugos de Colombia y Venezuela (Solifugae,

Ammotrechidae). Journal of Arachnology 10:123-143.

Maury, E.A. 1984. Las familias de sohTugos Americanos y su
distribucion geografica (Arachnida, Solifugae). Physis (Buenos
Aires), secc. C 42(103):73-80.

Maury, E.A. 1987 (1985). Consideraciones sobre algunos sohTugos de
Chile (Solifugae: Ammotrechidae, Daesiidae). Revista de la
Sociedad Entomologica Argentina 44:419^32.

Maury, E.A. 1998. Solifugae. Pp. 560-568. In Biodiversidad de
Artropodos Argentinos. (J.J. Morrone, S. Coscardn, eds.).
Ediciones SUR, La Plata, Argentina.

Mello-Leitao, C. 1938. SohTugos de Argentina. Anales del Museo
Argentino de Ciencias Naturales “Bernardino Rivadavia” 40:1-32.

Rocha, L.S. & M.C. Carvalho. 2006. Description and ecology of a
new solifuge from Brazilian Amazonia (Arachnida, Solifugae,
Mummuciidae). Journal of Arachnology 34:163-169.

Shultz, J.W. 1989. Morphology of locomotor appendages in
Arachnida: evolutionary trends and phylogenetic implications.
Zoological Journal of the Linnean Society 7:1-56.

Simon, E. 1879. Essai d’une classification des Galeodes, remarques
synonymiques et description d’especes nouvelles ou mal connues.
Annales de la Societe Entomologique de France, Series 5:93-154.

Van der Meijden, A., F. Langer, R. Boistel, P. Vagovic & M.
Heethoff. 2012. Functional morphology and bite performance of
raptorial chelicerae of camel spiders (Solifugae). Journal of
Experimental Biology 215:3411-3418.

Xavier, E. & L.S. Rocha. 2001. Autoecology and description of
Mummucia mauryi (Solifugae, Mummuciidae), a new solifuge from
Brazilian semi-arid Caatinga. Journal of Arachnology 29:127-134.

Manuscript received 12 February 2016, revised 28 March 2016.

2016. Journal of Arachnology 44:227-234

The first New World species of the pseudoscorpion family Feaellidae (Pseudoscorpiones: Feaelloidea)

from the Brazilian Atlantic Forest

Mark S, Harvey’, Renata Andrade^ and Ricardo Pinto-da-Rocha^: 'Department of Terrestrial Zoology, Western

Australian Museum, Locked Bag 49, Welshpool DC, WA 6986, Australia. Research Associate: Division of Invertebrate
Zoology, American Museum of Natural History, 79th Street at Central Park West, New York, New York 10024-5192,
USA. Research Associate: Department of Entomology, California Academy of Sciences, Golden Gate Park, San
Francisco, California 94103-3009 USA. Adjunct: School of Animal Biology, University of Western Australia, Crawley,
Western Australia 6009, Australia. School of Natural Sciences, Edith Cowan University, Joondalup, Western Australia
6027, Australia. E-mail: mark.harvey@museum.wa.gov.au; “Terradentro Estudos Ambientais, Caixa Postal 5367,
31011-970, Belo Horizonte, MG, Brazil. ^Depto de Zoologia, Instituto de Biociencias, Universidade de Sao Paulo, Rua
do Matao, travessa 14, 321, 05508-900, Sao Paulo SP, Brazil

Abstract. The first American species of the pseudoscorpion family Feaellidae is named from specimens collected in the
Atlantic Rainforest biome of southern Brazil. The lack of specialized setae on the movable chela! finger suggests that it
belongs to a new genus and new species, which we name Iporaugellci gen. nov. and Iporcmgella orchama sp. nov.,
respectively. The only known population of /. orchama is located near Iporanga, Sao Paulo, and juveniles of an
unidentified species are recorded from llha da Queimada Grande.

Keywords: New genus, new species, Brazil, Serra do Mar, Feaellci, morphology

Members of the pseudoscorpion family Feaellidae can be
instantly recognized by their raptorial pedipalps with oppos¬
ing processes on the trochanter and femur, large teeth on the
chela, including some facing prolaterally, and two, four or six
tubercles on the anterior margin of the carapace (e.g.,
Ellingsen 1906; Chamberlin 1931; Beier 1932, 1955, 1966;
Harvey 1992, 2013). Feaellidae is one of the smallest
pseudoscorpion families with only a single recognized genus,
Feaella Ellingsen, 1906, and 12 described Recent species from
Africa, the Indian region, the Seychelles Islands and north¬
western Australia (Harvey 2013), as well as a recently
discovered fossil species from Eocene Baltic amber deposits
(Henderickx & Boone 2014). A second genus has been found
in southeast Asian caves which differs from Feaella in several
ways including the morphology of the coxal region which is
highly modified (Harvey unpublished data; M. Judson, in
litt.). The genus Feaella is divided into three subgenera: F.
(Feaella) for those species with six anterior carapaceal lobes,
F. (Tetrafeaella) with four lobes, and F. (Difeaella) with two
lobes (Beier 1955, 1966; Harvey 2013). An alternative generic
classification was developed in an unpublished Ph.D. thesis by
Mark Judson (1992) and will be published in his forthcoming
review of the family.

The first record of an American feaellid was made by
Andrade (2003) who reported specimens from the Serra do
Mar ecoregion of the Atlantic Forest biome in southern
Brazil. The Atlantic Forest region has been heavily cleared
and is now highly fragmented, although the Serra do Mar
ecoregion is the most intact of the Atlantic Forest (Ribeiro et
al. 2009). The Serra do Mar is listed as Critical/Endangered by
the World Wildlife Fund (http://www.worldwildlife.org/
ecoregions/nt0160; accessed 7 January 2016).

The specimens reported by Andrade (2003) are the subject
of the present study, and are found to differ from other
feaellids in the lack of specialized setae on the movable chelal

finger. This difference is sufficient to warrant the formation of
a new genus to accommodate the new species. We also record
a population of the same genus from a nearby island, llha da
Queimada Grande. These specimens cannot be identified to
species level due to the lack of adult specimens.

The discovery of members of the family Feaellidae raises the
number of pseudoscorpion families recorded from South
America to 20, with only six families - Pseudogarypidae,
Hyidae, Neobisiidae, Parahyidae, Larcidae and Sternophor-
idae - absent or not yet discovered from the region (Harvey
2013).

METHODS

The specimens examined during this study are lodged in the
Museu de Zoologia da Universidade de Sao Paulo (MZSP),
and the Instituto Butantan, Sao Paulo (IBSP). They were
examined by preparing temporary slide mounts by immersing
the specimen in 75% lactic acid at room temperature for one to
several days, and mounting them on microscope slides with 10
or 12 mm coverslips supported by small sections of nylon
fishing line. Specimens were examined with a Leica MZ16
dissecting microscope, a Leica DM2500 or Olympus BH-2
compound microscope, and illustrated with the aid of a
drawing tube. Measurements were taken in mm at the highest
possible magnification using an ocular graticule. After study
the specimens were rinsed in water and returned to 75%
ethanol with the dissected portions placed in 12X3 mm glass
genitalia microvials (BioQuip Products, Inc.). Some specimens
(which have since been lost) were examined using a ZEISS
DSM 940 scanning electron microscope located in the
“Laboratorio de Microscopia Eletronica da Universidade de
Sao Paulo”.

Terminology and mensuration largely follow Chamberlin
(1931), with the exception of the nomenclature of the

227

228

JOURNAL OF ARACHNOLOGY

pedipalps, legs and with some minor modifications to the
terminology of the trichobothria (Harvey 1992), chelicera
(Harvey & Edward 2007; Judson 2007) and faces of the
appendages (Harvey et al. 2012).

SYSTEMATICS

Family Feaellidae Ellingsen, 1906
Genus Ipomngella gen. nov.
http://zoobank.org/?lsid=urn:lsid;zoobank.
org:act:C15233BC-C02E4798-948A-8A992C94712E

Type species. — Iporcmgella orchama sp. nov.

Diagnosis. — Iporangella differs from all other feaellids by
the lack of specialized setae on the retrolateral face of the
movable chelal finger.

Description. — Adult: most setae short, inconspicuous, slight¬
ly curved and acuminate.

Chelicera (Fig. 4D): hand with 5 large and several small
setae; is and Is adjacent to each other; movable finger with 1
subdistal seta; with 2 dorsal and 1 ventral lyrifissures; rallum
of 1 long, slender blade (Fig. 4E); lamina exterior absent;
movable finger short.

Pedipalp (Figs. 2H, 4F): trochanter with prolateral conical
protuberance, femur without prolateral process, chela tubular.
Fixed chelal finger and hand with 8 trichobothria, movable
chelal finger with 4 trichobothria (Fig. 4G): esb and est
situated midway on retrolateral face; ib, isb and ist situated
basally in straight row; eb and it situated subdistally, very
close to each other; et situated distally, much closer to diploid
trichobothrium (dt) than to it; dt situated distally; st situated
sub-basally; t slightly closer to sb than to b. Movable chelal
finger without specialized setae. Venom apparatus absent.
Chelal teeth large and diastemodentate.

Carapace (Figs. 1C, 2D, 2E, 4A): anterior margin with 2
broad lobes; with 2 pairs of eyes situated on tubercles away
from anterior carapaceal margin; all eyes with tapetum; with
posterior furrow; without postero-lateral processes.

Coxal region (Figs. 21, 4B): median maxillary lyrifissure
situated basally near clivus; posterior maxillary lyrifissure
absent. Coxa I without depression, each with 1 small coxal
spine, situated basally (Fig. 4C); coxa II without coxal spines.

Legs (Fig. 5A): patellae with shallow dorsal depression;
femora III and IV shorter than patellae III and IV; femora III
and IV not solidly fused with patellae III and IV, respectively;
metatarsi and tarsi fused; subterminal tarsal setae acuminate;
sub-ungual spine present; arolium slightly shorter than claws.

Abdomen (Figs. I A, IB, ID, 2A-C ): very broad, nearly
circular; tergite XI and sternite XI fused (Fig. 3F); tergite XII
and sternite XII (anal sclerites) strongly sclerotized; tergite XII
with 2 setae; anal region with raised circular rim. Sternite II of
female absent (Fig. 5C); sternite III of male and female slender
(Figs. 5B, 5C). Pleural membrane with numerous sclerotized
pleural platelets in two rows (Figs. ID, 3G), most platelets
with a single seta.

Genitalia: details not visible.

Tritonympb: Pedipalp: fixed chelal finger with 7 major
trichobothria, plus diploid trichobothria (dt), movable chelal
finger with 3 trichobothria (Fig. 4H); isb and sb absent; esb
and est situated midway on retrolateral face; ib and ist situated
basally; eb and it situated medially; et situated closer to diploid

trichobothrium (dt) than to it; dt situated distally; t situated
closer to st than to b; movable finger without specialized setae.
Carapace: with 2 anterior lobes; with 2 pairs of eyes.

Protonymph: Pedipalp: Fixed chelal finger with 2 major
trichobothria, eb and ist, plus a single trichobothrium {dt);
movable chelal finger with 1 trichobothrium, t (Fig. 41);
movable finger without specialized setae. Carapace: with 2
anterior lobes; with 2 pairs of eyes.

Remarks. — The new genus most closely resembles Feaella
(Difeaella) krugeri Beier, 1966 from South Africa, the only
species currently included in the subgenus Difeaella, due to the
presence of two lobes on the anterior margin of the carapace,
and the lack of a basal prolateral process on the pedipalpal
femur (Beier 1966). Iporangella orchama differs from all other
feaellids, including F. (D.) krugeri, by the lack of specialized
setae on the retrolateral face of the movable chelal finger.
Although the presence of these setae was not mentioned in the
original description by Beier (1966), they were reported by
Judson (1992). Iporangella orchama further differs from F.
(D.) krugeri by the location of the trichobothria: ist is situated
basal to esb in F. krugeri (Beier 1966, fig. 5), but is distal to esb
in Iporangella (Fig. 4G).

Etymology. — The generic epithet is derived from the type
locality Iporanga, which is a municipality in Sao Paulo. The
name comes from the Brazilian Indian language Tupi and
means ‘beautiful river’. The generic name is feminine in
gender.

Iporangella orchama sp. nov.
http://zoobank.org/?lsid=urn:lsid:zoobank.

org:act:F0AB5DDE-3IDD-4CC5-AB98-BB93ED21D327
Figs. 1-5

Material examined. — Holotype male. BRAZIL: Sdo Paulo:
Iporanga, Vale do Ribeira, 24°33'34"S, 48°40'02"W, 19 March
2002, Winkler extraction, “raizes e folhigo” (roots and leaf
fitter), R. Andrade (MZUSP 67821).

Paratypes: BRAZIL: Sdo Paulo: 1 6 , same data as holotype
except 8 March 2002 (MZUSP 67822); 1 ?, same data
(MZUSP 67817); 1 tritonymph, same data (MZUSP 67818); 1
tritonymph, same data (MZUSP 67819); 1 protonymph, same
data (MZUSP 67820).

Diagnosis. — As for genus.

Description (adults). — Color: all sclerotized portions deep
red-brown (Figs. lA-E). All sclerotized portions coarsely
tuberculate and reticulate.

Setae: most setae short, inconspicuous, slightly curved and
acuminate.

Cerotegument: most surfaces covered with conspicuous
cerotegument.

Chelicera (Fig. 4D): hand with 5 large and several small
setae; is and Is adjacent to each other; movable finger with 1
subdistal seta; galea very thick, without rami; hand coarsely
tuberculate, except for finger and basal third; fingers without
teeth; rallum with 1 long, thin blade (Fig. 4E); serrula exterior
with ca. 18 blades; lamina exterior absent.

Pedipalp (Figs. 2H, 4F): trochanter with long, curved
prolateral conical protuberance, 1.78-2.06 (cj), 1.90 ($),
femur very robust, without triangular process on prolateral
corner, 2.02-2.05 (c3), 1.97 (2), patella conical 2.91 (d), 2.73
(2), chela tubular, chela (with pedicel) 3.88-4.00 (<5), 3.75 (2),

HARVEY ET AL.— FIRST NEW WORLD FEAELLIDAE

229

Figure 1. — Iporangella orchama sp. nov., holotype male (MZUSP 67821): A, body, dorsal view; B, body, ventral view; C, cephalothorax,
dorsal view; D, body, lateral view; E, cephalothorax, ventral view.

chela (without pedicel) 3.58-3.73 (<?), 3.56 (9), hand (without
pedicel) 0.43-0.45 (c?), 0.42 (9) x longer than broad, movable
finger 5.80-6.31 (J), 6.27 (9) x longer than hand. Fixed chelal
finger and hand with 8 trichobothria, movable chelal finger
with 4 trichobothria (Fig. 4G): esb and est situated midway on
retrolateral face; ib, isb and ist situated subbasally in straight
row; eb and it situated subdistally, very close to each other; et
situated distally, much closer to diploid trichobothrium {dt)
than to //; dt situated distally; st situated sub-basally; t slightly
closer to sb than to b. Venom apparatus absent. Chelal hand
very small; retrolateral condyle small and rounded; with dorsal
protuberance at base of finger. Chelal teeth large, retrorse and
diastemodentate: fixed finger with 12 (cJ), 13 (9) retrorse
marginal teeth, 12 ((?, 9) prolateral, 16 (c?), 18 (9)
retrolateral teeth, and 6-7 distal teeth; movable finger with
10 (<?, 9) curved, marginal teeth and 12 (d, 9) prolateral
teeth. Movable chelal finger without specialized setae.

Carapace (Fig. 1C, 2D, 2E, 4A): anterior margin with 2
broad lobes and deep antero-median depression; with 2 dorsal
lobes on level with eyes; lateral margins nearly parallel;
broadest basally; 1.26-1.27 (c?), 1.25 (9) x longer than broad;
with 2 pairs of well-developed eyes situated on tubercles away
from anterior carapaceal margin; all eyes with tapetum; with
numerous inconspicuous setae; posterior furrow present.

Coxal region (Figs. IE, 21, 4B): pedipalpal coxa: tubercu-
late lateral processes situated near pedipalpal foramen; with
scattered small setae; manducatory process with 2 stout, long
acuminate setae; median manducatory lyrifissure basal,
situated near clivus; posterior manducatory lyrifissure absent.
Coxa I without depression, and each with 1 small, anteriorly
directed coxal spine situated basally (Fig. 4C); coxa II without
coxal spines; coxa III meeting in mid-line.

Legs (Fig. 5 A): patellae I and II slightly shorter than
femora I and II; femora III and IV shorter than patellae III
and IV; femora III and IV not solidly fused with patellae III
and IV, respectively; all patellae with shallow dorsal depres¬
sion; metatarsi and tarsi fused; tarsi long and slender, without
tactile seta; subterminal tarsal setae acuminate; sub-ungual
spine present; claws smooth (Figs. 2K, 2L); arolium about
same length as claws, with fimbriate distal margin (Figs. 2K,
2L).

Abdomen (Figs. lA, IB, ID, 2A-C): broadly ovate; tergites
II-X and sternites IV-X with distinct median suture lines;
most tergites gently curved; tergite XI and sternite XI fused
(Fig. 3F); tergite XII and sternite XII (anal sclerites) strongly
sclerotized; most segments with several setae, generally
arranged in a single irregular row along posterior margin of
sclerite; tergite XII and sternite XII each with 2 setae; anal
region with raised circular rim. Tergites and sternites with

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Figure 2. — Iporaiigella orchanui sp. nov., scanning electron micrographs of adult specimens: A, body, dorsal view, specimen 1; B, body, dorsal
view, specimen 2; C, body, ventral view, specimen 2; D, cephalothorax, dorsal view, specimen 1; E, cephalothorax, dorsal view, specimen 2; F,
junction between cephalothorax and abdomen, dorsal view, specimen 1; G, detail of carapace near left anterior eye, specimen 1; H, right
pedipalp, dorsal view, specimen 1; I, cephalothorax, ventral view, specimen 1; J, genital region, ventral view, male; K-L, tarsus, claws and
arolium, unknown specimen. Scale lines = 1 mm (A-C); 0.4 mm (H); 0.2 mm (D-F, I); 0.04 mm (J); 0.02 mm (G, K); 0.01 mm (L).

HARVEY ET AL.— FIRST NEW WORLD FEAELLIDAE

231

Figure 3. — Iporangella orchama sp. nov., scanning electron micrographs of adult specimens: A, right chela, retrolateral view; B, right chelal
fingers, retrolateral view; C, left chela, tip of fingers; D, left chela, prolateral view; E, left chela, tip of fingers; F, abdominal sternites VII-XII,
ventral view; G, pleural membrane; H-I, tergite, dorsal view. Scale lines = 0.2 mm (A, F); 0.1 mm (B, D, G, H); 0.02 mm (C, E, I).

posteriorly-directed micro-spinules (Fig. 3F, 3H, 31). Most
setae very small and inconspicuous; setae of male sternite II
longer (Fig. 5B). Pleural membrane with 2 lateral rows each
composed of 9 platelets, the dorsal ones larger (Fig. ID);
platelets with setae. First pair of spiracles connected to
anterior margin of sternite IV, spiracular opening round and
conspicuous; second pair of spiracles connected to anterior
margin of sternite V, spiracular opening slit-like. Sternite II of
male small and poorly sclerotized (Fig. 5B), of female absent
(Fig. 5C). Sternite III of male (Fig. 5B) and female (Fig. 5C)
slender and anteriorly curved.

Genitalia: details not visible.

Dimensions (mm): Males; holotype, followed by other male
(when measured): Body length 2.24 (2.15); abdomen width
(without pleura) 1.25 (1.15). Pedipalp: trochanter 0.32/0.18
(0.32/0.155), femur 0.635/0.315 (0.625/0.305), patella 0.495/
0.17 (0.48/0.165), chela (with pedicel) 0.64/0.165 (0.60/0.15),
chela (without pedicel) 0.59 (0.56), hand (without pedicel)
length 0.075 (0.065), movable finger length 0.435 (0.41).
Chelicera 0.235/0.14; movable finger 0.085. Carapace 0.625/
0.495 (0.615/0.485); anterior eye diameter 0.055, posterior eye
diameter 0.06. Leg I: femur 0.30/0.08, patella 0.23/0.085, tibia

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Figure 4. — Iporangella orchama, sp. nov., male holotype (MZUSP 67821), unless stated otherwise: A, carapace, dorsal view; B, cephalothorax,
ventral view; C, coxae I, ventral view (cs = coxal spine); D, left chelicera, dorsal view; E, left rallum; F, right pedipalp, dorsal view; G, left chela,
lateral view; H, left chela, lateral view, tritonymph paratype (MZUSP 67818); I, left chela, lateral view, protonymph paratype (MZUSP 67820).
Scale lines = 0.2 mm (A, B, F-I); 0.1 mm (C, D).

HARVEY ET AL.— FIRST NEW WORLD FEAELLIDAE

233

Figure 5. — Iporcmgella orchcmui sp. nov., male holotype (MZUSP 67821), unless stated otherwise: A, left leg IV, lateral view; B, genital
sclerites (sternites II and III), ventral view; B, genital sclerite (sternite III), ventral view, female paratype (MZUSP 67817). Scale lines = 0.25 mm
(A); 0.2 mm (B, C).

0.21/0.065, tarsus 0.33/0.055. Leg IV: femur 0.28/0.10, patella
0.32/0.115, tibia 0.425/0/075, tarsus 0.425/0.06.

Females: paratype: Body length 2.38; abdomen width
(without pleura) 1.42. Pedipalp: trochanter 0.38/0.20, femur
0.74/0.375, patella 0.52/0.19, chela (with pedicel) 0.675/0.18,
chela (without pedicel) 0.64, hand (without pedicel) length
0.075, movable finger length 0.47. Chelicera 0.25/0.155;
movable finger 0.09. Carapace 0.675/0.54; anterior eye
diameter 0.055, posterior eye diameter 0.05. Leg I: femur
0.31/0.09, patella 0.25/0.09, tibia 0.23/0.07, tarsus 0.35/0.06.
Leg IV: femur 0.325/0.10, patella 0.35/0.12, tibia 0.45/0.075,
tarsus 0.435/0.095.

Description (tritonymph). — Chelicera: movable finger with 1
seta.

Pedipalp: femur 2.04, patella 2.75, chela (with pedicel) 4.21,
chela (without pedicel) 3.89, hand 0.68 x longer than broad.
Fixed chelal finger with 7 major trichobothria, plus diploid
trichobothria {dt), movable chelal finger with 3 trichobothria
(Fig. 4H); ish and sh absent; esb and est situated midway on
retrolateral face; ib and ist situated basally; eb and it situated
medially; et situated subdistally; dt situated distally; t situated
closer to st than to b; movable finger without specialized setae.

Carapace: 1 .23 x longer than broad; with 2 anterior lobes;
with 2 pairs of eyes.

Coxal region: coxa I without depression, and each with 1
small coxal spine situated basally.

Legs: much as in adult.

Dimensions (mm): Body length 2.28. Pedipalp: femur 0.57/
0.28, patella 0.11/0.16, chela (with pedicel) 0.59/0.14, chela

(without pedicel) 0.545, hand length 0.095, movable finger
length 0.39. Carapace 0.57/0.465.

Description (protonymph). — Chelicera: movable finger with¬
out seta.

Pedipalp: femur 2.06, patella 2.77, chela (with pedicel) 3.67,
chela (without pedicel) 3.43, hand 0.62 x longer than broad.
Fixed chelal finger with 2 major trichobothria, eb and ist, plus
a single trichobothrium {dt)-, movable chelal finger with 1
trichobothrium, t (Fig. 41); movable finger without specialized
setae.

Carapace: 1.06 x longer than broad; with 2 anterior lobes;
with 2 pairs of eyes.

Coxal region: coxa I without depression, and each with 1
small coxal spine situated basally.

Legs: much as in adult.

Dimensions (mm): Body length 1.25. Pedipalp: femur 0.35/
0.17, patella 0.36/0.13, chela (with pedicel) 0.385/0.105, chela
(without pedicel) 0.36, hand length 0.065, movable finger
length 0.265. Carapace 0.365/0.345.

Remarks. — The type series of Iporcmgella orchama consists
of two adult males, a female and three nymphs. All were
collected using Winkler extraction methods from leaf litter
among the tangled thin roots of a tree. The site is situated near
the lower slope of a mountain within the Serra do Mar
ecoregion within the Atlantic Forest biome of southern Brazil.
The Atlantic coastal forest has been heavily cleared since
European settlement with only 11-16% of the original forest
remaining (Ribeiro et al. 2009). The remaining forest is highly
fragmented and only 9% is situated in protected areas (Ribeiro
et al. 2009). The Serra do Mar ecoregion has been less heavily

234

JOURNAL OF ARACHNOLOGY

cleared, probably due to its hilly topography, with an
estimated 36.5% of the original forest remaining.

The scanning electron micrographs were obtained from
specimens that have since been lost, and which are not
regarded as part of the type series.

Etymology. — The specific epithet refers to this species being
the first described from the New World (orchamos, Greek,
first) (Brown 1956).

Iporangella sp. indet.

Material examined. — BRAZIL: Sao Paulo: 2 protonymphs,
Ilha da Queimada Grande, Itanhaem Municipality, 24°29'S,
46°41'W, 30 April 2003, in pitfall traps, R. Indicati, C. Souza
(IBSP 1620, 1623).

Description (protonymph). — Chelicera: movable finger with¬
out seta.

Pedipalp: femur 1.85, patella 2.09, chela (with pedicel) 3.80,
chela (without pedicel) 3.50, hand 0.75 x longer than broad.
Fixed chelal finger with 2 major trichobothria, eh and ist, plus
a single trichobothrium {dt)', movable chelal finger with 1
trichobothrium, /; movable finger without specialized setae.

Carapace: 1 .20 x longer than broad; with 2 anterior lobes;
with 2 pairs of eyes.

Coxal region: coxa I without depression, and each with 1
small coxal spine situated basally.

Legs: much as in adult.

Dimensions (mm): Body length 1.11. Pedipalp: femur 0.305/
0.165, patella 0.23/0.165, chela (with pedicel) 0.38/0.10, chela
(without pedicel) 0.35, hand length 0.075, movable finger
length 0.255. Carapace 0.365/0.305.

Remarks. — Although these two protonymphs were collected
only 125 km from the type locality of I. orchama, it is not
possible to confirm whether they are conspecific with I.
orchama until adult specimens are collected from Ilha da
Queimada Grande.

ACKNOWLEDGMENTS

We gratefully acknowledge the assistance of Mark Judson
for sharing his Ph.D. dissertation with MSH. Antonio
Brescovit kindly allowed MSH access to the collections at
Institute Butantan, Sao Paulo. R. Pinto-da-Rocha has been
funded by Funda9ao de Amparo a Pesquisa do Estado e Sao
Paulo (FAPESP, 2012/02969-6 and 2013/50297-0), the Na¬
tional Science Foundation (NSF/DOB 1343578) and NASA.

LITERATURE CITED

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Brescovit, eds.). Sao Pedro, SP, Brasil.

Beier, M. 1932. Pseudoscorpionidea 1. Subord. Chthoniinea et
Neobisiinea. Tierreich 57:i-xx, 1-258.

Beier, M. 1955. Pseudoscorpionidea, gesammelt wahrend der
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Beier, M. 1966. Erganzungen zur Pseudoscorpioniden-Fauna des
siidlichen Afrika. Annals of the Natal Museum 18:455M70.

Brown, R.W. 1956. Composition of Scientific Words. Revised edition.
Smithsonian Institution Press, Washington, D.C.

Chamberlin, J.C. 1931. The arachnid order Chelonethida. Stanford
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Ellingsen, E. 1906. Report on the pseudoscorpions of the Guinea
Coast (Africa) collected by Leonardo Fea. Annali del Museo
Civico di Storia Naturale di Genova, series 3, 2:243-265.

Harvey, M.S. 1992. The phylogeny and classification of the
Pseudoscorpionida (Chelicerata: Arachnida). Invertebrate Taxon¬
omy 6:1373-1435.

Harvey, M.S. 2013. Pseudoscorpions of the World, version 3.0.
Western Australian Museum, Perth. Accessed 22 June 201 5. Online
at http://museum.wa.gov.au/catalogues-beta/pseudoscorpions

Harvey, M.S. & K.L. Edward. 2007. A review of the pseudoscorpion
genus Ideoblothrus (Pseudoscorpiones, Syarinidae) from western
and northern Australia. Journal of Natural History 41:445-472.

Harvey, M.S., P.B. Ratnaweera, P.V. Udagama & M.R. Wijesinghe.
2012. A new species of the pseudoscorpion genus Megachernes
(Pseudoscorpiones: Chernetidae) associated with a threatened Sri
Lankan rainforest rodent, with a review of host associations of
Megachernes. Journal of Natural History 46:2519-2535.

Henderickx, H. & M. Boone. 2014. The first fossil Feaella Ellingsen,
1906, representing an unexpected pseudoscorpion family in Baltic
Amber (Pseudoscorpiones, Feaellidae). Entomo-Info 25:5-11.

Judson, M.L.I. 1992. African Chelonethi. Studies on the systematics,
biogeography and natural history of African pseudoscorpions
(Arachnida). Ph.D. thesis. University of Leeds, Leeds.

Judson, M.L.I. 2007. A new and endangered species of the
pseudoscorpion genus Lagynochthonius from a cave in Vietnam,
with notes on chelal morphology and the composition of the
Tyrannochthoniini (Arachnida, Chelonethi, Chthoniidae). Zoo-
taxa 1627:53-68.

Ribeiro, M.C., J.P. Metzger, A.C. Martensen, F.J. Ponzoni & M.M.
Hirota. 2009. The Brazilian Atlantic Forest: How much is left, and
how is the remaining forest distributed? Implications for conser¬
vation. Biological Conservation 142:1141-1153.

Manuscript received 25 January 2016, revised 10 February 2016.

2016. Journal of Arachnology 44:235-244

Microhabitat use and behavior differ across sex-age classes in the scorpion Bmchistostermis feirugineus

(Scorpiones: Bothriuridae)

M.F. Nime’’^, F. Casanoves^ and C.I. Mattoni*; *Laboratorio de Biologia Reproductiva y Evolucion, Institute de
Diversidad y Ecologia Animal (IDEA), CONICET, and Facultad de Ciencias Exactas, Fi'sicas y Naturales, Universidad
Nacional de Cordoba, Argentina. E-mail: monicanime@yahoo.com.ar; “Unidad de Bioestadistica del Centro
Agronomico Tropical de Investigacion y Ensenanza (CATIE), Turrialba (7170), Costa Rica

Abstract. Infra- and interspecific coexistence has been recorded in several species of scorpions, reflecting different levels
of aggregation and sociability. Some species of scorpions avoid temporal or spatial overlap of their surface activities, which
may differ depending on species, age group or gender, and thus reduce intra- and interspecific competition and predation.

We examined the surface activity of males, females and juveniles (sex-age class) of the scorpion Brachistosternus fen ugineiis
(Thorell, 1876) in an area of Arid Chaco, and also its microhabitat preference and behavior by each sex-age class. The
month-by-month activity of each sex-age class was different, but all the classes were observed each month. The most
frequently used microhabitat was soil (64.8%), while leaf litter and vegetation were used in similar proportions. The
behavior most frequently observed was ambush (68.3%), followed by walking and less frequently /eer//ng, doorkeeping and
courting. Each sex-age class performed one particular behavior with more frequency than the others. Analyzing
combinations of microhabitat, behavior and sex-age class, we found the juveniles were associated with feeding on
vegetation, males with walking on leaf litter, while females were related to ambush on soil. No marked temporal distribution
between sex-age classes was observed. However, the spatial distribution and frequency of behaviors were highly dependent
on developmental stage and sex. These differences may facilitate understanding of the coexistence of different age-sex
classes of B. fei rugineus.

Keywords: Arachnids, intraspecific coexistence, surface activity. Arid Chaco

Scorpions are primarily solitary and sedentary arthropods.
They are excellent predators, preying on a wide variety of
populations of insects, spiders and even other scorpions, and
in turn are prey, mostly of vertebrates (Williams 1987;
McCormick & Polis 1990; Polis 1990; Brownell & Polls
2001). They occur in a variety of terrestrial habitats and may
be divided into two general groups based on microhabitat
preference: ground-dwelling species, which live in burrows or
under surface debris such as rocks or logs, and arboreal
species, found at various heights on the vegetation (Polis 1990;
Brown & O’Connell 2000). In deserts and semiarid regions,
where scorpions are common, the majority of species are
ground-dwellers (Polis 1990; Polis & Yamashita 1991). During
the day, these species are relatively inactive and remain in their
shelter or burrow. Individuals emerge from these diurnal
retreats shortly after sunset to forage or engage in other
activities, usually remaining on the ground surface near the
burrow or shelter (Polis 1979; Polis et al. 1985).

Intra- and interspecific coexistence has been recorded in
several species of scorpions (McReynolds 2004, 2008; Kaltsas
et al. 2009; Shehab et al. 2011; Lira and De Souza 2014),
reflecting different levels of aggregation and sociability (Polis
& Lourengo 1986; Polis 1990). Some species of scorpions
avoid temporal or spatial overlap of their surface activities,
which may differ depending on species, age group, gender or
body size. This is probably due to the presence of conspecifics
and heterospecifics in the environment, which would result in
substantial competition for food and shelter resources and

^ Current address: Centro de Relevamiento y Evaluacion de
Recursos Agricolas y Naturales - (IMBIV). Consejo Nacional de
Investigaciones Cienti'ficas y Tecnicas (CONICET)/ Universidad
Nacional de Cordoba. Av. Valparaiso s/n, Ciudad Universitaria,
CP 5000, Cbrdoba, Argentina.

may decisively influence habitat selection (Polis 1980, 1984;
Due & Polis 1985; Polis & McCormick 1987; McReynolds
2008; Kaltsas et al. 2009; Lira et al. 2013). Habitat selection by
an intermediate predator often means a balance between
success in foraging and risk of predation (Murdoch & Sih
1978; Luttbeg & Schmitz 2000). Foraging success in scorpions
may be associated with seasonal changes in prey availability
(Polis 1980; Polis & McCormick 1986) and in the risk of
predation by nocturnal predators, which may also be
associated with the lunar cycle (Hadley & Williams 1968;
Polis 1980; Polis et al. 1981; Nime et ai. 2013). Thus, to
increase predation success and to avoid being preyed upon,
scorpions may modify their nocturnal activity or microhabitat
use on nights with high illumination (Skuteisky 1996). For
example, they may feed on trees or shrubs with high prey
availability which, in turn, could provide protection against
predators (McReynolds 2004).

The behavior and the substrate used by many species of
scorpions are related, among other factors, with the sex-age
class (Skuteisky 1996; Brown & O’Connell 2000; McReynolds
2004; Yamashita 2004). For example, walking behavior in
several scorpion species is more associated with males than
with females and juveniles (Polis & Farley 1979; Polis 1980;
Yamashita 2004). Major displacement has even been observed
in the males of many species, especially in the breeding season,
as they actively search for females to mate with (Polis & Farley
1979; Polis & Sissom 1990; Araujo et al. 2010). Males of
Smeringurus mesaensis (Stahnke, 1957) can travel more than
100 m in a night (with an average of 34.7 m), meanwhile, the
females were observed up to one meter from their burrows
(Polis & Farley 1979). Likewise, the behavior of climbing
vegetation has been observed more frequently in juveniles than

235

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JOURNAL OF ARACHNOLOGY

in adults of several species of scorpions (Bradley 1988; Polls
1990; Skutelsky 1996).

No previous studies have examined habitat use by
Brachistosternus fernigineus (Thorell, 1876) or its behavior in
the months of activity. Only a few studies have addressed the
ecology of scorpions in Arid Chaco regions (Acosta 1995;
Nime et al. 2013, 2014). This lack is particularly surprising in
view of the high biodiversity of such environments and the
evidence that environmental change is transforming the
ecology of arid regions, which are often predicted to be
among the ecosystems most responsive to global climatic
change (Hamerlynck et al. 2000; Whitford 2002).

The aims of this study were to analyze whether the surface
activity of males, females and juveniles (sex-age class) of the
scorpion B. ferritgineus varies from month to month in an area
of Arid Chaco, and to examine whether each sex-age class has
a preferred microhabitat and behavior. Our hypothesis is that
sex-age classes of B. fernigineus use different microhabitats
and shows temporal displacement. The findings of this study
on the ecology and behavior of this widespread and abundant
species (Acosta 1995; Ojanguren-Affilastro 2005; Nime et al.
2013) are therefore important for understanding the processes
of intra-specific coexistence and can contribute to a greater
understanding of the structure of arthropod communities in
the Argentine Arid Chaco.

METHODS

Study site. — The study was conducted in the Parque
Provincial y Reserva Forestal Chancanf (Cliancani Reserve,
3r22T3.21" S 65°27T3.75" W, 4,960 ha). The reserve is
located in the southernmost portion of the Chaco (Arid Chaco
ecoregion, NT0701 in Olson et al. 2001) in Cordoba province,
Argentina. Vegetation in the reserve is dry xerophilous
woodland. The canopy is discontinuous and ~15 m high,
dominated by Aspidosperina qiiehracho-blanco and Prosopis
flexuosa trees. The shrub stratum (~4 m high) is thorny, dense,
and almost continuous (Carranza et al. 1992; Cabido & Pacha
2002). The reserve supports forest stands that are close to
climax conditions. The climate is highly seasonal, with a
pronounced dry season. Annual rainfall averages 450 mm,
concentrated during the summer (October-March). In the dry
winter season (April-September), the water balance is
negative, resulting in a soil humidity deficit. Mean annual
temperature is 18 °C, with a mean value of 25 °C in the
warmest month (January, with maximum temperatures
reaching 45 °C during the day) and 10 °C in the coldest
month (July) (Cabido & Pacha 2002).

There are four different ecological-type sites within the
study site. (1) The mature forest site shows forest formations
that are close to climax conditions, mainly with trees such as
A. quehracho-hlanco and P. flexuosa. The shrub layer is
dominated by Larrea divaricata, Mymozyganthus carinatus
and Acacia furcatispina (Carranza et al. 1992). (2) In
December 1994, a high-intensity wildfire affected about
32,000 ha of Chaco forest, 230 ha of which are within the
western boundaries of the Chancanf Reserve. Alongside the
mature forest area, the fire generated a secondary forest area in
which the vegetation is dense and homogeneous, dominated
by high grasses (about 1 m tall) and shrubs about 2.5 m in
height. In this area, young trees are common and dead trees

are still standing (Pelegrin & Bucher 2010). (3) The Jarillal site
is located in the central area of the reserve. In the past, the
area was used for the logging of large trees for fuel and
charcoal. Currently, it is dominated by shrubs of the Larrea
genus (“jarilla”); the area is not used for any activities and is
recovering. (4) The forest with livestock is a private area facing
the reserve that has been used for years for raising livestock, so
the site is much degraded with low, scattered shrubs.

Study animal. — The genus Brachistosternus Pocock, 1893 is
second in richness within the family Bothriuridae Simon, 1880,
containing 43 species described so far (Rein 2015; Ojanguren-
Affilastro et al. 2016). The species of this genus inhabit arid
and semi-arid environments from northern Ecuador to
southern Patagonia in Argentina. Within Argentina, Brachis¬
tosternus species are dominant in xeric environments of the
west and south, where they form large populations and are
generally more abundant than other sympatric species (Acosta
1995; Ojanguren-Affilastro 2005; Nime et al. 2013; Ojanguren-
Affilastro et al. 2016). They are ground-dwelling, living in
burrows or under surface debris such as rocks or logs
(Ojanguren-Affilastro 2005). Most species of Brachistosternus
are active during the summer (from November to April), with
generally a more extended activity period than other
bothriurid genera (Ojanguren-Affilastro 2005). Brachistoster¬
nus fernigineus are small to medium size scorpions (males
between 27 to 43 mm, females between 35 to 56 mm)
(Ojanguren-Affilastro 2005). Most of the localities where B.
ferritgineus have been collected in Argentina are within the
Chaco phytogeographic province, where it is the most
common species (Acosta 1995; Ojanguren-Affilastro 2005).
This species lives at low or medium altitudes, from 100 to 1500
meters above sea level (Ojanguren-Affilastro 2005).

Experimental design. — Three sites were selected within the
Chancanf Reserve representative of “mature forest” (Fig. la),
“secondary forest” (Fig. lb) and “Jarillal” (Fig. Id). An
additional site, “forest with livestock”, was added adjacent to
the reserve (Fig. Ic) in order to cover the landscape’s
heterogeneity.

At each site, 15 transects (50 X 6 m) were established,
separated from each other by 70 m. Transects, one after the
other, passed along and on the main roads in each sector, due
to the difficulty of accessing and observing scorpions inside the
dense forest.

Sampling consisted of walking along each transect with a
portable ultraviolet (UV) lamp. Irradiation with UV causes
scorpions to fluoresce, which enables all stages of the active
scorpion to be easily detected in the dark (Honetschlager
1965).

Sampling lasted about 2 h after dusk (from 21:00 to 23:00),
as these were the main activity hours of scorpions at the study
site. Sampling was conducted for three nights a month at each
site, always close to the new moon phase, to avoid the effect of
moonlight on scorpion activity (Nime et al. 2013). Sampling
was conducted during the periods November 2009 through
January 2010 and November 2010 through February 2011.
The sampling effort was the same at each site on each
recording night (2 hours/night), but the months in which each
site was sampled were not the same (Table 1). February was
sampled in only one year (2010) but sampling was carried out

NIME ET AL.^MICROHABITAT USE AND BEHAVIOR OF A SCORPION SPECIES

237

Figure 1. — Sampling sites within and outside the Chancani reserve, Cordoba, Argentina, a. mature forest; b. secondary forest; c. forest with
livestock; d. Jarillal.

in the other months in both seasons. Every month had the
same number of sampling nights.

When a scorpion of B. fernigineus species was located, it
was observed and classified by sex-age class (male, female and
juvenile). When necessary, scorpions were temporarily cap¬
tured with forceps for identification. Every observation was
made avoiding disturbances to other scorpions nearby.
Specimens were not marked, since on a preliminary study
the recovery of marked individual was too low (less than 2%).
At the time of observation, we recorded the behavior of each
scorpion and the microhabitat in which it was found. The
behaviors recorded were ambush, feeding, walking, doorkeeping
and courting. Ambush was when the individual was at rest,
with extended pedipalps (waiting for prey) or resting with the
appendages in contact with the body. We attempted to record
both behaviors (ambush and rest) separately according to the
position of the pedipalps, but if they perceived our vibrations
when we approached, they moved their pedipalps to the body,
so we prefer not to discriminate between these two behaviors.
The scorpion was classified as feeding when it was feeding on

prey or when it had consumed the prey leaving only a small
part between the chelicerae. The scorpion was classified as
walking when it was observed moving. Doorkeeping behavior
was when the animal was seen at the entrance of the burrow,
either in ambush or entering. Finally, courting behavior is
performed by scorpions prior to copulation (the male holding
the female with their pedipalps), so only adults exhibit this
behavior (Fig. 2).

The microhabitats considered were grouped into the
following categories: soil, leaf litter and vegetation. Soil was
considered when the individual was on bare ground, usually
packed soil (Fig. 2). In leaf litter, the individual was on the leaf
litter composed mainly of dry leaves (mostly from A.
quebracho-bianco trees) and small branches. In vegetation,
the individual was observed climbing vegetation, usually at the
end or in the middle, not above 30 cm in height. Three
scorpions were observed on cattle feces at the site with
livestock, and this was considered as leaf litter.

We surveyed the availability of each microhabitat in the
total area and in each site (with exception of jarillal). Three

Table 1. — Number of nights sampled for scorpions with UV light in the Chancani reserve. * = no data.

Site Nov.’09 Dec.’09 Jan. ’10 Nov.’lO Dec.’lO Jan. ’ll Feb.’ll Total

Mature forest 3

Secondary forest 3

Forest with livestock
Jarillal
Total

3

3

3

3

3

3

3

3

3

3

12

3

3

3

3

12

3

3

3

3 26

3 26

3 18

6
75

6

9

9

9

9

238

JOURNAL OF ARACHNOLOGY

Figure 2. — Brachistostemus ferrugineus couple, male on the right
and female on the left, courting on soil, in the secondary forest of
Chancanf reserve, Cordoba, Argentina. Photo courtesy of Maria
Eugenia Romero Lebron.

quadrats (0.50 X 0.50 m) were placed to left, right and center
of each transect (9 quadrats per transect) and the percentage
(%) of vegetation, leaf litter and soil was recorded. The average
of each microhabitat was then calculated at each site and for
all of these.

Statistical analysis. — Temporal distribution of the sex-age
class: Surface activity of scorpions (measured as the number of
individuals active on the ground surface) of each sex-age class
(males, females and juveniles) of the species B. ferrugineus and
its variation between months were analyzed. Scorpion counts
were modeled with a generalized linear mixed model (GLMM)
using the glmer function of the lme4 library through the
interface with R-packages (R Core Team 2013) implemented
in InfoStat (Di Rienzo et ai. 2013). We used a Poisson
distribution (with a log-link function) because this is most
appropriate for counts. The overdispersion was evaluated to
ensure that the Poisson assumptions held. Sex-age class and
month and their interaction were considered fixed effects. In
order to take into account the repeated measures in months,
site and transect were considered random effects. Significance
level was set at 0.05. To compare means, we used the Fisher’s
least significant difference test (LSD).

Microhabitat preferences and behaviors associated with each
sex-age class: First, contingency tables were used to evaluate if
there were significant differences in proportions of microhab¬
itat use by B. ferrugineus without discriminating by sex-age
class. The same was done for the differences between
proportions of behavior observed. Subsequently, a GLMM
was performed to determine whether there were differences in
proportions between classes within each microhabitat, using
site, month and sex-age class as classification variables; sex-
age class was selected as a fixed factor and site and month as
random factors. The same analysis was performed for
behavior.

Finally, a bi-plot obtained from multiple correspondence
analyses (MCA) was performed to explore the associations
between microhabitat, behavior and sex-age class. MCA takes
multiple categorical variables and seeks to identify associa¬

tions between levels of those variables. MCA allows exploring
contingency tables by means of the decomposition of chi-
square matrix. The contributions of each axis are indicated as
a percentage of inertia (Abdi & Valentin 2007). The MCA
results allow all the variables (behavior, microhabitat, sex-age
class) to be observed together. Analyses were performed for
the total sample and for each site separately to detect changes
in associations due to the effects of site. Microhabitat,
behavior and sex-age class were selected as classification
criteria and frequency as the combination of these variables.

RESULTS

Temporal distribution of sex-age class. — We found a
significant interaction between sex-age class and month for
scorpion counts (Wald test, = 25.60, P = 0.0003). Due to the
presence of the interaction, we performed a Fisher’s LSD test
for the combination of sex-age class and month. Females and
juveniles had a higher surface activity than males in
November. In January, the majority of active individuals that
we saw were juveniles. In February, we observed more males
and juveniles than females (Fig. 3). December showed no
significant differences between the three classes. In general, the
average number of active individuals of all three sex-age
classes decreased from November through January, rising
again in February (Fig. 3).

Microhabitat preference and behaviors associated with each
sex-age class. — Microhabitat availability: Soil was the most
abundant microhabitat in the study area (54.9%), while leaf
litter (23.7%) and vegetation (21.3%) were similar in propor¬
tion. Soil was also the most abundant microhabitat in the
three sampled sites (from 50.4 to 60.2%, see Table 2), followed
by leaf litter in the mature forest site, and vegetation in the
secondary forest. In the forest with livestock, there were
similar percentages of both vegetation and leaf Utter (Table 2).

Microhabitat preference: The results showed a significant
difference between proportions of microhabitat used for total
scorpions (Chi-square test, x" = 702.32, P < 0.0001). The most
frequently used microhabitat for total scorpions was soil
(64.8%), while leaf litter (18.1%) and vegetation (17.1%) were
used in similar proportions, matching microhabitat availabil¬
ity. Table 3 shows the number of individuals of each sex-age
class in different microhabitats at each site.

However, we detected significant differences between counts
of sex-age classes in leaf Utter and vegetation microhabitats
(Table 4, Fig. 4). Males and females were significantly more
abundant than juveniles in the leaf litter microhabitat.
Juveniles were significantly more abundant in vegetation,
followed by females and then the males (Table 4). No
significant differences were found between the classes in the
use of soil (Table 4).

Frequencies of behavior: The results showed a significant
difference in proportions of observed behavior for total
scorpions (Chi-square test, x^ = 3693.46, P < 0.0001). Ambush
behavior was the most frequently observed (68.3%), followed
by walking (20.6%) and less frequently feeding (7.6%), door¬
keeping (2.7%) and courting (0.8%). Table 5 shows the number
of individuals in each sex-age class performing different
behaviors at each site.

In the analysis of sex-age classes and behaviors, significant
differences were detected for some behaviors (Table 6, Fig. 5).

NIME ET AL.— MICROHABITAT USE AND BEHAVIOR OF A SCORPION SPECIES

239

0.90

E

o

'a.

L.

o

o

m

N—

O

<u

Q

c

c

3

eg

Month

Figure 3. — Number of Brachistostenms ferrugineiis scorpions, males (squares), females (circles) and juveniles (triangles), each month in the
Chancani reserve. Mean expressed as average number of scorpions by transect (± SE). Common letters are not statistically different (Fisher’s
LSD test, p > 0.05).

Ambush behavior was performed more frequently by females
and juveniles than males. Juveniles were observed feeding
more frequently than adults (males and females), and male
adults did so less frequently than females. Females were
observed more doorkeeping than males and juveniles. Most of
the time, the females were found with the metasoma outside
the burrow, as if entering the burrow (in 93% of observations).
Courtship behavior was observed in six pairs of scorpions.

Combination of microhabitat, behavior and sex-age class. —
Significant differences were observed between proportions of
microhabitats used and sex-age class (Chi-square test, =
777.38, P < 0.0001), and between behavior and sex-age class
(Chi-square test, — 3188.31, P < 0.0001). The bi-plot
obtained by multiple correspondence analyses, performed for
the total sample using sex-age class, behavior and microhab¬
itat, reveals that juveniles were more likely to be feeding on
vegetation (Fig. 6). While the majority of males were observed
walking on the leaf litter, the females were related to ambush
on soil (Fig. 6). Even when the four sites were analyzed
separately, the pattern observed was very similar.

Table 2. — Percentage of microhabitat available (mean ± SD) in
three sampled sites of the Chancani reserve.

Site Soil Leaf litter Vegetation

Mature forest 50.4 ± 16.1 30.6 ± 14.4 18.7 ± 13.5

Secondary forest 53.9 ± 14.3 20.0 ± 12.9 26.0 ± 13.9

Forest with livestock 60.2 ± 14.3 20.6 ± 11.5 19.2 ± 8.6

DISCUSSION

Temporal distribution of sex-age class. The surface activity
of males, females and juveniles was higher in November, and
declined over the months in all three classes. However, all sex-
age classes remained active during all the months sampled.
The decrease was more marked in females; they did not have
the increase in February that was observed in the other classes
(Fig. 3). This may be because, after getting pregnant, the

Table 3. — Counts of Brachistosternus fernigineus occurrence in

each microhabitat within each site in Chancani
period of sampling.

reserve, during the

Site

Sex-age

class

Soil ]

Leaf litter

Vegetation

Total

Mature forest

Males

126

43

15

184

Females

108

41

47

196

Juveniles

104

43

83

230

Secondary forest

Males

93

24

5

122

Females

125

38

19

182

Juveniles

118

12

54

184

Forest with

Males

42

16

2

60

livestock

Females

64

27

7

98

Juveniles

73

7

20

100

Jarillal

Males

53

12

0

65

Females

30

9

3

42

Juveniles

56

5

7

68

Total

992

277

262

153!

240

JOURNAL OF ARACHNOLOGY

Table 4. — Generalized linear mixed models. Relationship between the surface activity of males, females and juveniles of Brachistosternus
ferriigineiis and each microhabitat used in the Chancani reserve. Average monthly scorpions ± standard error of the mean. Means with a letter in
common are not significantly different (Fisher’s LSD, P > 0.05).

Microhabitat

T

P

Males

Females

Juveniles

Soil

2.12

0.3463

40.99 ± 6.83 A

42.68 ± 7.10 A

45.82 ± 7.59 A

Leaf litter

12.93

0.0016

12.92 ± 2.08 A

]5.64± 2.42 A

9.11 ± 1.58 B

Vegetation

124.89

<0.0001

2.93 ± 0.74 C

10.12 ± 1.82 B

21.83 ± 3.48 A

females stay sheltered in their burrows (Mahsberg 2001;
Kaltsas et al. 2008).

The higher surface activity for the three classes in November
could be because, in this region of the southern hemisphere,
scorpions generally begin to leave their shelters or burrows
and surface during spring when temperatures begin rising
(Warburg & Polis 1990; Ojanguren-Affilastro 2005; Yamaguti
& Pinto-da-Rocha 2006; Nime et al. 2013) to begin feeding
after the winter and probably find a partner. We did not
observe marked differences in temporal distribution between
sex-age classes, as exists in many species of scorpions to avoid
intra- and interspecific competition and predation (Polis 1980,
1984; Due & Polis 1985; Polis & McCormick 1987).

Microhabitat preference and behaviors associated with each
sex-age class. — The microhabitat most used in all classes of B.
ferrugineus was soil, and ambush was the most common
behavior. When we examined all sex-age classes of scorpions
together, we found that microhabitats were used in proportion
to their availability. On the another hand, the most common
behavior expected was ambush, because active looking for prey
is not common in scorpions (McCormick & Polis 1990) since it
involves a significant expenditure of energy (Kaltsas et al.
2008). Kaltsas et al. (2008) found that the most common
behavior (over 84%) in looking for food for all sex-age classes

360-

Microhabitat

Figure 4. — Surface activity of males (black bars), females (dark
grey bars) and juveniles (light grey bars) of Brachistostenuis
ferrugineus in different microhabitats in the Chancani Reserve. Bars
represent total number of scorpions observed.

of Mesobuthus gibbosiis (Brulli, 1832) species was “sit-and-
wait” {ambush), coinciding with our observations.

However, the correspondence analysis clearly demonstrated
one combination of a behavior and microhabitat was
associated with each sex-age class. Male scorpions were
mostly seen walking on leaf litter, females were more likely
to be seen engaging in ambush behavior on soil, and juveniles
were associated with feeding in vegetation.

That walking behavior is more associated with males was
also observed in the species Centruroides vittatus (Say, 1821)
(Polis 1980; Yamashita 2004). Males of this species are more
active (54.4% walking on the soil surface) than females
(34.9%). Also, males had a greater displacement, with marked
individuals being found many meters away from the initial
site, while females were found a few meters from the site, even
after several weeks (Yamashita 2004). This increased move¬
ment of males is observed mainly in the breeding season as
they actively search for females to mate (Polis & Farley 1979;
Polis & Sissom 1990; Araujo et al. 2010).

In the present study, doorkeeping behavior was performed
more often by females than by males and juveniles. Similar
results were observed in females of M. gibbosus; these fed and
looked for prey at the entrance of their burrows {“door¬
keeping'' strategy) more than males and juveniles, who were
preferably near or far from their burrows (Kaltsas et al. 2008).

420

Ambush Feeding Walking Doorkeeping Courting
Behavior

Figure 5. — Surface activity of males (black bars), females (dark
grey bars) and juveniles (light grey bars) of Brachistosternus
ferrugineus in the performing of different behaviors in the Chancani
Reserve. Bars represent total number of scorpions observed.

NIME ET AL.— MICROHABITAT USE AND BEHAVIOR OF A SCORPION SPECIES

24!

Table 5. — Counts of Brachistosternus ferrugineus performing each behavior in each study site of the Chancani reserve.

Site

Sex-age class

Ambush

Feeding

Walking

Doorkeeping

Courting

Total

Mature forest

Males

114

9

57

1

3

184

Females

144

11

31

7

3

196

Juveniles

161

31

34

4

0

230

Secondary forest

Males

81

1

36

3

1

122

Females

119

9

41

12

1

182

Juveniles

125

22

36

1

0

184

Forest with livestock

Males

39

2

19

0

0

60

Females

59

10

22

7

0

98

Juveniles

65

15

17

3

0

100

Jarillal

Males

55

0

7

1

2

65

Females

29

2

7

2

2

42

Juveniles

54

4

9

1

0

68

Total

1045

116

316

42

12

1531

In the “doorkeeping’' hunting strategy, the scorpion is located
at the entrance to the burrow or refuge and waits for prey to
approach (Benton 2001). Most females of M. gibbosus had
recently mated. Therefore, staying close to their burrows is
probably due to the maternal protective instinct (Mahsberg
2001; Kaltsas et al. 2008). Furthermore, foraging far from the
burrow requires tolerance to adverse environmental condi¬
tions and inter- and intraspecific competition. In the open
area, where males and juveniles of M. gibbosus feed mainly
using the “sii and wait" strategy, temperature and relative
humidity are comparatively lower, wind speed is higher and
the moon is an important factor, while in the burrows of
females, generally under the vegetation, environmental condi¬
tions are better (Kaltsas et al. 2008). Probably staying at the
entrance is advantageous as there is a microclimate inside and
environmental conditions are more favorable, and they have a
shelter near to hide from predators. The females of M.
gibbosus observed at the entrance of their burrows had only
their pedipalps and sometimes their prosoma visible. In our
study, the most common behavior of females of B. ferrugineus
when doing doorkeeping, was at the entrance of their burrows
with their metasoma outside, as if entering the burrow (in 93%
of observations). The reasons why scorpions prefer a
backwards position at the burrow entrance are unknown;
they may have been entering in their burrows in flight after
sensing the approach of collectors. Burrowing scorpions are
very sensitive to vibrations in the ground (Warburg & Polis
1990).

We found the juveniles associated with feeding in vegetation.
The behavior of climbing the vegetation has been previously
observed in other species of scorpions (Williams 1970; Polis

1979; Bradley 1988; Cao 1993; Skutelsky 1996; Brown &
O’Connell 2000; McReynolds 2004, 2008). In Paruroclonus
utahensis (Williams, 1968), more juveniles than adults were
observed in the vegetation (Bradley 1988). Juveniles of Buthus
occitanus (Amoreux, 1789) were found in the bushes at a rate
ten times higher than that of adults (Skutelsky 1996).

Although the reason for climbing behavior in the vegetation
is unclear, there are some hypotheses such as decreasing the
risk of predation, or increasing feeding success by foraging in
an area with higher prey availability (Bradley 1988; Polis 1990;
Brown & O'Connell 2000). The first hypothesis suggests that
climbing is a behavior to avoid predation and assumes that the
risk of predation is lower in vegetation than on the soil. Two
observations are consistent with this hypothesis (Brown &
O’Connell 2000). First, climbing has been seen generally in
small species and juveniles of larger species (Polis 1979;
Bradley 1988; Skutelsky 1996). In this study, B. ferrugineus is
one of the smallest species in the area and the only one that we
observed to climb vegetation. As these species or individuals
probably have a wide range of predators that live on the soil
(including larger intra- and interspecific scorpions; Polis &
McCormick 1987), climbing could reduce meeting potential
predators. Also, it was noted that C. vittalus moves on to
vegetation during the phase of the moon with greater light
intensity (50-100%) (McReynolds 2004). In open areas,
scorpions are more visible to predators at night and so such
a change in microhabitat use during the lunar cycle behavior
may reduce the risk of predation when the illumination of the
moon is high (McReynolds 2004). The present study could not
test this, because sampling was always performed on moonless
nights. Second, some species have been seen to take prey

Table 6. — Generalized linear mixed models. Relationship between the surface activity of males, females and juveniles of Brachistosternus
ferrugineus in each behavior observed in the Chancani reserve. Average monthly scorpions ± standard error. Means with a letter in common are
not significantly different (Fisher’s LSD, P > 0.05).

Behavior

P

Males

Females

Juveniles

Ambush

19.51

0.0001

38.48 ± 5.94 B

46.74 ± 7.12 A

53.93 ± 8.15 A

Feeding

49.33

<0.0001

1.53 ± 0.53 C

4.09 ± 1.08 B

9.19 ± 2.11 A

Walking

2.73

0.2548

11.67 ± 5.19 A

9.90 ± 4.42 A

9.41 ± 4.21 A

Doorkeeping

19.19

<0.0001

0.60 ± 0.30 B

3.24 ± 1.00 A

1.08 ± 0.44 B

Courting

9.73

0.0077

0.32 ± 0.26 A

0.32 ± 0.26 A

0 ± 0 B

242

JOURNAL OF ARACHNOLOGY

Figure 6. — Bi-plot obtained by multiple correspondence analysis. The principal axes represents the gradient in the relationship among the
categorical variables sex-age class, behavior and microhabitat of Brachistostemus ferrugineiis., categories close in the graph are more related.

captured on soil to vegetation before consumption, perhaps in
order to reduce the chance of encountering a predator that
might cause death or the loss of the prey while trying to escape
(Poiis 1979; Cao 1993; Brown & O’Connell 2000). In
Smeringiirus mesaensis this was age-specific behavior, with a
significantly higher proportion of juveniles than of adults
consuming prey in the vegetation (Poiis 1979). In the present
study, after the juveniles, females of B. ferrugineiis were more
frequently observed in the vegetation. The same occurs in C.
viltatiis, with females climbing more than males (Brown &
O’Connell 2000; Yamashita 2004).

The second hypothesis suggests that prey abundance is
higher in the vegetation, so feeding there would be more
energy efficient (Bradley 1988; Poiis 1990). However, although
scorpions have been observed feeding while on vegetation,
evidence of active foraging there is scarce (Poiis 1990;
Skutelsky 1996).

Possibly the vegetation, which creates a more complex
environment than environmental deserts, offers juveniles more
opportunities for hiding and reducing overlap with adults
(Hofer et al. 1996). Habitat complexities reduce niche overlaps
and may reduce the need of temporary displacement
(Yamashita 2004). Based on our results, we hypothesize that
the behavior of climbing into vegetation is performed by
juveniles for the purpose of feeding without risk of losing the
prey and avoiding being preyed on in turn. However, this
hypothesis requires testing.

Our hypothesis that sex-age classes of B. ferrugineiis use
different microhabitats while showing temporal displacement
is rejected; sex-age classes did use different microhabitats, but
without temporal displacement. We conclude that the
microhabitat use and behavior frequencies were highly
dependent on developmental stage and sex. The differences
observed may facilitate the age-sex class coexistence of B.
ferrugineiis and may reduce the need of temporary displace¬
ment due to the risk of predation and competition for feeding.
Also, this species is the only one that uses vegetation in the
study area, and this possibility of using different niches could

be one reason why B. fernigineus is the most abundant and
conspicuous species in the area, despite being physically one of
the smallest (Nime et al. 2013). The findings of this study on
the ecology and behavior of this widespread species are
therefore important for understanding the processes of intra¬
specific coexistence and can contribute to a greater under¬
standing of the structure of arthropod communities in the
Argentine Arid Chaco, and to general knowledge of scorpion
ecology.

ACKNOWLEDGMENTS

We are grateful to the Secretaria de Ambiente (Gobierno de
la Provincia de Cordoba), for allowing access to work in the
Chancam' Reserve. We thank Jose Gonzalez for assisting us in
the field and Joss Heywood for help with the English language.
We thank Eugenia Romero for the picture of a B . ferrugineus
couple. We thank two anonymous reviewers for comments
that improved the manuscript. This research was supported by
a doctoral grant from the Consejo Nacional de Investigaciones
Cientificas y Tecnicas, Argentina (CONICET) to MN.
Fieldwork was supported by a Rufford Small Grants
Foundation award to MN, and by SECYT (UNC) grant
214/10 to CIM. CIM is a CONICET researcher.

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Araujo, C.S., D.M. Candido, H.F.P. Araujo, S.C. de Dias & A.
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2016. Journal of Arachnology 44:245-246

SHORT COMMUNICATION

Leucism in Titym pmillus (Scorpiones: Buthidae): Report of a rare event in scorpions

A.F.A. Lira', L.M. Pordeus' and C.M.R. Albuquerque^: 'Universidade Federal de Pernambuco, Centro de Ciencias
Biologicas, Departamento de Zoologia, Programa de Pos-Graduagao em Biologia Animal. Rua Prof. Moraes Rego S/N,
Cidade Universitaria, 50570-420, Recife, PE, Brazil. E-mail: andref.lira@gmail.com; “Universidade Federal de
Pernambuco, Centro de Ciencias Biologicas, Departamento de Zoologia. Rua Prof. Moraes Rego S/N, Cidade
Universitaria, 50570-420. Recife, PE, Brazil

Abstract. Leucism is a congenital disorder in which the individual is born with partial hypopigmentation. It is quite
common in vertebrates, but rare in invertebrates, especially in arachnids like scorpions. This paper presents the first record of
this congenital disorder to be observed in the order Scorpiones. During field studies in the Area de Conservafdo Aldeia-
Beberibe, a set of Atlantic forest fragments of 31,634 hectares, we collected a pregnant leucistic female Tityus pmillus Pocock,

1 893. In this female, the variegated pattern described for the species was a lighter color than normal. The animal produced 10
normal juveniles (not leucistics). In addition, we analyzed 1,164 specimens from 17 populations deposited in the CA-UFPE to
verify the frequency of leucism; there were no scorpions with leucism within the analyzed populations. Thus, a break in
variegated pattern, as with the leucism described in this study, may increase the mortality rate due to predation.

Key words: Brazilian Atlantic Forest, color pattern, depigmentation degree

Color patterns in scorpions have often attracted the attention of
researchers, principally for ecological and taxonomical studies
(Harington 1984; Lourenfo & Cloudsley-Thompson 1996; Lourenfo
2002; Vignoli et al. 2005; Olivero et al. 2012). Coloration patterns in
these arachnids can vary from pale yellow to black, and the presence
of sub-cuticular pigments form different configurations such as
longitudinal or confluent bands (Louren^o 2002). Variations from
these patterns are well documented for these arachnids, producing
different morphology types (‘morphs’) for the same species (Lamoral
1979; Williams 1980; Flarington 1984; Vignoli et al. 2005).

Environmental factors seem to play a role in many scorpions’ color
variations (Louren^o & Cloudsley-Thompson 1996; Lourengo 2002).
For example, with the buthid scorpions Tityus costatus (Karsch, 1879)
found in the southern portion of the Brazilian Atlantic forest, those
that live at sea level (dry environment) are ‘light morph,’ whereas
individuals that occur at 1000 m above sea level (wet environment) are
‘darker morph’ (Lourengo 2002). Olivero et al. (2012) also found
color variation within six Argentinean populations of the bothriurid
scorpion Bothriwus honarieusis (C.L. Koch, 1842); these authors also
reported that scorpions from wet sites are darker than specimens
found in drier sites. Another form of variation in scorpion coloration
is a complete absence of pigmentation, commonly found in animals
that inhabit caves (Mitchell 1968, 1972; Francke 1977, 1978).

In some cases, however, atypical coloration can occur without
environmental influences, due to an excess (melanism) or an absence
(albinism) of color pigment in part or all of the body (Sanchez-
Hernandez et al. 2012). Albinism is a very rare event in scorpions,
being reported in the literature only for the Australian scorpion
Urodacus yascbenkoi (Birula, 1903) (Locket 1986). This author
reported the presence of two albino specimens from the south of
Australia, and also compared the eye structure between normal and
albino animals, finding abnormalities in the eyes of albino scorpions.
Albinism is an extreme form of an absence of color pigmentation in
the body, but some animals show a partial hypopigmentary
congenital disorder called ‘leucism’ (Herreid & Davies 1960), which
is very common in vertebrates (Sanchez-Hernandez et al. 2012) but
not in invertebrates. Here, a case of leucism in the scorpion Tityus
pusillus Pocock, 1893 is described, together with data on its offspring
and the frequency of occurrence in the population.

This is a small ambush predator and the most common scorpion
species in the northeast Brazilian Atlantic forest (Louren90 2002; Lira
et al. 2013; Lira & Albuquerque 2014). Tityus pusillus is typically found
inhabiting the forest floor, with its abundance correlated with climatic
conditions and microhabitat structure (Lira et al. 2013, 2015). The
species possesses a yellow basal color and brownish variegated
coloration (Louren^o & Cloudsley-Thompson 1996; Louren90 2002).

Leucism in T. pusillus was recorded in a pregnant female collected
during a nocturnal field study conducted in the Area de Preserva9ao
Ambiental Aldeia-Beberibe, a set of Atlantic forest fragments of 31,634
hectares (7° 54' 48"S 35° 2' 36' W) (CPRH 2015). The leucistic individual
was maintained in laboratory conditions and gave birth to 10 normal
(non-leucistic) young. Confirmation of female gender was carried out
according to Louren90 (2002), following death after the young dispersed
from her dorsum. The specimen was then deposited in the Arachnolog-
ical Collection of Universidade Federal de Pernambuco, Brazil.

Leucism (Fig. lA) was characterized by a lighter scale of
pigmentation of the variegated pattern of coloration common for
the species in relation to the normal color range of individuals (Fig.
IB & C). To verify the frequency of leucism in this species, we
examined 1,164 individuals from 17 different populations deposited in
the Arachnological Collection (Table 1). Except for the leucistic
female, no other record of leucism among individuals from the
different populations analyzed was registered.

Scorpions’ coloration constitutes the first line of defense of these
animals, whose color patterns primarily have cryptic significance (Polis
1990; Louren90 & Cloudsley-Thompson 1996). These authors also
suggest that most scorpion species that inhabit forests present two
patterns of coloration, darker (darker brownish or black) and
variegated (mottled). These colorations are camouflage within the
darker environment found in forest interiors (Cloudsley-Thompson
1993a, b). Thus, the sedentary behavior shown by T. pusillus specimens
(Lira et al. 2013) associated with variegated coloration may work as an
effective defense against potential predators. Consequently, lighter
morph types, such as the leucistic coloring described in the present
study, would be more exposed to predation due to the lack of cryptic
protection. In conclusion, our study describes for the first time the
occurrence of leucism in scorpions; this event is rare, corresponding to
a rate of just 0.06% in the population examined.

245

246

JOURNAL OF ARACHNOLOGY

Figure 1. — Variation of color pattern of the scorpion, Tityus pusillus Pocock, 1893. A) Leucistic individual. B) Lighter individual. C) Normal
individual.

Table 1. — Number of
populations.

scorpions examined in the

seventeen

Location

Coordinates

Scorpions

examined

Paudalho

7°54'48"S, 35°02'36"W

60

Agua Preta

8°41'31.4'’S, 35°29'49"W

2

Jaqueira

8°43'03.9"S, 35°50'2i.6"W

104

Ipojuca

8°31'48"S, 35°01'05"W

24

A,breu e Lima

7N6'55"S, 35°09'02"W

101

Recife

8°00'00"S, 34°56'0'0"W

50

Tamandare

8°43'43"S, 35°10'39.8"W

60

Moreno

8°06'38.1"S, 35°06'56.4"W

165

Timbauba

7°36'36.6"S, 35°22'46.6"W

80

Igarassu

8°00'05.8"S, 34°52'23.1"W

85

Bui'que

8°35'08.2"S, 37'I4'29.3"W

48

Gravata

8°11TI.8"S, 35°33'51.9"W

37

Sao Bento do Una

8°31'45.7"S, 36°27'23.8"W

6

Sirinhaem

8°38'59.0"S, 35°I0'26.6"W

96

Jaboatao dos Guararapes

8°02'34"S, 35°02'22"W

86

Rio Formoso

8°32'07.6"S, 35°05'53"W

150

Caruaru

8°22'09"S, 36°05'00"W

10

ACKNOWLEDGMENTS

We are very grateful to Coordenagao de Aperfei^oamento de
Pessoal de Mvel Superior (CAPES) for a doctoral scholarship to
AFAL and Conselho Nacional de Desenvolvimento Cientifico e
Tecnologico (CNPq) for a masteral scholarship to LMP. We are also
very grateful to Juarez Pordeus for permission to use the area to
collect samples.

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Manuscript received 13 January 2016, revised 14 April 2016.

2016. Journal of Arachnology 44:247-250

SHORT COMMUNICATION

Egg sac parasitism: how important are parasitoids in the range expansion of the wasp spider

Argiope bmennichP.

Wioletta Wawer* and Agata Kostro-Ambroziak^; ’Museum and Institute of Zoology Polish Academy of Sciences, Wiicza
64, 00-679 Warszawa, Poland. E-mail; wawer@miiz.waw.pl; "Department of Invertebrate Zoology, Institute of Biology,
University of Bialystok, Ciolkowskiego IJ, 15-245 Biaiystok, Poland

Abstract. During recent decades, the wasp spider, Argiope hruennichi (Scopoli, 1772), has expanded relatively quickly
towards north Europe. As a consequence of its spreading, it is newly exposed to various factors of selection. We studied the
impact of egg sac parasitoids on the mortality of A. bruennichi in three regions differing in climate conditions and time of
settling by this spider. Parasitism of wasp spider egg sacs was relatively low (0-3.9%) and no significant differences between
studied regions were found. One primary parasitoid, Tromatobia onuita, was reared; in approximately 60% of these
! parasitized cocoons, the entire content of the egg sac was destroyed.

• Keywords: Pseudohyperparasitoid, host, Tromatobia onuita, Pediobius brachycerus

‘ The wasp spider, Argiope bruennichi (Scopoli, 1772) is a Palaearctic
species that has been expanding northward from the Mediterranean
(Kumschick et al. 2011); its European range currently also includes
Scandinavia (Jonsson & Wilander 1999; Bratli & Hansen 2004;

Koponen et al. 2007). In Poland, the wasp spider was first recorded in
1935 (Urbanski 1935), and, for several decades, its area was restricted
only to the west and south-eastern parts of the country. Since the
1990s, A. bruennichi has rapidly spread towards the north. Now, the
wasp spider occurs throughout Poland and is numerous everywhere,
i except for the higher elevation of the mountains (Wawer 2012). The
expansion of some European species to the north may be connected
with global warming (e.g., Hughes 2000; Walther et al. 2002; Hickling
et al. 2006). In the case of the thermophilous wasp spider, this may be
. one of the most important factors (Ivinskis et al. 2009), but other
factors may also favor its spreading, e.g., prolongation of the growing
: season, increasing the area of fallow lands or transport intensification

; causing passive dispersion (Guttmann 1979).

As a consequence of expansion, A. hruennichi is exposed to new
factors of selection (Leborgne & Pasquet 2005). Natural enemies have
a significant mortality impact on local spider populations (Polis et al.

1998), and parasitoids might be the most important of these (Foelix
2011). Among parasitoids, there are some that develop individually
within eggs (e.g., Scelonidae), or feed on spider egg masses (e.g.,

Mantispidae, Ichneumonidae, Phoridae), as well as ectoparasitoids
(e.g., Ichneumonidae) and endoparasitoids (e.g., Acroceridae) of
post-embryonic spiders — both spiderlings and mature spiders (Austin
1985; Fitton et al. 1987; Schlinger 1993; Allard & Robertson 2003;

Finch 2005). The mortality of eggs and spiderlings is considered to be
the most significant factor regarding the spider life cycle (Topping
1997). Parasitoids that are associated with the wasp spider are species
of the Ichneumonidae and Eulophidae (Hymenoptera). Among the
ichneumonid wasps, Tromatobia ornata (Gravenhorst, 1829) from the
Pimplinae (Rollard 1985, 1990) and Buathra tarsoleucos (Schrank,

1781) and Thaumatogelis gallicus (Seyrig, 1928) from the Cryptinae
have been recorded as parasitizing A. bruennichi (Fahringer 1922;

Schwarz 2001). These species develop by feeding on cocooned spider
eggs, but none of them are specific to A. bruennichi (Fitton et al. 1987;

Yu et al. 2012). The parasitic wasp from the Eulophidae, Pediobius
brachycerus (Thomson, 1878) is an obligatory hyperparasitoid
(secondary parasitoid) of spider egg sacs, which necessarily parasitizes
a spider’s primary parasitoids, including some species of ichneumonid
parasitoid wasps (Fitton et al. 1987; Kostro-Ambroziak & Wawer

2015). Here we studied the influence of egg sac parasitoids on the
mortality of A. bruennichi in regions differing by time of settling of
this spider and climatic conditions, based on populations from
Poland.

Investigations were carried out from 201 1 to 2013. Egg sacs of A.
hruennichi were collected from nine localities in three regions of
Poland: the Suwalki Lake District (SLDi: 54°8'14.88"N,
22°55'58.94"E; SLD2: 54°8'32.29"N, 22°50'55.37"E; SLD3:
54°5'25.23"N, 22°59'12.55"E), the Mazovian Lowland (MLl:
52°19'5.39"N, 20°52'32.29"E; ML2: 52°22'46.67"N, 20°47'47.04"E;
ML3: 52°0'29.66''N, 21°22T2.58"E), and the Sandomierz Valley
(SVl: 50°10'38.01"N, 21°43'12.09"E; SV2: 50°1 0'48.08 "N,
21°42'31.02"E; SV3: 50°8'57.20"N, 2r40'36.67"E) (Fig. 1).

In south-east Poland, the first individuals of A. bruennichi were
discovered in the 1960s (the Low Beskids) (Bednarz 1966). In the
Mazovian Lowland, A. bruennichi was observed for the first time in
1998 (Kajak & Luczak 2003). At that time, this species was considered
to be rare and endangered in Poland, which led to its protection by
law. In northern Poland (the Suwalki Lake District), the wasp spider
was observed for the first time in 2005 (W. Wawer, unpubl.).
Meanwhile, recent years have brought a wave of expansion of unusual
intensity, causing species dispersal and establishment across virtually
the entire country. The number of known locations doubled from
1990 to 2007 (Wawer 2014). The three regions mentioned above
(SLD, ML, SV) differ in climatic conditions — the region furthest to
the north is the coldest and probably due to this factor, it is
characterized by fewer A. bruennichi.

Egg sacs overwintered in natural conditions on plant leaves, ca. 20
cm above the ground, in open areas, mainly in meadows and on
agricultural wastelands. In April, they were collected and transported
in a plastic jar and kept at a constant temperature of 8 °C until the
dissection began. The egg sacs were opened at the end of April. Each
egg sac was examined under a microscope and cut by medical scissors.
Parasitized egg sacs were stored at room temperature on a piece of
cotton wool in a plastic jar (50 ml), and every day, a few drops of
water were added to preserve humidity. The adult parasitic wasps
emerged after about 10 days (April/May).

Adult wasps were identified by morphological characteristics with
reference to the taxonomic literature (Boucek 1965; Fitton et al.
1988). Additionally, pupal cases of parasitoids were confirmed by
DNA barcode sequences. Reference Sequences (RefSeq) were
obtained in this study. Genomic DNA was extracted using a Genomic

247

248

JOURNAL OF ARACHNOLOGY

Figure 1 . — Distribution of localities in Poland where the egg sacs
of Argiope bruennkhi were collected: the Suwalki Lake District
(SLD), the Mazovian Lowland (ML) and the Sandomierz Valley
(SV).

Mini Kit (A&A Biotechnology). The primers LepFl (5'-A.TTCAAC-
CAATCATAAAGATATTGG-3') and LepRl (5-TAAACTTCTG-
GATGTCCAAAAAATCA-3') were used for COI mtDNA fragment
amplification (650 pz) (Smith et al. 2009). PCR consisted of an initial
activation step at 95 °C for 1 5 min; 45 cycles of denaturation at 94°C
for 30 sec, annealing at 52 °C for 90 sec and extension at 72 °C for 60
sec and a final extension at 60 °C for 30 min. The sequence data for
the COI gene sequences were submitted to GenBank with the
accession number KU870312. All voucher specimens are deposited in
the collection of Museum and Institute of Zoology Polish Academy of
Sciences.

In total, 560 egg sacs of A. bruennkhi were examined. The degree of
parasitism of wasp spider egg sacs was 3.9% (Table 1). In the central
region ML, we noticed no infested egg sacs. No significant differences
between the other two regions (SLD and SV) were found (Fisher’s
Exact test, P = 0.21). One primary parasitoid, Tromatobia ornata, was
reared from 22 egg sacs and the secondary parasitoid Pediobius
brachycerus from 11 egg sacs (Table 1). The former parasitoids were
noticed as pupae (1-3 per sac) and the latter as larvae inside pupal
casings (1-8 per sac). Only two egg sacs produced two or three
individuals of T. ornata. The reproductive fitness of the A. bruennkhi
female was greater than zero in all cases of parasitization. In 41% of
parasitized cocoons, an average of 148 living nymphs of spiders per
egg sac were detected (SD =100.1; range = 1-220). The unparasitized

egg sacs either contained up to 642 living nymphs of the wasp spider,
or were damaged, empty or with undeveloped embryos. Both in
parasitized and unparasitized egg sacs, a portion of the spider eggs
failed to develop (59% and 35.3%, respectively), a statistically
significant difference (Chi-square test, X^i = 475.5, P < O.OOi).

The egg-sac of A. bruennkhi is under maternal care for a few days
(Leborgne & Pasquet 2005). The layer structure of the wasp spider
cocoon protects eggs and spiderlings from fluctuating temperatures
and desiccation, as well as acting as a mechanical barrier against
parasitoids and parasites (Hieber 1985, 1992; Bergthaler 1995).
Tromatobia ornata lays eggs into spider cocoons in reddish threads
close to the outer layer (Rollard 1990). Parasitism of the egg sac of A.
bruennkhi by T. ornata in Poland is distinctly lower (0-3.9%) than in
Germany (0-50%) (Sacher 2001) or France (8^4%) (Leborgne &
Pasquet 2005), where T. ornata is also a main parasitoid in egg sacs of
this spider species. The low degree of parasitism in Poland may be the
effect of a shift in the time of oviposition of host and parasitoid
caused by the recent spread of A. bruennkhi. Rollard (1987) revealed
that egg sacs were parasitized by T. ornata only by the time juveniles
emerged. The spider embryos develop in approximately 2 to 3 weeks
and nymphs hibernate during winter in the egg sac (Von Becker 1983;
Rollard 1987). Because of this, females of A. bruennkhi that lay their
eggs early avoid parasitoids and have higher reproductive success
(Leborgne & Pasquet 2005). Because T. ornata is relatively
widespread in Poland, it is probable that here it parasitizes other
hosts, both other spiders and moths (Yu et al. 2012), and the wasp
spider is not a limiting factor for this parasitoid.

Argiope bruennkhi lays on average 800 eggs per sac (Rollard 1985;
Kohler & Schaller 1987; Miyashita 1996). Larvae of T. ornata feed on
spider eggs and develop very quickly to fifth instars, and in this
inactive stage they overwinter inside the spiders’ egg sacs (Rollard
1985). Little is knov/n about the phenology of this parasitoid from
spring to autumn. Oehlke & Sacher (1991) indicated that T. ornata is
univoltine, but based on the biology of its other hosts (Yu et al. 2012),
e.g., Nuctenea umbratka (Clerck, 1757), it is highly probable that it is
at least bivoltine. Although T. ornata is known as a gregarious
parasitoid (Fitton et al. 1988; Sacher 1988, 2001), we recorded mainly
single specimens of it in egg sacs. We also noticed that not all of the
parasitized egg sacs were destroyed. Rollard (1985) indicated that
total destruction of spider eggs occurred only when there was more
than one larva of T. ornata in a cocoon. According to Cortes et al.
(2000), Tromatobia sp., as a parasitoid of Araneus granadensis
(Keyserling, 1864), also destroys only a portion of the contents of
the egg sac.

We recorded P. brachycerus as a gregarious parasitoid inside the
pupae of T. ornata. According to these data and the earlier suggestion
of Fitton et a!. (1987) that P. brachycerus attacks the primary
parasitoid during the pupal stage, we think it should be labelled as a
pseudohyperparasitoid. In contrast to hyperparasitoids, which
parasitize the larvae of other parasitoids while they are feeding on
or in the primary host, pseudohyperparasitoids attack the primary
parasitoid after it has completed feeding on its host (Quicke 2015). Of
course, detailed studies on the biology of this parasitoid wasp are
needed. Pediobius brachycerus is a parasitoid of some species of
Ichneumonidae which parasitize spider eggs (Kostro-Ambroziak &

Table 1. — Egg sac parasitism of Argiope bruennkhi in three regions differing in climate conditions and time of settling by the spiders: the
Suwalki Lake District (SLD), the Mazovian Lowland (ML) and the Sandomierz Valley (SV).

Region

N of studied egg sacs

N and (%) of egg sacs
parasitized by T. ornata

N and (%) of T. ornata pupae
parasited by P. brachycerus

Suwalki Lake District (SLD)

220

15 (6.8%)

6 (33.3%)

Mazovian Lowland (ML)

220

0

0

Sandomierz Valley (SV)

120

7 (5.8%)

6 (75%)

Total

560

22 (3.9%)

12 (46.15%)

WAWER & KOSTRO-AMBROZIAK— EGG SAC PARASITISM

Wawer 2015), but T. onuita is recorded for the first time as its
I secondary host.

To summarize, in our study the overall mortality of A. hruennichi
induced by its egg parasitoids was relatively low. Additionally, a high
level of undeveloped eggs both in the parasitized and unparasitized
egg sacs suggests that other factors, such as unfavorable wintering
conditions (temperature, humidity) or fungal penetration, may have a
significant impact on the mortality and life history of this range
expanding spider.

ACKNOWLEDGMENTS

• We would like to express special thanks to Beata Ostrowiecka
(Institute of Biology, University of Bialystok) for her help in
laboratory work. We are grateful to Dr. Giselher Grabenweger
(Agroscope, Institute for Sustainability Sciences, Zurich) for deter¬
mination of Eulophidae specimens.

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2016. Journal of Arachnology 44:251-253

SHORT COMMUNICATION

Endosymbiotic Rickettsiales (Alphaproteobacteria) from the spider genus Amaurobioides (Araneae:

Anyphaenidae)

F.S. Ceccarelli', C,R. Haddad^ and M.J. Ramirez’: 'Division de Aracnologia, Museo Argentine de Ciencias Naturales,
Av. Angel Gallardo 470, C1405DJR - Buenos Aires, Argentina; E-mail: saracecca@hotmail.com; ^Department of
Zoology & Entomology, University of the Free State, P. O. Box 339, Bloemfontein 9300, South Africa

Abstract. Endosymbiotic bacteria are commonly found in terrestrial arthropods and their effects have been studied
extensively. Here we present the first recorded case of endosymbiotic bacteria found in the spider family Anyphaenidae. A
fragment of the cytochrome oxidase c subunit I “barcoding” region belonging to unidentified Rickettsiales, presumably
belonging to the genus Rickettsia, was sequenced from six individuals of Amaurobioides africana Hewitt, 1917.

Keywords: Bacteria, barcoding, intertidal

The gram-negative proteobacteria are one of the most widespread
and diverse groups of bacteria, including medically important
pathogens, free-living nitrogen-fixing organisms, and the order
Rickettsiales, which contains obligate intracellular bacteria such as
Wolhachia and Rickettsia that can be found living inside the cells of
terrestrial arthropods (Ferla et al. 2013). These two genera have been
the subject of numerous studies in arachnids, as they represent
important pathogens in some cases (e.g.. Paddock et al. 2010), while
in others they are endosymbionts that manipulate their host’s
physiology, behavior and/or bias the host’s sex ratio to favor their
transmission (Rowley et al. 2004; Goodacre et al. 2006; Duron et al.
2008; Gunnarsson et al. 2009; Wang et al 2010; Vanthournout et al.
2011; Goodacre & Martin 2012, 2013). R/rA'ctti/a-infected spiders
have also been shown to display increased long-distance dispersal
tendencies (Goodacre et al. 2009). Bacterial endosymbionts are
usually transferred vertically in spiders, although there is evidence
for horizontal transfer in closely related taxa (Baldo et al. 2008).

Early methods of detection of bacterial endosymbionts in insects and
spiders relied on staining techniques (Cowdry 1923). With the advent of
PCR-sequencing techniques, molecular detection of specific endosym¬
bionts was made possible with relative ease, in the case of spiders
resulting in targeted studies (e.g., Baldo et al. 2008; Jin et al. 2013) or
sometimes as a byproduct of a study with a different aim (e.g., Rezac et
al. 2014 in Dysdera microdouta Gasparo, 2014). With bacteria-specific
primers, amplification of endosymbiont DNA from potential host
tissue provides positive results only for infected hosts. On the other
hand, more “universal” primers may find the annealing sites in both
host and symbiont, if said sites are conserved enough for the genomic
region. One such case appears to be the “barcoding” fragment of the
Cytochrome Oxidase C subunit I (COI), a protein-coding gene that
appears to have its origins deep within the origins of life on the planet
(Castresana et al. 1994). The fact that COI has highly conserved
regions means that certain primers can be used across a wide range of
organisms to amplify the same gene region. This is advantageous if the
tissue used for DNA extraction exclusively belongs to one species.
However, in the case of organisms hosting endosymbionts, this could
be seen as a complication, although for large-scale barcoding studies, it
has been shown to be manageable (Smith et al. 2012).

As part of a larger study on the intertidal anyphaenid genus
Amaurobioides O. Pickard-Cambridge, 1883 (Araneae: Anyphaeni¬
dae) (Ceccarelli et al. in prep.), DNA was extracted from leg tissue of
19 individuals of A. africana Hewitt, 1917 and approximately 630
base-pairs of the COI gene fragment were amplified and sequenced
using the primers LCOI 1490 (Folmer et al. 1994) and HCOoutout

(Prendini et al. 2005). For six out of the 19 individuals, the sequenced
COI region did not belong to the targeted host species, but to an
unknown species of the order Rickettsiales, presumed to be an
intracellular symbiont. There was no variation in the nucleotides of
the six sequences obtained, indicating that the A. africana individuals
in this study were all infected with the same bacterial species. The
sampling localities of the six infected specimens are shown in Fig. la
and the COI sequences have been deposited in GenBank (accession
numbers KU600819-KU600824).

The identification of the Rickettisales COI sequences amplified in
this study was based on comparisons to sequences available in the
public databases International Barcode of Life Database (BOLD
systems; http://www.boldsystems.org/) and GenBank (http://www.
ncbi.nlm.nih.gov/genbank/). The information available from BOLD
was minimal (the level of identification provided was to the order
Rickettsiales) and there were cases of misidentification in GenBank,
as the BLAST-quened sequences from this study were 99% identical
to COI sequences labelled as “Hymenoptera sp.”, while the correct
identification as proteobacteria started at 90% identity.

At this point, questions relating to why non-bacterial primers
preferentially amplified COI regions of endosymbionts rather than host
DNA in this study — even resulting in clean sequences (rather than a mix
of host and symbiont amplicon) — remain largely unanswered. A visual
inspection of the priming sites revealed that 9 Rickettsiales COI
sequences downloaded from GenBank had 80-90% identity in the last
10 base-pairs towards the 3' end of the forward and reverse primers. This
base-pair identity, coupled with the possibility of a very high number of
endosymbiotic Rickettsiales, may have been enough to give initial
preference and later exclusivity to the bacterial over the host DNA for
primer annealing and DNA amplification during PCR. As mentioned
earlier, the presence of endosymbionts is not thought to interfere with
DNA barcoding of arthropods when using universal primers (Smith et
al. 2012). However, the possibility still exists that COI sequences of
endosymbionts are obtained when in fact the target organism is the host,
as shown in this study, along with other isolated cases (e.g., Rezac et al.
2014) and the presence of misidentified Rickettsiales COI sequences in
GenBank (where the target organism was the host and thus the
identification was placed as Hymenoptera sp.).

Of the COI sequences in GenBank from Rickettsiales (all belonging
to the genus Rickettsia) with an identity score >75% for the sequences
from this study, ten were selected for Bayesian phylogenetic analyses,
along with a sequence from a closely related genus (Orientia, based on
Weinert et al. 2009), four sequences of Wolhachia and a sequence
belonging to Anaplasma, to root the tree. The COI sequences were

251

252

JOURNAL OF ARACHNOLOGY

South Africa

iKiniiaBgi

30°Wi

qiWolbachia sp. (gb JN625825)
W^Wolbachia sp. (gb JN625g52)
Ti Wolbachia sp. (gb KF490376)

Wolbachia sp. feb JN625947)

R felis (gb CP000053)

feR monacensis (emb LN794217) •

^R rickeUsii (gb CP006010)
lR a/far/ (gb CP000847)
gR australis (gb CP003338) i

» R prowazekii (emb AJ235271)i
■-R. canadensis (gb CP003304)
R£ie///7(gbCP000849)

NEW SEQUENCE (gb KU600822)
lEW SEQUENCE (gb KU600824)

EW SEQUENCE (gb KU600823)

EW SEQUENCE (gb KU600820)

EW SEQUENCE (gb KU600821)

New sequence (gb KUSOOSIS)

Rickettsia sp. (gb KF005604)

Rickettsia sp. (emb HE583223),

/ (emb AM494475) "

0.09 subst./site

Anapfasma marginale (gb CP001079)

Figure 1. — a. Map showing localities where Rickettsiales-infected specimens of Amaurobioides africana were collected; b. Bayesian
phylogenetic tree of COI sequences for selected Rickettsia species and specimens from closely related genera, obtained from the NCBI database.
Nodal support in Bayesian posterior probability (PP) represented by filled (0.95 < PP <= 1) and empty {0.9 < PP <= 1) circles. GenBank
accession numbers are shown after taxon names in brackets. Terminal taxa labelled as NEW SEQUENCE are from this study. Colored boxes
around taxon names represent host classes (blue = Insecta; red and yellow = Arachnida; arachnid orders: red = Acari; yellow = Araneae); c.
Genera! habitus of A. africana', d-e. Rock faces in the intertidal zone at De Hoop Nature Reserve, showing abandoned retreats (arrows) of A.
africana (d), and at Jeffrey’s Bay, showing sealed retreats of A. africana (e). Photos: C.R. Haddad.

aligned using TranslatorX (Abascal et a!. 2010) and a partitioning
strategy along with nucleotide substitution models for each partition
(TrNef+I, TrN+G and TrN+I-K5 for COI T', 2"^^ and 3''*^ codon
positions, respectively) chosen by PartitionFinder v.l.l.I (Lanfear et al.
2012). A Bayesian phylogenetic tree was obtained by forming a
consensus of 20,000 trees (minus 10% burn-in) from 20 million
generations of Markov Chain Monte Carlo simulations performed in
MrBayes v. 3.2.3 (Ronquist et at. 2012). Based on the phylogenetic tree
obtained (Fig. lb), the sequences from this study belong to an

unidentified Rickettsia species, closely related to a Rickettsia species
infecting the spider Dysdera microdonta. Apart from being confident
that A. africana can harbor the endosymbiont Rickettsia, a more in-
depth study is required to fully understand the distribution, ecology
and biology of the endosymbionts detected in this study.

Of particular interest in this relationship is the biology of the host
spiders, which are exclusively found in the intertidal zone of rocky
shores in marine habitats (Fig. !c). The spiders regularly construct
their silken retreats in rock faces (Fig. Id, e), which they seal with silk

CECCARELLI ET AL.— RICKETTSIALES IN AMAUROBIOIDES AFRICAN A

253

during high tide to avoid immersion in salt water, emerging at low
tide to forage (Lamoral 1968). Therefore, transmission of the
symbionts through the water medium in which the spiders occur
seems unlikely. Apart from the most likely transmission pathway of
the endosymbionts in A. africana being vertical transmission, the
possibility of horizontal transmission should not be ruled out at this
stage; a plausible additional explanation may be the transmission of
the endosymbionts during ingestion of prey tissues, such as isopods,
amphipods and dipterans that occur in the intertidal zone (Lamoral
1968). Further, the possibility that the same endosymbionts may
infect various other spiders and pseudoscorpions occurring in the
^ intertidal zone in South Africa (Lamoral 1968; Haddad & Dippenaar-
: Schoeman 2009; Larsen 2012; Owen et al. 2014), and the platygastrid
wasp egg parasitoid of the only other truly exclusive intertidal spider
in South Africa, Desis fonnidabilis (O. Pickard-Cambridge, 1890)

: (Desidae), viz. Echthrodesis lamorali Masner, 1968 (Owen et al. 2014),
requires further investigation. Nevertheless, this study represents the
first record of Rickettsiales in anyphaenids and is a contribution
towards a broader understanding of proteobacteria in spiders.

, ACKNOWLEDGMENTS

Candice Owen (Rhodes University, Grahamstown) and Dawn
Larsen (Iziko South African Museum, Cape Town) are thanked for
the loan of recently collected specimens that contributed towards this
study. This study was supported from grant PICT 2011-1007 from
ANPCyT and a postdoctoral fellowship from CONICET.

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Manuscript received 15 December 2015, revised 25 January 2016.

2016. Journal of Arachnology 44:254-255

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Binford, G. 2013. The evolution of a toxic enzyme in sicariid
spiders. Pp. 229-240. In Spider Ecophysiology. (W.
Nentwig, ed.). Springer-Verlag, Heidelberg.

Cushing, P.E., P. Casto, E.D. Knowlton, S. Royer, D. Laudier,
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Harvey, M.S. & G. Du Preez. 2014. A new troglobitic
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World Spider Catalog. 2015. World Spider Catalog. Version
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Roewer, C.F. 1954. Katalog der Araneae, Volume 2a. Institut
Royal des Sciences Naturelles de Belgique, Bruxelles.
Rubio, G.D., M.O. Arbino & P.E. Cushing. 2013. Ant
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Volume 44

CONTENTS

Journal of Arachnology

SKrTHSONIAN LIBHAHIE8

3 9088 01878 7838

Number 2

Featured Articles

Revision of the Nearctic Eratigena and Tegenaria species (Araneae: Agelenidae)

by Angelo Bolzern & Ambros Hinggi . . . ..... . . . . . . 105

The Mediterranean recluse spider Loxosceles rufescens (Dufour, 1820) (Araneae: Sicariidae) established in a natural
cave in Thailand

by Narin Chomphuphuang, Sureerat Deowanish, Chaowallt Songsangchote, Varat Sivayyapram,

Panupong Thongprem & Natapot Warrit. . . . . . . . . . 142

Chromosomal analyses of Salticinae and Lyssomaninae reveal a broad occurrence of the 2nd= 28, X^X^O karyotype
within Salticidae

by Douglas Araujo, Mariana Bessa Sanches, Juliane da Silva Gonsalves Santana Lima,

Erica Vanessa JuMao do Nascimento, Andre Marsola Giroti, Antonio Domingos Brescovit,

Doralke Maria Celia & Marielle Cristina Schneider. . . . . . . . . . . . . . 148

The hemolymph vascular system in Araneus diadematus with special focus on intraspecific variability in artery
systems

by Jens Runge & Christian S. Wirkner. . . . . . . . . . . . . 153

Limb loss and limb regeneration of crab spiders Misumena vatia

by Douglass H. Morse . . . . . . . 165

Female feeding history impacts gonad development and reproductive timing in the wolf spider Schizocosa ocreata
(Hentz, 1844)

by Brian Moskalik & George W. Uetz . . . 171

Influence of predator cues on terminal investment in courtship by male Schizocosa ocreata (Hentz, 1 844) wolf spiders
(Araneae: Lycosidae)

by Benjamin Nickley, Diana Saintignon & J, Andrew Roberts . . . . . . 176

Spatial patterns and environmental determinants of community composition of web-building spiders in understory
across edges between rubber plantations and forests ji

by Booppa Petcharad, Tadashi Miyashita, George A. Gale, Sunthorn Sotthibandhu & Sara Bumruegsri . . 182

First record of a representative of Ballarrinae (Opiliones: Neopilionidae), Americovibfne remota sp. nov., from New
Zealand

by Christopher K. Taylor . . . . . . . . j . . . 194

Mechanical properties of male genitalia in Leiobunum harvestmen (Opiliones: Sclerolomatidae)

by Mercedes Burns & Jeffrey W. Shultz . . ;..... . 199

Mating behavior of the solitary neotropical harvestman Pachyloides thorellii (Arachnida: Opiliones)

by Estefania Stanley, Gabriel Francescoli & Carlos A. Toscano-Gadea . i . 210

The smallest known solifuge: Vempironiella aguilari, new genus and species of sun-slider (Solifugae: Mummuciidae)
from the coastal desert of Peru

by Ricardo Botero-Trujillo . . . . . . . . . 218

The first New World species of the pseudoscorpion family Feaellidae (Pseudoscorpiohes: Feaelloidea) from the
Brazilian Atlantic Forest

by Mark S. Harvey, Renata Andrade & Ricardo Pinto-da-Rocha . . . . . . . 227

Microhabitat use and behavior differ across sex-age classes in the scorpion Brachistosternus ferrugineus (Scorpiones:
Bothriuridae)

by M.F. Nime, F. Casanoves & C.I. Mattoni . . . . . . . 235

Short Communications

Leucism in Tityus pusillus (Scorpiones: Buthidae): report of a rare event in scorpions

by A.F.A. Lira, L.M. Pordeus & C.M.R. Albuquerque. . 245

Egg sac parasitism: how important are parasitoids in the range expansion of the wasp spider Argiope bruennichil

Wioletta Wawer & Agata Kostro-Ambrozlak . . . . . . . 247

Endosymbiotic Rickettsiales (Alphaproteobacteria) from the spider genus Amaurobioides (Araneae: Anyphaenidae)

F.S. Ceccarelli, C.R. Haddad & M.J. Ramfrez . . . 251

Instructions to Authors . . . . 254

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