(ISSN 0161-8202)

.A65?

Journal of
ARACHNOLOGY

PUBLISHED BY THE AMERICAN ARACHNOLOGICAL SOCIETY

VOLUME 42

2014

NUMBER 1

THE JOURNAL OF ARACHNOLOGY

EDITOR-IN-CHIEF: Robert B. Suter, Vassar College

MANAGING EDITOR: Richard S, Vetter, University of California-Riverside

SUBJECT EDITORS: Ecology — Stano Pekar, Masaryk University; Systematics — Mark Harvey, Western Aus-
tralian Museum and Matjaz Kuntner, Scientific Research Centre of the Slovenian Academy of Sciences and Arts;
Behavior — Elizabeth Jakob, University of Massachusetts Amherst; Morphology and Physiology — Jason Bond, Au-
burn University

EDITORIAL BOARD: Alan Cady, Miami University (Ohio); Jonathan Coddington, Smithsonian Institution;
William Eberhard, Universidad de Costa Rica; Rosemary Gillespie, University of California, Berkeley; Charles
Griswold, California Academy of Sciences; Marshal Hedin, San Diego State University; Marie Herberstein,
Macquarie University; Yael Lubin, Ben-Gurion University of the Negev; Brent Opel!, Virginia Polytechnic Insti-
tute & State University; Ann Rypstra, Miami University (Ohio); William Shear, Hampden-Sydney College; Jef-
frey Shultz, University of Maryland; Petra Sierwald, Field Museum; Soren Toft, Aarhus University; I-Min Tso,
Tunghai University (Taiwan).

The Journal of Arachnology (ISSN 0161-8202), a publication devoted to the study of Arachnida, is published
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THE AMERICAN ARACHNOLOGICAL SOCIETY

PRESIDENT: Charles Griswold (2013-2015), California Academy of Science, San Francisco, California, USA.
PRESIDENT-ELECT: Marshal Hedin (2013-2015), San Diego State University, San Diego, California, USA.
MEMBERSHIP SECRETARY: Jeffrey W. Shultz (appointed), Department of Entomology, University of Maryland,
College Park, Maryland, USA.

TREASURER: Karen Cangialosi, Department of Biology, Keene State College, Keene, New Hampshire, USA.
SECRETARY: Alan Cady, Department of Zoology, Miami University, Middletown, Ohio, USA.

ARCHIVIST: Lenny Vincent, Fullerton College, Fullerton, California, USA.

DIRECTORS: Jonathan Coddington (2013-2015), IngiAgnarsson (2013-2015), Richard S. Vetter (2013-2015)
PARLIAMENTARIAN: Brent Opell (appointed)

HONORARY MEMBERS: C.D. Dondale, H.W. Levi, A.E Millldge.

Cover photo: A displaying male Coastal Peacock Spider, Maratus speciosus (Salticidae), from coastal dune habitats near Perth in

Western Australia. Photo by Jurgen Otto.

Publication date: 10 April 2014

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

2014. The Journal of Arachnology 42:1-15

REVIEW

New sequencing technologies, the development of genomics tools, and their applications in

evolutionary arachnology

Michael S. Brewer'’^, Dark© D. Cotoras^, Peter J. P. Croucher’’- and Rosemary G. Gillespie*'-: ‘Department of

Environmental Science, Policy, and Management, University of California, Berkeley, California 94720 USA. E-mail:
michaelsbrewer@gmail.com; ^Essig Museum of Entomology, University of California, Berkeley, California 94720 USA;
^Department of Integrative Biology, University of California, Berkeley, California 94720 USA

Abstract. Molecular genetic tools have been a boon to arachnologists for decades and used to study many unique aspects
of arachnid biology including genomics, phylogenetics, population genetics, and biogeography. These tools have evolved
over time and now provide myriad methods for exploring evolutionary questions. Early tools, while still useful under the
proper circumstances, are giving way to a new generation of DNA sequencing technologies. These new platforms yield
impressive amounts of data at a fraction of the cost of traditional techniques. Herein, we discuss the history and future of
molecular evolutionary arachnology in terms of available genetic/genomic tools and their potential applications, strengths,
weaknesses, and relative costs. Next-generation sequencing (NGS) platforms are varied in their methods and potential uses,
making high-throughput sequencing studies focusing on a wide array of questions tractable. To date, relatively few studies
have employed NGS technologies using arachnids, but many could benefit from using them. Because no model species exist
within the class Arachnida, we have a limited understanding of arachnid genomics. With the ever-advancing nature of
sequencing technologies and bioinformatics, arachnologists can relatively easily implement NGS studies to bridge the gaps
in our understanding and open avenues for deeper and more powerful experiments. To this end, we discuss examples of
applications of NGS technologies focusing on arachnid taxa. Despite the allure of acquiring massive quantities of sequence
data, we should recognize the limitations of existing NGS technologies and not forsake pre-NGS methods when these
technologies could adequately address our questions.

Keywords: Next-generation sequencing, genome, transcriptome, phytogeny, population genetics, genomics, adaptation,
selection

TABLE OF CONTENTS

1. Introduction. ................................

2. Traditional markers of nuclear genetic variation .......

2. 1 . Allozyme electrophoresis

2.2. Satellite and microsatellite DNA ...............

2.3. Random amplified polymorphic DNA (RAPD) . . . .

2.4. Restriction fragment length polymorphisms (RFLPs)

2.5. Amplified fragment length polymorphisms (AFLPs) .

3. Sequencing methods . . .

3.1. Sanger sequencing of mitochondrial DNA ........

3.2. Sanger sequencing of nuclear DNA .............

3.3. Sanger sequencing versus NGS methods .........

3.4. NGS versatility in sequencing targets. ...........

4. Next generation technology platforms ..............

4.1. Second-generation NGS technologies. ...........

4.2. Compact persona! genome sequencers .

4.3. Third-generation NGS technologies . . .

5. Arachnid genome efforts ........................

5.1. Published genomes

5.2. Genomes in progress - what we have learned ......

Mitochondrial genomes. ...................

Nuclear genomes ........................

5.3. A cautionary note .........................

6. Applications of NGS technologies in arachnology ......

6.1. Functional genomics - adaptation and selection . . . .

Measures of selection .....................

Molecular basis for adaptation ..............

6.2. Phylogenetics. ............................

6.3. Population genetics and phylogeography .........

7. Conclusion.

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THE JOURNAL OF ARACHNOLOGY

1. INTRODUCTION

Since Linnaeus and before, scientists have sought to put
order into the diversity of life, the thirst for information
increasing with the recognition of the role of evolutionary
processes in shaping that diversity. The advent of molecular
techniques in the 1980s introduced a huge diversity of novel
markers for assessment of phylogenetic affinities. Moreover,
with the growth of the human genome project, the potential
use of vast numbers of genes across the genome was soon
recognized (Jones 1991). Analytical tools were developed that
could use the almost limitless data to address questions
ranging from historical and recent demographic and migrato-
ry patterns to identifying signatures of recent natural selection
(Nielsen 2010; Rasmussen et al. 201 1; Lohmueller 201 1 ). Here,
we examine where the field of arachnology stands within the
genomic revolution.

In recent years, advances in sequencing technology have led
to great increases in genomic resources for many non-model
species. The arthropod class Arachnida, encompassing over
100,000 nominal species classified into 12-13 traditional
orders (Krantz and Walter 2009; Blick and Harvey 2011),
comprises a diverse array of taxa that serve key functions in
terrestrial ecosystems as important predators and decompos-
ers. Much of their diversity is unique to particular arachnid
taxa, including complex silk production (spiders and mites),
venom composition (spiders, scorpions, and pseudoscorpions)
and detoxification of plant compounds (Grbic et al. 2011), and
has long been of particular interest to researchers. As an
example, spiders have been used to study behavior (reviewed
by Herberstein and Hebets 2013), development (e.g., Ka-
nayama et al. 2010; Wolff and Hilbrant 2011; Mittmann and
Wolff 2012), sexual selection (e.g., Kuntner et al. 2009; Su et
al. 201 1), genetics (reviewed by Goodacre 2013), evolutionary
ecology (reviewed by Moya-Laraho et al. 2013) and biogeog-
raphy (reviewed by Gillespie 2013), among other fields.
However, relationships within and among arachnid taxa are,
in many cases, not presently resolved (Giribet and Edgecombe
2013), and a paucity of genomic resources has hindered efforts
in various fields of arachnology. Molecular techniques have
been successfully employed to investigate a number of these
issues, but some problems require data at a scale heretofore
unavailable to arachnologists.

Here we first briefiy review how more “traditional”
molecular tools have been applied to arachnid biology and
then go on to discuss emerging next-generation sequencing
(“NGS”) applications and their potential impact within the
field. Instead of deeply discussing each area of arachnology
and thoroughly reviewing the literature, we provide a brief
background with select citations and proceed to describe some
ways in which the rapidly multiplying set of NGS tools may be
used.

2. “TRADITIONAL” MARKERS OF NUCLEAR
GENETIC VARIATION

Prior to genomic tools, multiple techniques were used for
examining variation in nuclear DNA and hence to assess
geographic and population structure, the most widely used
being randomly amplified polymorphic DNA (RAPDs),
restriction fragment-length polymorphisms (RFLPs), and
satellite and microsatellite DNA.

2.1. Allozyme electrophoresis. — Allozyme electrophoresis
has proven very useful for the analysis of geographic structure
of arachnids. Some early studies focused specifically on
population structure (Porter & Jakob 1990; Steiner et al.
1992; Smith & Engel 1994; Hudson & Adams 1996; Smith &
Hagen 1996; Boulton et al. 1998), but allozymes have also
been used to examine questions of relatedness among colonies
of social spiders (Johannesen et al. 1998; Johannesen &
Lubin 1999, 2001; Johannesen & Veith 2001; Evans &
Goodisman 2002; Yip et al. 2012), paternity (Schafer & Uhl
2002), species boundaries and speciation (Piel & Nutt 2000;
Ramirez & Chi 2004), dispersal (Pedersen & Loeschcke 2001;
Schafer et al. 2001), the effects of forest fragmentation,
whether natural (Vandergast et al. 2004) or manmade
(Ramirez & Haakonsen 1999; Gurdebeke et al. 2000), and
to estimate selection on color polymorphisms (Tso et al.
2002; Oxford 2005; Oxford & Gunnarsson 2006; Croucher et
al. 2012) as well as patterns of diversification within rapidly
diversifying lineages (Pons & Gillespie 2004; Baert et al.
2008; De Busschere et al. 2010).

The primary limitations of allozyme electrophoresis are: a)
Organisms must generally be alive or deep-frozen before use;
b) when bands co-migrate, they are assumed to be homolo-
gous; c) only a very small subset of the genetic variation at a
given locus is revealed; and d) it is not possible to distinguish
ancestry and descent among different alleles. The technique is
inexpensive, fast, and can give insight into multiple loci and so
is useful for addressing questions of geographic structure.

2.2. Satellite & microsatellite DNA. — Tandem repeats
include three subclasses: satellites, minisatellites and micro-
satellites. Satellites range in size from 100 kb to over 1 Mb
with repeat units of ca. 100-200 bp; most are located at the
centromere. Minisatellites range from 1 kb to 20 kb in size
with shorter repeats (9-80 bp), while microsatellites (also
known as short tandem repeats, STR), are repeats of
sequences less than about five base pairs in length (an
arbitrary cutofO. Among spiders, satellite DNA has proven
very useful in assessing relationships among species within a
radiation of spiders in Hawaii; this was because the tandem-
arranged units show a high intraspecific sequence identity due
to concerted evolution (Pons & Gillespie 2003, 2004). As a
result, the length of the branches and corresponding support
were much greater for satellite DNA than for mtDNA
sequence data.

Microsatellites are repeated short sequences of DNA that
occur throughout the genomes of many organisms, including
spiders. Because repeat units are readily added to or lost from
microsatellite DNA, the sequence length of these regions
evolves rapidly. Microsatellites offer a valuable pool of genetic
variation that has proven very useful for looking at paternity
and relatedness among spiders, including social species (Ji
et al. 2004; Bilde et al. 2009; Duncan et al. 2010), as well as
understanding geographic structure between closely related
populations (Rutten et al. 2001; Reed et al. 2007, 2011;
Krehenwinkel & Tautz 2013; Parmakelis et al. 2013).
However, compared to many other fields, the development
and application of microsatellites in spider ecology and
evolution has been limited. A potential cause for the paucity
of microsatellite studies in arachnids is the apparent difficulty
in finding reliable loci. However, the low % GC (percentage of

BREWER ET AL.— GENOMICS IN EVOLUTIONARY ARACHNOLOGY

3

guanine and cytosine residues in DNA sequences) in some
lineages, as discussed below, could potentially play a role.

23. Random amplified polymorphic DNA (RAPD). — In the
RAPD procedure, a single nucleotide primer (8-10 base pairs
long) is used to amplify random sections of nuclear DNA,
with differences in band sizes being used to provide
information on relationships. The method has been used in
spiders (e.g., A’Hara et al. 1998; Gurdebeke et al. 2003).
However, although the approach provides a lot of variability,
RAPDs suffer from poor repeatability, lack of codominaiice,
and the possibility of non-heritable or non-homologous
bands.

2.4. Restriction fragment length polymorphisms (RFLP). —
For generating RFLPs, regions of nuclear DNA isolated
through PCR or other means can be digested with restriction
enzymes that cut samples of homologous DNA at specific
four- or six-base sequences, differences arising from the
locations of restriction enzyme sites. This technique could,
compared to other technologies of the time, exploit an
enormous amount of genetic variation. However, although
used in mites (e.g., Osakabi & Sakagami 1994), it was never an
important technique in other arachnid groups.

2.5. Amplified fragment length polymorphisms (AFLP). —
AFLPs use restriction enzymes to digest genomic DNA, with
the fragments then amplified and separated, providing
markers across many loci that are highly variable and are
also reproducible. Like RAPDs, however, they are also both
anonymous and dominant and may produce non-homologous
bands. They have been used in studies of geographic structure
among spider populations (Jung et al. 2006; Lambeets et al.
2010; Croucher et al. 2011a, b), where they provided fine
resolution of population differentiation and subdivision. They
have also been used in assessments of inbreeding and sociality
(Bilde et ai. 2005).

3. SEQUENCING METHODS

3.1. Sanger sequencing of mitochondrial DNA. — Mitochon-
drial DNA, notably the cytochrome oxidase 1 (COl), NADH
dehydrogenase 1 (NDl) and 16S rRNA genes (Agnarsson et
al. 2013) proved particularly useful in the earliest studies of
biogeography and species differentiation in spiders (Gillespie
et al. 1994; Hedin 1997a, b; Johaiinesen et al. 2002). The
reason for this is simply because of the abundance of
mitochondrial DNA relative to nuclear DNA, making it
much easier to amplify. However, problems with mtDNA that
affect recently evolving lineages include the lack of recombi-
nation, as a result of which it behaves as a single locus, making
it of limited value for analytical approaches requiring multiple
loci. This makes its use in species delimitation particularly
problematic (Hamilton et al. 2014). Moreover, the haploid
nature of mitochondrial DNA means that the marker is more
sensitive to small population sizes than is nuclear DNA, and
the maternal inheritance means that biases in movement
between sexes cannot be recovered. For this reason, recent
studies that have used mtDNA sequences have generally
included various nuclear markers (e.g., Vandergast et al. 2004;
Starrett & Hedin 2007; Croucher et al. 201 la & b, 2012; Satler
et al. 2013). Mitochondrial DNA has been applied to
questions at deeper phylogenetic levels where the microevolu-
tionary problems mentioned above are less severe. However,

the rapid evolution of the marker means that it tends to
become saturated rather quickly (Brewer et al. 2013).

3.2. Sanger sequencing of nuclear DNA. — Because of the
issues of amplification, the most reliable, and hence useful,
nuclear genes have tended to be those that occur in multiple
copies such as Histone 3, and the ribosomal 18S and 28S
genes, and these have been of particular impact in the realm of
phylogenetic reconstruction (reviewed by Agnarsson et al.
2013 and Giribet and Edgecombe 2013). At the population-
species level, attention has focused on nuclear introns —
noncoding sequences within nuclear genes, as these are not
subject to the same selective constraints as exons and tend to
evolve faster (Garb & Gillespie 2009; Hedin et al 2010). ITS
(internal transcribed spacer) regions within the ribosomal
RNA genes can frequently provide sufficient variability at
shallow levels (Hormiga et al. 2003), though paralogy can
often make the identification of homologous DNA difficult
or impossible. Indeed, the problem of generating multilocus
(nuclear) data has remained. Thus, researchers have looked
increasingly toward modern, high-throughput (or “next-
generation”) sequencing technologies as a potential means
to generate large amounts of multilocus data by increas-
ing the amount of data per monetary cost by orders of
magnitude.

3.3, Sanger sequencing versus NGS methods. — In the past,
DNA sequence data were primarily collected using dideoxy-
ribonucleotide (ddNTP) termination methods (i.e., Sanger
sequencing). This approach provides long, high-quality
sequences but suffers from a number of limitations (Table 1),
including the ability to sequence only a single locus per
reaction. In addition, reactions typically require taxon (or
even population) specific oligonucleotide “primers” — short
fragments of DNA (ca. 20-25 bp) of known sequence for
polymerase chain reaction amplification and the sequencing
reaction. Lastly, the cost of collecting data is much higher than
in NGS approaches. Sanger sequencing methods are quite
scalable in that one can easily obtain data for a single locus to
hundreds of loci with a concomitant change in cost, but one
cannot sequence massive amounts of genomic data from
numerous specimens in a cost effective (in terms of time and
money) manner.

In contrast to Sanger sequencing, NGS techniques provide
vastly larger quantities of data much faster and for far less
money. NGS approaches achieve this in two ways. The first
involves ligating or attaching adaptors (synthesized DNA
strands of known sequence) to the ends of fragments of target
DNA. These adaptors allow identical sets of PCR or
sequencing primers to be used for all the DNA fragments (a
form of “shotgun” sequencing). From an operational point of
view, the major differences between the various NGS
approaches are in the size of the DNA fragment (the “insert”)
and the number of base pairs of sequence data that can be
recovered from the end of the fragment. The second way that
NGS approaches dramatically reduce costs is by miniaturiza-
tion and parallelization — millions of sequencing reactions take
place in small reaction chambers or flow cells (Shendure & Ji
2008). Consequently, high-throughput methods can sequence
numerous DNA fragments concurrently and in a single
reaction/run with full-length sequences typically being assem-
bled after the fact.

4

THE JOURNAL OF ARACHNOLOGY

Table 1. — Summary of the strengths, weaknesses, starting materia! and applications of select molecular data sources. Pre- and post-next-
generation sequencing molecular data sources discussed here are listed. Several positives and negatives are given for each technique, along with
differences in starting material required. Additionally, several historical and potential applications in arachnology are provided for each method.

Method

Raw sequence output

Cost per Mb

Positives

Negatives

Potential in arachnids

Traditional Sanger
sequencing

1.9-84 Kb

$2,400

Long, high quality
reads; scalability

Very high cost per Mb

Traditional phylogenetics,
population genetics and
studies of few genes

454 pyrosequencing

0.7 Gb

$10

Long reads

Problems with
homopolymers; high
cost per Mb (for
next-gen technology)

Genomes, transcriptome and
microbiome studies

Illumina (Solexa)
sequencing

600 Gb
(HiSeq 2000)

$0.05-$0.15

High output; relatively
low cost; widely used
and supported

Short reads

Genome, transcriptome,
massively barcoded
ampiicon and microbiomes
studies

SOLiD sequencing

120 Gb

$0.13

Highly accurate

Short reads; not as
widely supported

Genome, transcriptome,
epigenetic and resequencing
studies

Ion Torrent
sequencing

20 Mb-1 Gb

$1

Scalability

Short reads

Transcriptome and barcoded
ampiicon studies

MiSeq sequencing

1.5-2 Gb

$0.50

Longer reads and
smaller scale than
older Illumina
technologies

Output too low for
some applications

Transcriptome, barcoded
ampiicon and microbiome
studies

Single molecule real
time (SMRT)

400 Mb

$0.75-$1.50

Very long reads

High error rate; best
combined with
other technologies

Genome and transcriptome
studies

3.4= NGS versatility in sequencing targets. — The “shotgun”
nature of NGS, using ligated universal adaptors, gives these
approaches tremendous versatility in terms of what can be
sequenced. This versatility facilitates genomic data collection
for organisms about which little or no prior genetic
information is known. Sequenced DNA targets can therefore
theoretically consist of any source of DNA from total genomic
DNA for genome sequencing (see Section 5) to cDNA (derived
from total RNA by reverse transcription of expressed mRNA)
(Mortazavi et al. 2008) from whole organisms or specific
tissues that may have experienced different “treatments”
(“transcriptomics” and “differential expression”). RNAseq
libraries used for transcriptome sequencing target only the
transcribed portion of the genome and are therefore a type of
“reduced representation library” (RRL) (Van Tassell et al.
2008), and we discuss this approach along with other RRL
approaches such as Exon Capture (Bi et al. 2012) and RADseq
(Miller et al. 2007) below. RRL approaches target specific loci
and produces fewer unique reads per individual than whole
genome libraries and on certain NGS platforms, especially
Illumina (see below), may produce highly redundant amounts
of data per individual. This has resulted in methodologies that
multiplex numerous individuals using small, unique oligonu-
cleotide indices (i.e., tags or barcodes). These are typically
incorporated into the adaptors and allow the sequences from
each individual to be post-hoc sorted computationally.
Barcoding therefore permits NGS approaches to act as high-
throughput variant detection and genotyping platforms (e.g.,
Dahl et al. 2007; Meyer et al. 2008). Barcoding also comes into
its own when the DNA targets originate from amplicons
generated by traditional PCR approaches. Amplicons might
be derived from long-range PCR of mitochondrial genomes,
for example, or from standard molecular markers such as

bacterial 16S, fungal i8S, or metazoan COL Such massive
barcoding approaches with ampiicon sequencing are per-
mitting community-wide metagenomic/microbiome analyses
(Amaral-Zettler et al. 2009; Gloor et al. 2010; Caporaso et al.
2012) and large-scale phylogenetic studies (see below).

4. NEXT-GENERATION TECHNOLOGY PLATFORMS

Modern, high-throughput (or “next-generation”) sequenc-
ing technologies have made many questions more tractable by
increasing the amount of data per monetary cost by orders of
magnitude. Although a number of NGS sequencing tech-
niques have a steep learning curve (in terms of both wet-
laboratory work and bioinformatics), much can be out-
sourced, and myriad computational resources (many of which
may be used free of charge) are readily available. Some of
these techniques are widely used, while others are still more
limited in their availability. All such methods have inherent
strengths and weaknesses that can be leveraged to address a
wide range of questions. As more molecular data are collected
for arachnid taxa, these groups may begin to approach
“model” organism status in terms of a foundational under-
standing of genetics. This will allow more in-depth studies,
using complex and powerful genetic and genomic techniques,
to understand the basis of arachnid-specific traits.

4.1. Second-generation NGS technologies. — The basic logic
behind several NGS technologies has been reviewed in recent
works (Liu et al. 2012; McCormack et al. 2013; Quail et al.
2012). Therefore, we do not delve into specifics of the
approaches, instead choosing to highlight strengths and
weaknesses of the platforms. Many of the points below are
summarized in Tables 1 and 2.

The first mainstream high-throughput technology was the
Roche 454 system. This method relies on pyrosequencing

BREWER ET AL.— GENOMICS IN EVOLUTIONARY ARACHNOLOGY

5

Table 2. — Comparison of DNA sequencing technologies with an emphasis on uses in arachnology. Several aspects of common sequencing
technology are compared. The raw sequence output is highly variable between platform and shows the potential scalability provided by using
different sequencing platforms. The cost per one million base pairs of data between methods also differs substantially and must be considered
when attempting a study requiring sequencing. To help with choosing between platforms, we provide select positives and negatives for each.
Finally, some potential high-level applications in arachnology are given.

Data source

Positives

Negatives

Starting material

Applications in arachnids

Pre next-gen sequencing

Allozyme electrophoresis

Ease of use; widely used
in arachnology

Requires fresh or frozen
material; uncertain
homology

Fresh or frozen
specimens

Population genetics and
biogeography

Variable nucleotide
tandem repeats
(VNTR; satellites)

More certain homology;
widely used outside
of spiders (many
analytical tools)

Difficult to design; may
not work between even
closely related taxa

gDNA

Paternity, population

genetics and biogeography

Random amplified
polymorphic DNA
(RAPD)

Easier to implement than
satellite techniques

Lack of repeatability;
codominance;
uncertain homology

gDNA

Population genetics and
biogeography

Restriction fragment
length polymorphisms

(RFLP)

Ease of use

Fragments of same
size may not be
homologous; not widely
used in arachnology

gDNA

Population genetics and
biogeography

Amplified fragment
length polymorphisms
(AFLP)

Widely used

Anonymous and
dominant; uncertain
homology

gDNA

Assessments of inbreeding;
population genetics and
biogeography

Termination (Sanger)
sequencing

Sequence data; homology
easier to infer; highly
scalable compared to
other non-NGS
techniques

Much more expensive
than NGS at a per
base level; taxon/
population specific
primers needed

gDNA

Assessments of inbreeding,
population genetics,
biogeography,
phylogenetics and
single gene studies

Post next-gen sequencing

Genome sequencing

Full sequence data,
creates foundation for
many future studies

Costly, difficult, and
unnecessary for
many projects

High quality, high
quantity gDNA

Genome studies, genetic
mapping and developing
model organisms

Transcriptome sequencing

Easy to sequence and
serves a wide range
of projects

Costly and biased
towards coding
regions of genome

High quality RNA
from fresh or
frozen specimens

Studying coding sequencing,
differential expression,
identifying isoforms, evolution
of gene families, functional
genes, and deep phylogenomics

RAD Tags

Powerful for genetic
mapping, population
genetics and
phylogeography

Requires large amount
of high-quality DNA

High quality, high
quantity gDNA

Phylogeography, population
genetics, species-level
phylogeny and genomic
mapping

Target Capture

Targets portions of the
genome for wide
array of projects

Requires additional
genomic information

gDNA from fresh
or preserved
specimens (quality
depends on
application)

Studies of specific regions of
the genome, functional
genes, population genetics,
species-level phylogeny
and phylogeography

Anchored enrichment

Orthology certainty and
phylogenetics at various
taxonomic levels

Not developed in
all groups

gDNA from fresh
or preserved
specimens (quality
depends on
application)

Phylogenetics of various
taxonomic depths

chemistry to obtain millions of unique reads. Roche’s 454
approach provides relatively long reads (—700 bp) at a much
lower cost (— $I0/Mb) than traditional sequencing methods
(—$2, 400/Mb). The 454 sequencing technology, although
expensive per base of data in comparison to other NGS
methods and yielding fewer unique sequence reads, is still
widely used in genome and transcriptome sequencing and
metagenomics because of the relatively long length of
individual reads. However, other techniques (namely Illumina
technologies, see below) can now serve many of the same

functions as 454 sequencing at a much lower cost while
providing many more unique sequence reads.

The second NGS platform to become widely used was
Applied Biosystems sequencing by oligo ligation detection
(SOLiD). This method uses the ligation of short probes to the
template DNA. Each probe’s extension relies on two-base
matches, yielding highly accurate results at a lower cost-point
(—$0.1 3/Mb) and in higher quantities than 454 sequencing (see
below). A minor downside to the SOLiD sequencing platform
is that the output is in a format unlike other technologies and

6

THE JOURNAL OF ARACHNOLOGY

requires computationally expensive algorithms to assemble.
Nonetheless, this method can be used to efficiently study
genomes, transcriptomes, and epigenetics (i.e., non-genetic
modifications of the DNA sequence that affect expression
such as methylation of CpG “islands”, areas of the genome
containing high frequencies of cytosine and guanine residues).

The last of the commonly used second-generation technol-
ogies, and the most frequently used NGS platform, is the
Illumina system, lllumina chemistry relies on fixed flow-cell
binding site oligonucleotides and complementary adaptors
that also contain sequencing primer sites, and that are ligated
to the DNA fragments to be sequenced. This technology yields
a vast quantity of raw data (600 Gb) for relatively low cost
($0.05 - $0.1 5/Mb), and much effort has gone into developing
novel ways of applying the method to a wide array of studies.
These range from tweaking protocols to creating new
algorithms and software for analyses. The main downside of
the Illumina technology is that the sequence reads are
relatively short. Early versions of the platform yielded reads
that were only 36 bp in length, but read length as well as
throughput continue to increase for all the NGS technologies,
and the Illumina platform, for example, although still short in
comparison to Sanger and 454 approaches, can now generate
reads in excess of 150 bp. Short reads lead to complications in
genome sequence assembly efforts and in commimity/micro-
biome sampling where assembly of sequences from a mixed
pool of taxa is problematic. Fortunately, several approaches
have been developed to address these problems including
combining Illumina data with other sources, using large insert
sizes for scaffolding, and overlapping reads for metagenomic
amplicon sequencing (Masella et al. 2012). Therefore, Illumina
sequencing is often used in genome sequencing efforts,
transcriptomics, community sampling, resequencing, target
enrichment, and many more techniques.

4.2. Compact personal genome sequencers. — In an attempt to
down-scale high-throughput sequencing technologies to pro-
vide a more manageable amount of data for less money,
“personal genome sequencers” have been developed. These
machines are less expensive to buy, use, and maintain; hence
individual labs may realistically own these machines for smaller
scale and exploratory sequencing experiments. The first of
these, the personal genome machine (PGM), was released by
Ion Torrent (Rothberg et al. 2012). This platform is unique in its
scalability. Sequencing takes place on individual disposable chips
that can collect variable amounts of sequence data for differing
levels of cost. This method is used for small genome sequencing
(e.g., organellar or prokaryotic genomes) and transcriptomes.
The Illumina MiSeq is similar in application to the larger Illumina
platform, but at a smaller scale. Sequencing and data analysis are
integrated into a single machine and can yield analyzed data in a
single day. This method is commonly applied in highly
multiplexed amplicon sequencing, small genome sequencing,
microbial community analysis (Caporaso et al. 2012) and for the
identification of transcription factors (i.e., ChiP-Seq).

4.3. Third-generation NGS technologies. The newest high-
throughput sequencing platforms, or single molecule sequenc-
ing, include two main technologies — single molecule real-time
(SMRT) sequencing by Pacific Biosciences (PacBio) and the
unreleased Nanopore platform (Oxford Nanopore Technolo-
gies, Oxford, UK). These methods are characterized by two

main features: 1) no PCR prior to sequencing (limiting
artifacts) and 2) sequences are recorded in real-time (i.e.,
during the polymerase reaction or depolymerization). These
methods can each yield very long reads (>5 Kb and up to 13-
14 Kb for PacBio) making them useful in de novo genome
sequencing efforts. Short reads from the PacBio SMRT
technology can be highly accurate since the platform has the
ability to resequence the circularized molecule repeatedly until
base confidences are high; however, long reads have a very
high endemic error rate (ca. 15%). Approaches are being
developed to correct the SMRT data using large quantities of
accurate but short-read Illumina data (English et al. 2012;
Koren et al. 2012). The Nanopore technology is currently not
widely available, so much is still unknown concerning its
performance. Moreover, although SMRT methods provide
much longer reads than earlier NGS approaches with
considerable simplification of library preparation, neither is
currently well supported by common NGS bioinformatics
tools.

5. ARACHNID GENOME EFFORTS

NGS technologies allow the sequencing and reconstruction
or “assembly” of whole genome sequences. Accurate genome
assembly in model organisms (organisms that are amenable to
genetic study, have short generation times, breed in large
numbers, and can inform about other organisms) has
traditionally relied upon an edifice of classical genetics
resources including inbred lines to minimize genetic variation,
genetic linkage maps generated from laboratory crosses
among inbred lines, and the sequencing and hierarchical or
clone-based assembly of large 40-200 kb genome fragments
called “bacterial artificial chromosomes” (BACs) large-insert
libraries (Lander et al. 2001). Arachnids, like most non-model
organisms, lack most of these resources. They often have long
generation times and can be very small (forcing pooling of
individuals and an increase in heterozygosity, making
assembly difficult). Moreover, they are generally difficult to
breed in captivity and, except for some mite species, no inbred
lines are available, with the possible exceptions of naturally
inbred social species such as the eresid Stegodyphus mimo-
sarian (J.S. Bechsgaard & T. Bilde pers. comm.) and theridiid
Anelosinuis e.xiniiiis (1. Agnarsson pers. comm.).

5.1. Published genomes. — The three presently available
arachnid genomes are from highly derived acarine species:
the two-spotted spider mite Tetranychus urticae (Grbic et al.
2011), the honey bee ectoparasitic mite Vairoa destructor
(Cornman et al. 2010) and the deer tick Ixodes scapidaris
(http://iscapularis.vectorbase.org). The choice of these arach-
nids as early targets for genome sequencing is perhaps
unsurprising; Tetranychus and Varroa are of tremendous
agricultural and economic importance, and Ixodes is of great
importance as a vector of numerous livestock and human
diseases including Lyme disease. In addition to its economic
importance, Tetranychus urticae was selected as a candidate
for genome sequencing as it has the smallest known genome of
any arthropod at a mere 89.6 Mbp (Grbic et al. 2011), is easily
cultured in the laboratory and has inbred lines available. The
small Tetranychus genome was sequenced using traditional
Sanger sequencing methods to a depth of 8.05X, resulting in
640 scaffolds: 70,778 EST sequences plus RNA-seq data (see

BREWER ET AL.— GENOMICS IN EVOLUTIONARY ARACHNOLOGY

7

below) were mapped to the genome and supported 15,397 of
18,414 gene models. The genome of the ectoparasitic mite
Varroa destructor, which has emerged as the primary pest of
domestic honey bees (Cornman et al. 2010), was “surveyed”
using 4.3X coverage of 454 sequence data from the DNA of

1.000 pooled mites. This 2.4 Gbp was clearly insufficient to
provide a comprehensive de novo assembly of this moderately
sized genome (at 294 Mbp still far bigger than most sequenced
insects) and yielded 184,094 contigs (assembled contiguous but
not “scaffolded” sequences) with an N50 (weighted median of
contig lengths) of 2,626 bp; however, the data were sufficient
to permit the prediction of 31.3 Mbp of gene sequence,
information about the integration of microbes into the
genome and the occurrence of single nucleotide polymor-
phisms (Cornman et al. 2010). Finally, the genome of the deer
tick Ixodes scapularis, which is very large compared to
Tetranychus and Varroa at 2.1 Gbp, was shotgun sequenced
using Sanger sequencing to a coverage of 3-6X. Although
many data on the expressed gene sequences (i.e., the
transcriptome) are available in the public databases, the
genome sequence remains highly fragmented (e.g., ca. 571,000
contigs with a contig N50 of 3000 bp) and has not been
officially published (http://iscapularis.vectorbase.org). Of the
three acarine species, Tetranychus provides the most complete
genome reconstruction, with genome assemblies for Varroa
and Ixodes remaining highly fragmented.

5.2. Genomes in progress - what have we learned? — Apart
from the three acarine species discussed above, our knowledge
of the nuclear DNA structure of arachnids remains extremely
limited. Most knowledge about arthropods conies from
insects — a reflection of biological diversity, societal impact
and economic and medical importance, and the scale of the
research community, among other factors. From an evolu-
tionary and phylogenetic perspective, this bias of course
does not reflect relative importance. However, efforts such
as the research community-driven 15K project (http://www.
arthropodgenomes.org/wiki/Main_Page) that aims to sequence

5.000 arthropod genomes over five years should redress the
balance to some extent. Even so, of 787 species currently
nominated for sequencing, there are 702 Hexapoda (89%), 64
Chelicerata (8%), only 20 Crustacea (2%), and 6 Myriapoda
( 1 %) (http://www.arthropodgenomes.org/wiki/i5K_nominations).
Several arachnids have been included in the pilot sequencing
project of the I5K, and these are discussed below, together with
our own efforts on Theridion (Theridiidae) and other efforts on
Stegodyphus (Eresidae) and Acanthoscurria (Theraphosidae).
In addition, the genome of Limidus has been sequenced, and a
preliminary assembly is about to be publicly released (Nipam
Patel pers. comm.). However, pre-NGS we have revealed much
about arachnid mitochondrial genomes, and we briefly review
this here before going on to examine nuclear genomes.

Mitochondrial genomes: Although knowledge of the arach-
nid nuclear genome remains in its infancy, several decades of
research, based upon traditional PCR and Sanger sequencing,
have yielded detailed knowledge of arachnid mitochondrial
genomes. This work has revealed lineage-specific gene order
rearrangements in Opiliones (Masta 2010) and pseudoscorpi-
ons (Ovchinnikov & Masta 2012), and most interestingly has
revealed truncated mitochondrial tRNA (and rRNA) second-
ary structures among most arachnid lineages (Masta & Boore

2004, 2008; Masta et al. 2008; Fahrein et al 2009; Masta 2010;
Ovchinnikov & Masta 2012). NGS technologies can poten-
tially greatly increase our understanding of the sequence
diversity, variation and transcriptional mechanisms among
arachnid mitochondria since 1) whole mitochondrial genomes
can rapidly be sequenced from many barcoded and pooled
individuals using amplicon sequencing (e.g., on small scale
MiSeq or lonTorrent systems) (see below); 2) mitochondrial
genomes can be assembled from total genome sequence data
(lorizzo et al. 2012); and 3) RNA-seq reads (Illumina-based
method of sequencing cDNA obtained via reverse transcrip-
tion of niRNA extractions) can be mapped to mitochondrial
genes to explore expression differences among genes and taxa
and post-transcriptional modification and editing of gene
sequences (Smith 2013).

Nuclear genomes: Since no non-acarine genomes have been
published so far, detailed discussion of their structure is not
yet possible. Our own efforts at sequencing a spider genome
have focused on the Hawaiian happy face spider Theridion
graUator and primarily have used Illumina paired end data
based upon a variety of insert sizes. Initial assemblies were
highly fragmented (resulting in many contigs of short length;
i.e., a low “contig N50”). Although this is partly due to
heterozygosity (no inbred lines are available), the main
complication appears to be that this species has a low average
% GC across the genome (ca. 28%) (Fig. 1). Although most
arthropod genomes are somewhat “Ad'-rich” (e.g., the honey
bee Apis mellifera has 34.8% GC; The Honeybee Genome
Sequencing Consortium 2006), the only arthropod genome
with a % GC in the range we have found is that of the pea
aphid Acyrthosiphum pisum at 29.6% GC (The International
Aphid Genomics Consortium 2010).

A potential extreme % GC bias in arachnid genomes is both
intriguing and technically challenging from both an informatic
and molecular biological point of view. In order to investigate
this further, we have examined the assembled contigs data,
where available, from the pilot runs for the I5K project (http://
www.arthropodgenomes.org/wiki/Main_Page). In total we
have examined the contig N50 length and % GC from two
I5K sequenced spiders, Latrodectus hesperus and Parasteatoda
tepidariorum (Theridiidae), and the Arizona bark scorpion
Centruroides sculturutus (Buthidae), together with 15 other
arthropods (14 insects and one copepod; ftp://ftp.hgsc.bcm.
edu/I5K-pilot/), and these are plotted in Fig. 1. In addition we
have included our data from Theridion graUator (PJPC
Linpubl. data) and data from Stegodyphus mimosarum
(Eresidae: J.S. Bechsgaard & T. Bilde, pers. comm.). The
scorpion and the three theridiid spiders (L. hesperus, P.
tepidariorum and T. graUator) all have less than 30 % GC and
correspondingly low contig N50 lengths. In general, the lower
the % GC, the shorter the contig N50 as a simple function of
decreased information content available to the assembly
algorithms. Interestingly, the P. tepidariorium sequenced had
been through five generations of inbreeding (A. McGregor
pers. comm.) — apparently sufficient to reduce heterozygosity
enough to substantially increase contig lengths despite a low %
GC.

Alternatively, S. mimosarum did not exhibit an extreme %
GC bias (34% GC) and as a social species (Mattila et al. 2012)
is somewhat naturally inbred — and has a correspondingly

THE JOURNAL OF ARACHNOLOGY

Contig N50 (bp)

Figure 1. — Assembly contig N50 length (bp) is negatively correlated with average genome-wide %GC bias among arthropod taxa. As average
%GC decreases so does the contig N50 (weighted median of contig lengths), since lower information content leads to more fragmented
assemblies. Open diamonds refer to arachnid genome projects and closed diamonds refer to other arthropods. Theridion grallator data are
calculated from the authors’ own (unpublished) preliminary genome assembly and have the lowest %GC to date (28.37%). Stegodyphus
mimosarum values from J.S. Bechsgaard (pers. comm.). C. darwini and N. clavipes values from L Agnarsson (pers. comm.). All other values
estimated from initial contig (not scaffolded) assemblies of the I5K initiative pilot genome assemblies; data and assembly parameters are
therefore similar among species (ftp.hgsc.bcm.edu/I5K-pilot/). Although all arthropods show a bias toward low %GC (<32%), the theridiid
spiders T. grallator, L. Hesperus and P. tepidariorum, as well as the scorpion C. sculpturatus, have very low %GC. 5. mimosarum has a more
moderate bias (34% GC), and both this species and P. tepidariorum show the benefit of inbreeding and low-heterozygosity and have longer contig
N50 lengths than the other arachnids. Additionally, the remaining non-theridiid spiders, C. darwini and N. clavipes, have moderate %GC values
but low contig N50s (<10,000 bases), possibly due to heterozygosity stemming from the lack of inbreeding. The included insects are 1) Athalia
rosae (turnip sawfly: Insecta: Hymenoptera); 2) Ceratitis capitata [Mediterranean fruitfly (medfly): Insecta: Diptera]; 3) Orussus abietinus
(parasitic wood wasp: Insecta: Hymenoptera); 4) Cimex lectularius (bed bug: Insecta: Hemiptera); 5) Anoplophora glabripennis (Asian long-
horned beetle: Insecta: Coleoptera); 6) Libellula fulva (scarce chaser: Insecta: Odonata); 7) Helicoverpa punctigera (Australian bollworm: Insecta:
Lepidoptera); 8) Ephemera danica (green drake mayfly: Insecta: Ephemeroptera); 9) Agrilus planipennis (emerald ash borer: Insecta, Coleoptera);
10) Copidosoma jloridamim (chalcid wasp: Insecta: Hymenoptera); 11) Homalodisca (glassy-winged sharpshooter: Insecta: Hemiptera);

12) Leptinotarsa decemlineata (Colorado potato beetle: Insecta: Coleoptera); 13) Eurytemora affinis [copepod: Maxillopoda (Crustacea):
Calanoida]; 14) Limnephilus lunatus (caddisfly: Insecta: Trichoptera); 15) Pachypsylla venusta (hackberry petiole gall psyllid: Insecta: Hemiptera).

much better assembly contiguity (J.S. Bechsgaard & T. Bilde
pers. comm.). Furthermore, initial sequencing of the huge
(6 Gbp) genome of the Brazilian white knee tarantula
Acanthoscurria geniculata (Theraphosidae) indicates that this
species has a ca. 40% GC genome content (J.S. Bechsgaard &
T. Bilde pers. comm.). Until more arachnid genome sequence
becomes available, the question as to how widespread %GC-
bias is among arachnids will remain unclear. From the above
data it may appear to be specific to theridiid spiders and
Centruroides scorpions; however it is tempting to speculate
that extreme %GC-bias may extend to other spider families
and other arachnid orders. This possibility should be
considered in future genome-sequencing efforts, and we note
that transcriptome assembly (RNA-seq) is unlikely to be so
impacted by %GC-bias, since coding regions typically do not
exhibit such extreme biases.

5.3. A cautionary note. — Despite the allure of NGS
technologies, some caution is needed before embarking on a
project to sequence an arachnid genome. Particular questions
a researcher working on a specific taxon should pose are: 1)

Do we need a genome sequence? And if so, 2) how complete
do we need it to be? And, perhaps more fundamentally, 3)
what level of completeness can we attain without spending an
unreasonable amount of resources? In reality, no genome
sequence (including human) is fully complete, and de novo
assembled and NGS derived genomes are even less so. De
novo assembly of short-read shotgun sequence data without
references, such as linkage maps or BAG libraries, remains
extremely challenging. However, as the Tetranychus, Varroa,
and Ixodes projects demonstrate, a fractured assembly may
still be useful if it is contiguous enough to build valid gene
models. In addition to life history and often body-size
considerations (i.e., the need for pooling individuals), intrinsic
features of arachnid genomes — in particular, the low % GC
content in some lineages mentioned above — might raise a
substantial barrier to whole genome de novo assembly
projects.

Even though the cost of NGS sequencing continues to drop
rapidly, depending upon the biological question, either
classical genetic marker-based approaches (Section 3 above)

BREWER ET AL.— GENOMICS IN EVOLUTIONARY ARACHNOLOGY

9

may be cheaper, easier, and sufficient, or NGS based
alternatives to genome sequencing may be more attainable
(e.g., transcriptome sequencing, and reduced representation
methods). Indeed these approaches may even be best used as a
means to rapidly develop numerous classical markers or
identify single nucleotide polymorphisms (as discussed in
Section 6). RNA-seq (the sequencing of cDNA libraries
derived from extracted mRNA and hence targeting tran-
scribed and therefore mainly coding regions — the transcrip-
tome) is rapidly becoming the tool of choice in genomic
studies. This is because RNA-seq data permits one both to
build gene models rapidly and to measure “digital” gene
expression among taxa and tissues; consequently the technique
has many potential applications.

Although “complete” genome sequences, even fragmented
ones, will yield fascinating information about genome
structure (repeats, transposons, translocations, etc.), to be of
greatest functional utility genomes must be annotated. While
computational annotation of gene models is possible (al-
though of course not optimized for arachnids), most
annotation schemes work best when supported by sequence
evidence. Again, RNA-seq and transcriptome data are of
greatest utility here and thus should also be generated for the
taxon whose genome is sequenced. Since RNA-seq data can be
assembled de novo, for example using software Trinity
(Grabherr et al. 2011), and annotated by homology searches
(at least for genes where known homologs exist) (e.g., using
BlastX and Blast2GO; Conesa et al. 2005), the experimenter
must again ask whether a full genome sequence is required at
all, and be cautious about assuming that this is a practicable
route.

6. APPLICATIONS OF NGS TECHNOLOGIES
IN ARACHNOLOGY

The number of possible applications using NGS technolo-
gies is vast and continues to grow. Here we provide examples
of their use, most of which do not require the sequencing of
entire genomes. There are many more potential applications
than those discussed below, and, as new platforms and
bioinformatic tools are developed, new avenues of research
will open.

6.1. Functional genomics: adaptation & selection. — Biologists
frequently seek to elucidate the relationship between environ-
mental parameters and organismal diversity. The potential for
detailing the genetic response of an organism to changes in the
biotic and abiotic environment are now in plain sight with the
availability of vast quantities of DNA sequence that can be
generated by NGS technologies, in particular through the
“assembly” of whole genome sequences. A review by Stapley
et al. (2010) discusses the potential of high-throughput
technologies in studies of adaptation. Whether focusing on
coding gene sequences, differential expression of transcripts,
identifying genomic regions experiencing linkage disequilibri-
um (LD), or quantitative trait locus (QTL) mapping to detect
genomic regions under selection, established methods using
high-throughput data exist.

Measures of selection: When studying protein-coding loci,
the most common method for measuring selection involves the
ratio of nonsynonymous to synonymous changes (dN/dS or
co). The resulting value potentially indicates the mode of

selection acting on the gene: co = 1 (neutral selection), co < 1
(stabilizing selection) and or > 1 (positive selection). By
employing a likelihood ratio test, R-values can be obtained to
differentiate between neutral and directional selection in
pairwise comparisons. Additionally, comparisons of co be-
tween branches in a multi-species/population phylogeny are
possible to identify genes or residues evolving differently or
similarly between clades. Inherently, co-based tests for selection
require coding data and are best served by transcriptomic
data. Commonly used tools for analysis of these data include
PAML (Yang 2007) and HyPhy (Pond et al. 2005). Some
studies have employed co tests in arthropods (e.g., Averof
2002; Porter et al. 2006; Viljakainen et al. 2009; Fort et al.
201 1), recently including spiders (Brewer et al. in review; Yim
et al. in prep).

To collect the data necessary for investigating selection in
coding sequences, RNAseq libraries are often generated. To
obtain the most nearly unique sequence possible in a single
run, the resulting cDNA libraries can be normalized by
removing excessive copies of highly expressed transcripts to
“equalize” the numbers with respect to the more poorly-
expressed transcripts (Zhulidov et al. 2005), but normalizing is
not essential. In addition to retrieving sequences, non-
normalized RNAseq libraries provide information concerning
the expression levels of transcripts. In order to leverage this
information, specimens must be treated to control all variables
so that the sources of differential expression (DE) can be
identified. Methods to analyze expression data using RNA-seq
data include the R packages “edgeR” (Robinson et al. 2009)
and “DEseq” (Anders & Huber 2010). Differences in
expression of transcripts between populations or species
indicate the evolution of coding loci involved in the expression
of a gene or non-coding regions of the genome that affect the
transcription (i.e., promoters, enhancers, and suppressors).
These methods are currently being employed in Hawaiian
Tetragnatha spiders to study differences in venom composition
between a lineage that builds webs compared to one that does
not build webs (Brewer et al. in review), building on earlier
work that used protein gel electrophoresis patterns to show
coarse differences between these lineages (Binford 2001). With
NGS techniques, we are now able to explore the individual
genes and relative changes in expression levels.

Selection can also be examined using LD approaches,
although this is necessarily limited to taxa where full genomes
are available. By mapping SNPs to a reference genome, data
obtained using reduced representation techniques (e.g., RAD-
seq) can be used to detect regions of the genome under strong
LD. This method has been used to identify regions of the
genomes of stickleback populations that are resistant to
introgression of outside genes (Hohenlohe et al. 2010).
Unfortunately, RAD-seq methods require high quality and
high quantity gDNA, which is often limited in small organisms
such as many spiders, even when freshly collected (Cotoras
unpubl. data).

Molecular basis for adaptation: Perhaps the most important
applications of NGS technologies in arachnids relate to silk
and venoms, two aspects of the biology of these organisms
that provide an almost endless variety of questions relating to
gene function. Both silks and venoms comprise complex
combinations of highly-derived, and often highly repetitive.

10

THE JOURNAL OF ARACHNOLOGY

proteins that serve myriad functions within and between
taxonomic groups. Both are linked to major ecological shifts
and evolutionary modifications in a number of clades. The
evolution of the forms and functions of spider silks has great
potential in evolutionary studies, as well as bioengineering
applications (Blackledge 2012; Garb 2013). Tools such as
SMRT and Nanopore, with their long reads, could help to
alleviate assembly issues associated with the highly repetitive
elements and allow more detailed exploration of the diversity
of spider silks at the genomic level. Venoms also vary greatly
across the Arachnida and are found in several orders (e.g.,
Araneae, Scorpiones, and Pseudoscorpiones). Beyond differ-
ential expression analyses, such as that described above,
characterization of venom cocktails and their molecular
evolution is lacking in most groups. Most work done so far
has focused on medically relevant species such as those in the
spider genera Latrodectus (e.g.. Garb & Hayashi 2013) and
Loxocdes (e.g., Zobel-Thropp et al. 2013) and the scorpion
genus Ceutniroides (e.g., Valdez-Velazquez et al. 2013). In an
applied context, these compounds have vast potential in
pharmacology and as pest control substances (reviewed by
King and Hardy 2013). Moreover, as mentioned above, these
compounds may also provide insights into the factors
underlying adaptation and how selection acts at the transcrip-
tional level (Binford 2001).

6.2. Phylogenetics. — To date, most molecular studies of
arachnids have sought to ascertain relationships between taxa.
Until recently, assessment of the phylogenetic affinities of an
organism required PCR amplification with degenerate primers
followed by amplicon sequencing to study loci in distantly
related taxa. The weakness of this approach is that rather few
loci can be examined, limiting the resolution of the Tree of
Life. Thus, the internal phylogeny of the subphylum
Chelicerata, class Arachnida, and lower taxonomic levels has
remained unresolved despite numerous efforts to ascertain the
relationships between taxa, including molecular phylogenetic
studies (recently reviewed in Agnarsson et al. 2013; Giribet
and Edgecombe 2013). Mitochondrial sequences have been the
most common data source. However, for the reasons
mentioned above (3.1), mitogenomic sequence data may not
be appropriate for reconstructing deep arthropod relation-
ships (Brewer et al. 2013). For example, although the
Euchelicerata (Xiphosura + Chelicerata) is almost unambig-
uously recovered using nuclear loci, datasets using mitochon-
drial genomic data often fail to support this relationship
(Masta et al. 2009; Rota-Stabelli 2010).

Within the Arachnida, most molecular phylogenetic studies
have focused on spiders, including the relationships within the
subclasses Mygalomorphae (Hedin and Bond 2006; Bond
et al., 2012) and Araneomorphae (Blackledge et al. 2009).
Molecular phylogenetic analyses within other orders exist,
including the Opiliones (Hedin et al. 2010; Hedin et al. 2012;
Burns et al. 2013), Acari (Klompen et al. 2007; Dabert et al.
2010; Pepato et al. 2010), Scorpiones (Salomone et al. 2007;
Borges et al. 2010; Prendini & Esposito 2010) and Amblypygi
(Esposito el al. in review). Representing a small sampling
of published works, all of these studies except Hedin et al.
(2012) use traditional Sanger sequencing approaches. Even
at these lower taxonomic levels, nuclear molecular markers
with appropriate phylogenetic signal are lacking, and primer

combinations for PCR often do not transfer between arachnid
groups, especially for species/population-level appropriate
loci.

High-throughput sequencing technologies provide a means
to collect vast amounts of molecular data for many taxa in a
timely manner and are currently used in various ways in
phylogenetics (see McCormack et al. 2013 and Rocha et al.
2013). The potential use of some NGS technologies in spider
systematics was recently discussed by Agnarsson et al. (2013)
and in Opiliones by Hedin et al. (2012). As for most non-
model organisms, the most common NGS data sources for
deep phylogenetics in arachnids are transcriptomes (Agnars-
son et al. 2013) and information generated from bait capture
techniques (for all taxonomic levels) such as anchored
enrichment (Lemmon et al. 2012). These approaches do not
require full genome sequences, which is especially useful given
the potential difficulties with arachnid genome efforts
mentioned above; moreover, the data generated provide loci
that are relatively easy to assign orthology and can be used at
deep taxonomic levels. Tools for the assignment of orthology
include HaMStR (Ebersberger et al. 2009), OrthoDB (Water-
house et al. 2012) and AGALMA (Dunn et al. 2013), while
PhyDesign (Eopez-Giraldez and Townsend 201 1) can be used
to investigate the phylogenetic signal of a locus across an
ultrametric tree. Recent molecular models of evolution (e.g.,
CAT. Lartillot and Phillippe 2004) and algorithms for
phylogeny reconstruction (e.g., Phylobayes, Lartillot et al.
2009; RAxML, Stamatakis 2006; and Fasttree 2, Price et al.
2010) have made phylogenomic studies much more tractable.
However, these analyses still can take weeks of computation
time, require large amounts of computer memory, and
demand a somewhat deep understanding of bioinformatics.

6.3. Population genetics & phylogeography. — NGS ap-
proaches have been widely celebrated for their potential in
providing large numbers of markers across the genome, which
is essential for population genetic and phylogeographic
studies. Since the per base cost is much lower than for Sanger
sequencing, it has become economical to apply NGS
techniques to generate traditional markers [e.g., microsatellites
in a tetragnathid species (Parmakelis et al. 2013)].

Among the most useful tools for population genetics, and
phylogenetics for that matter, are those based on reduced
representation libraries (RRLs), which attempt to recover a
small, random (i.e., unlinked) snapshot of the total genome.
As a result of focusing on a small sample of the genome, the
cost of sequencing a single individual is greatly reduced and
yet RRL methods can still identify many thousands of usable
single nucleotide polymorphisms (SNPs).

RADseq is a popular method for genome-wide marker
analysis because it reduces the complexity of the genome by
sub-sampling at certain restriction sites, assumed to be
homologous among taxa/specimens, to generate a single
nucleotide polymorphism (SNP) data set. The approach is
much like RFLPs and AFLPs, except that, instead of
separating the fragments on a gel to recover a DNA
fingerprint, they are sequenced (Davey et al. 2011). This
approach can provide several SNPs from each fragment,
multiplying the amount of data obtained from a single run. A
recent modification of this technique uses a double-digestion
and yields an increase in efficiency and a reduction in cost

BREWER ET AL.— GENOMICS IN EVOLUTIONARY ARACHNOLOGY

(Peterson et al. 2012). However, for many arachnid groups,
the RADseq method has requirements that may limit its use.
First, a large amount of high molecular weight DNA is
required (>2 micrograms per sample). Such high quality DNA
is essential in order to generate fragments that result only from
the restriction enzyme digest (i.e., library adaptors are not
ligated to the end of randomly sheared/degraded fragments).
Moreover, a high starting concentration of DNA is necessary,
since the protocol involves many steps that result in the loss of
DNA — typically only 7-15% of the starting material will be
recovered. The issue of DNA quality can be resolved by
preserving samples in 95% ethanol at -80 C, RNAlater at
— 80 C, or by using fresh specimens. Standard extraction kits
using ion-exchange columns or salt precipitation should work
well without causing undue shearing of the DNA. Ultimately,
DNA yield depends on the organism and although large-
bodied arachnids (e.g., many mygalomorphs, scorpions,
amblypigids, or solfugids) may yield sufficient DNA, smaller
taxa may require specimens to be “pooled” together, thus
losing individual-level data (Emerson et al. 2010).

An alternative RRL approach to RADseq is to use bait
capture methods, such as Exon Capture, which, in contrast to
RADseq, requires starting material to be randomly fragmented
(Bi et al, 2012). The basic approach is to sequence the
transcriptome of one individual and use those sequences to
design small, overlapping probes that are then attached to a
capture array (“chip”) or beads. The protocol starts with either
naturally (i.e., degraded or historical) or intentionally frag-
mented DNA, which is used to prepare DNA libraries following
a standard NGS protocol (e.g., Meyer & Kircher 2010). These
libraries are barcoded for each individual and used in a
hybridization experiment similar to a microarray. The number
of individuals that can be multiplexed in these experiments
depends on the target size in base pairs (i.e., the number of bases
printed on the chip), the desired depth of coverage and the
availability of barcodes. The number of single barcodes
commercially available is currently 96, but by using eight
double barcodes this can be increased to 768. This theoretically
allows the parallel sequencing of thousands of loci in hundreds
of individuals at the same time. One advantage of exon capture
for non-model organisms is that the sequences for the array are
obtained directly from a transcriptome and do not require a
previously sequenced genome. Moreover, the protocol is
suitable for historical museum samples, since it explicitly
requires randomly fragmented DNA, which is often the natural
state for museum-derived material.

There are two main limitations to the Exon Capture
approach. The first is that starting costs are high (reagents
and specialized equipment not common in most laboratories),
though they can be minimized by sharing among research
groups. Second, as in most NGS applications, sophisticated
expertise in bioinformatics is needed to manage the large and
complex data sets. Fortunately, user-friendly programs and
tools are becoming increasingly available for post-NGS
processing and analyses. Since exon capture targets exons,
most of the captured variation will correspond to synonymous
mutations in coding genes, allowing insights into population
variability. However, because genomic DNA is captured,
some of the non-coding flanking regions (e.g., untranslated
regions, introns) will also be recovered.

7. CONCLUSION

Arachnids have a rich history of molecular studies focusing
on many aspects of their biology. To date, few of these have
made use of recent advances in sequencing technology, but, as
we have outlined above, many future projects should benefit
from the use of next-generation sequencing platforms. These
technologies are diverse in their methods and applications,
and promising advances are on the horizon. However, it is
important to realize the strengths and weakness of NGS tools
and to embrace traditional techniques when more appropriate.

Although it is easy to be seduced by the amount of data that
can be generated by sequencing an entire genome, this is often
not necessary. In many cases, studies using transcriptomes
or reduced representation techniques can collect incredible
amounts of useful data to address any number of questions.
Regardless of the study, the number of potential avenues to
gather molecular data is large in terms of strategy and scale.
As arachnologists continue to amass novel data from diverse
lineages, our ability to identify loci, in terms of function and
homology, will increase and open more research opportuni-
ties. The unique biology and evolutionary history of arach-
nids, coupled with technological and bioinformatic advances,
will provide research opportunities for years to come.

ACKNOWLEDGMENTS

We would like to thank Douglass Morse for shepherding
this publication, two reviewers (Marshal Hedin and Ingi
Agnarsson) for greatly enhancing this work, and the I5k
initiative (J.S. Bechsgaard, J. Bechsgaard, A. McGregor and
T. Bilde) for providing preliminary data used herein. D.
Cotoras is funded by a Fulbright/CONICYT scholarship. The
Theridion grallator genome project is funded by NSF DEB
0919215.

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Manuscript received 3 October 2013. revi.sed 7 January 2014.

2014. The Journal of Arachnology 42:16-23

A new spider (Araneae: Haplogynae: Plectreuridae) from the Cretaceous Fossil-Lagerstatte of

El Montsec, Spain

Paul A. Selden: Paleontological Institute and Department of Geology, University of Kansas, 1475 Jayhawk Boulevard,
Lawrence, Kansas 66045, USA, and Natural History Museum, Cromwell Road, London SW7 5BD, UK. E-mail:
selden@ku.edu

Abstract. Two specimens of a new spider from the Cretaceous (ca. 129 Ma) El Montsec Fossil-Lagerstatte, northeastern
Spain, are described as Moiitseccirachne amicus gen. et sp. nov. and referred to the extant haplogyne family Plectreuridae.
Plectreurids are found today in only in the southwestern USA, Mexico, Central America and Cuba, but fossils show a more
widespread distribution in Eurasia. The new species adds an additional stratigraphic and biogeographical record. The
paleobiogeographical history of plectreurids is discussed, and it is concluded that the most likely scenario is that the family
was more widespread in the past, and has suffered extinction over much of its range, resulting in the present distribution.

Possibly, extant plectreurids represent a living remnant of a more diverse group of haplogynes that were widespread in the
Mesozoic.

Keywords: Mesozoic, paleobiogeography, Pholcoidea

Spiders from the Cretaceous Fossil-Lagerstatte (locality of
exceptional fossil preservation) of El Montsec, Spain, were
among the earliest Mesozoic Araneae to be described (Selden
1989, 1990). In these works, both araneoid and deinopoid
orbweavers were recognized, superfamilies that belong to the
Entelegynae Simon 1893 of the infraorder Araneomorphae
Smith 1902. Within this infraorder, the Haplogynae Simon
1893 are considered more primitive spiders, and the new
species described here belongs to that group. The two
specimens, conspecific adult males, come from the lithograph-
ic limestones of earliest Barremian age (ca. 129 Ma), from the
locality of La Cabrua in the Sierra de Montsec, northeast
Spain (Selden & Nudds 2012).

The fossils show distinctive characters, which allow them to
be placed in the modern haplogyne superfamily Pholcoidea
Koch 1850. This superfamily includes three families with the
sister-group relationship: Pholcidae Koch 1850 (Diguetidae
Pickard-Cambridge 1899, Plectreuridae Simon 1893) (Dunlop
& Penney 2011). Among these, the fossil species most closely
resembles the relatively plesiomorphic haplogyne family
Plectreuridae. The fossils are placed in a new genus and species,
Montsecarachne amicus gen. et sp. nov., in Plectreuridae.

Today, plectreurids are restricted in their distribution to
southwestern North America, Central America and the
Caribbean, but fossils show a more widespread distribution
in Eurasia. The new species described here, from the
Cretaceous of Spain, adds an additional biogeographical
record. It is possible that extant Plectreuridae represent a
living remnant of a group of haplogynes which were
widespread in the Mesozoic.

STRATIGRAPHY AND PALAEOECOLOGY

The specimens come from the outcrop known as La Cabrua,
which is an old quarry on the track between Rubies and Santa
Maria de Meia, just above the Sant Sebastia hermitage, in the
Sierra de Montsec mountain range (Bataller et al. 1953). They
were collected during an expedition from the Institut d’Estudis
Ilerdencs, Lleida, Spain.

The lithographic limestones of El Montsec were mapped as
a subunit of the Calcaires a Charophytes du Montsech
(Peybernes & Oertli 1972). Marine fossils such as rudist
bivalves and foraminiferans occur beneath and above the
Calcaires a Charophytes du Montsech. Studies on regional
stratigraphy, charophytes, and fossil assemblages have shown
that the age of the El Montsec lithographic limestones is
earliest Barremian (Gomez et al. 2002). The lithographic
limestones are rhythmically laminated carbonate mudstones
without any sign of currents or emersion features such as
raindrop marks or mud cracks. The exceptional preservation
of soft tissues, skeletons undisturbed by scavengers or
decomposers and random orientation of fossils, all point to
deep water, probably an anoxic lake bottom. The rhythmic
laminations are typical of lacustrine conditions and reflect
seasonal changes in the lake environment. Although the lake
was close to the sea, there is no evidence of a permanent
connection; e.g., in the form of marine fossils (De Gibert et al.
2000).

METHODS

The fossils are preserved on thin slabs of pale buff-grey
limestone. Grains are not visible in the rock, and the hackly
fracture and vitreous appearance under high-power micros-
copy suggest crystallization from a lime mud. Calcite-filled
cracks cross the specimens. The spiders are preserved as pieces
of cuticle on, and slightly within, the bedding surface. The
cuticle is brittle and brown: thicker parts are deep brown and
the thinnest cuticle pale buff. The cuticle is presumed to be
preserved as kerogen (Gupta et al. 2008). The best-preserved
parts are visible through a thin layer of translucent limestone,
but their morphological details are hazy due to the presence of
the overlying matrix. In such instances, 1-A% hydrochloric
acid was used, sparingly and with care, to remove the matrix
and thus to reveal fine structural details. The specimens are
fairly complete, with part and counterpart available for both.

The specimens were studied and photographed under 70%
ethanol (to enhance contrast) using a Leica MZ16 stereomi-
croscope, and photographed using a Canon EOS 5D Mklll

16

SELDEN— SPANISH CRETACEOUS PLECTREURID

17

digital camera attached to the microscope and DSLR
Assistant software (www.kaasoft.com) on an Apple MacBook
Pro computer. Photographs were manipulated using Adobe
Photoshop software, and final drawings were made from the
photographs using Adobe Illustrator. All measurements are in
millimeters and were made from the drawings using Photo-
shop’s analysis tools. Measurements of paired organs are
means of left and right of part and counterpart; i.e., maximally
four measurements if all are preserved.

Leg formula (e.g., 1423) indicates the length of each leg
relative to the others, from longest to shortest. Abbreviations:
car carapace, ch chelicera, cx coxa, fe femur, L length, lb
labium, mt metatarsus, mx maxilla, op opisthosoma, pa
patella, Pd pedipalp, sp spinnerets, st sternum, ta tarsus, ti
tibia, W width.

MORPHOLOGICAL INTERPRETATION

The two specimens are both adult males with identical
pedipalp morphology; they differ slightly in size (the holotype
is slightly larger), but otherwise any other morphological
differences can be explained by preservation, so they are
considered to be conspecific. Like other spider specimens from
this locality, and others from similar Mesozoic lacustrine
deposits (e.g., the Daohugou Lagerstatte of China (Selden &
Huang 2010), only cuticular structures are preserved, dorsal
and ventral superimposed. After splitting the rock, part and
counterpart may bear mainly dorsal or ventral structures, but
may show structures from both surfaces superimposed, or
some structures split between part and counterpart. For
example, LC-2936 B and LC-3780 B show mostly dorsal
features of the body (Figs. 1C, 3C), while their counterparts,
LC-2936 A and LC-3780 A (Figs. lA, 3A), show predomi-
nantly ventral body features. Nevertheless, chelicerae, pedi-
palps, and legs occur on all specimens, in some cases ventral,
others dorsal, and in places both sides superimposed. As with
most matrix-preserved fossil spiders, eyes are very difficult to
discern on the carapace. Commonly in fossil spiders, the
chelicerae splay apart during compression (e.g., in the
palpimanoids from Daohugou: Selden et al. 2008). This
phenomenon is not seen in the Montsec specimens, however,
which show a distinct overlap in the basal half; this suggests
cheliceral fusion, which is common in haplogyne spiders.

A peculiar feature of LC-2936 is an elongate piece of setose
cuticle running along the length of the tibia of leg 4 (on the
right in the part, LC-2936 A). The cuticle is identical in pattern
to that of the podomere and most likely belongs to the animal.
Since it is an adult male, which would not undergo post-adult
molting, the fossil represents a carcass, and so this feature can
best be explained as part of the podomere disrupted post
mortem, possibly by decay or scavenging.

SYSTEMATIC PALEONTOLOGY

Order Araneae Clerck 1757
Suborder Opisthothelae Pocock 1892
Infraorder Araneomorphae Smith 1902

Haplogynae Simon 1893

Remarks. — A number of features identify this species as a
member of the Haplogynae (see Table 1), including the
distinctive, pyriform pedipalpal bulb and embolus; swollen
male pedipalpal tibia; basally fused chelicerae and convergent

pedipalpal endites (maxillae). Also, the general habitus, with
legs of subequal length and low carapace, is haplogyne in
appearance.

Superfamily Pholcoidea Koch 1850
Family Plectreuridae Simon 1893

Remarks. — The characters of swollen male pedipalpal tibia,
chelicerae fused basally, and convergent ( parallel) maxillae
are indicative of the pholcoid-scytodoid branch of the
Haplogynae (Platnick et al. 1991; Griswold et al. 2012;
table 1). Plectreurid characters are unfused labium and
sternum (also shared with Periegopidae), and legs bearing
macrosetae. The distinctive tarsal bristles (Figs. 3E,F) are not
unique to plectreurids among haplogynes (e.g., figures in
Labarque & Ramirez 2012), but very similar bristles were
described and figured by Gertsch (1958, fig. 9). The new fossils
show particular resemblance to the extant Plectreurys castcmeci
Group, in having a short embolus, and the Jurassic
Eoplectreurys gertschi Selden & Huang 2010 in lacking
onychium, subsegmented tarsus and cheliceral stridulatory
ridges.

Genus Montsecarachne new genus

Diagnosis. — Distinguished from all other plectreurids,
except those belonging to the Plectreurys castanea Group
(Gertsch 1958), by its short embolus; all except Kihramoa
Chamberlin 1924 and Palaeoplectreiirys Wunderlich 2004 by
its lack of a spur on the tibia of leg 1 of the male; from
Kibramoa by the robust femur, as long as the carapace; and
from Palaeoplectreurys by the pedipalp and the presence of
additional spines.

Etymology. — After the type locality, El Montsec, and the
Greek cipdxvq (L. arachne), a spider.

Type species. — Montsecarachne anhcoritm n. sp. (monotypic).

Montsecarachne amicovum new species
Figs. 1-3

Etymology. — Latin amicus, a friend, in honor of the Amies
de la Paleontologia (Friends of Paleontology), who began the
series of organized excavations at the quarry of La Cabrua in
the 1970s and discovered so many of the exciting fossils from
the Fossil-Lagerstatte of El Montsec (Selden & Nudds 2012).

Type series. — Holotype adult male, LC-3780 lEI A,B (part
and counterpart); paratype LC-2936 lEI A,B (part and
counterpart) adult male; from lithographic limestones within
the Calcaires a Charophytes du Montsech, of Cretaceous
(earliest Barremian) age, in the quarry of La Cabrua, Sierra de
Montsec, northeast Spain; deposited in the Institut d’Estudis
Ilerdencs, Lleida, Spain.

Diagnosis. — As for the genus.

Description. — Holotype and paratype, adult males. Body
thickly clothed in fine setae. Macrosetae on legs, especially
distally. Carapace subcircular, very slightly longer than wide,
narrowed somewhat anteriorly; opisthosoma suboval, ca.
1.25X longer than wide (Figs. 1, 3). Sternum suboval, ca.
1.1 5X longer than wide, gently scalloped around coxae;
labium approximately pyriform, slightly longer than wide,
separate from sternum; maxillae longer than wide, convergent
and meeting in front of labium (Fig. 3A,B). Chelicerae
parallel, elongate, porrect, apparently conjoined in basal half;
fang short, curved; small tooth on paturon adjacent to fang tip

THE JOURNAL OF ARACHNOLOGY

18

Figure 1. — Montsecarachne amicorum gen. et sp. nov.: A. photograph of holotype part LC-3780 lEI A; B. explanatory drawing of A;
C. photograph of holotype counterpart LC-3780 I El B; D. explanatory drawing of C. Scale bars = 1 mm.

(Fig. 2A). Pedipalp approximately equal in length to leg 1
femur + patella; tibia swollen, bulb subspherical, embolus
narrows rapidly, from wide base, in short spiral to sharp
pointed apex. Leg formula 2134; legs subequal in length. Long
podomeres (femora, tibiae and metatarsi) approximately equal
in length (Figs. I, 3). Leg 1 femur slightly curved, approxi-
mately equal in length to carapace length. Few, thin

macrosetae on femora; distal femur of leg 1 with two strong,
curved macrosetae mesiodistally, another in midline, adjacent
to swollen pedipalp tibia (Fig. 1C,D). No macrosetae on
patellae; tibiae and metatarsi with numerous, stronger
macrosetae along their lengths; macrosetae at distal joints of
metatarsi. Tarsi without macrosetae but with strong bristles
distally, especially two or three large ones dorsally at distal

Table 1. — Comparison of morphological features of Montsecaraclme amicorum gen. et sp. nov. with other Plectreuridae and selected haplogyne families. See Ubick (2005), Labarque &
Ramirez (2012), and Griswold et al. (2012) for details. Notes: 'mostly, “blade-like morphology, ^flexibly according to Ubick (2005).

SELDEN— SPANISH CRETACEOUS PLECTREURID

19

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end above claws (Fig. 3E, F). Tarsal paired claws pectinate,
small unpaired claw present (Fig. 3E, F). Calamistrum absent.
Spinnerets subterminal on opisthosoma; short, one pair much
larger than the others (Figs. lA, 3A).

Measurements of LC-3780 lEI: body length (inc. ch) 5.36;
car L 2.13, W 1.99, ratio 1.07; op L 2.62, W 1.93, ratio 1.36;
St L 1.01, W 0.86, ratio 1.17; lb L 0.46, W 0.46; mx L 0.65,
W 0.50; ch L 0.89. Podomere lengths: Pd fe 0.75, pa 0.40,
ti 0.73, ta 0.46, bulb L 0.51, W 0.38, total 2.60; leg 1 cx 0.66,
fe 2.1 1, pa 0.72, ti 2.41, mt 2.42, ta 0.90, total fe-ta 8.56; leg 2
cx 0.73, tr 0.19, fe 2.32, pa 0.81, ti 2.54, mt 2.07, ta 0.89, total
fe-ta 8.62; leg 3 cx 0.62, tr 0.20, fe 2.02, pa 0.77, ti 1.68, mt
1 .94, ta 0.95, total L fe-ta 7.35; leg 4 cx 0.69, tr 0.22, fe 2.03, pa
0.76, ti 1.77, mt 1.85, ta 0.94, total L fe-ta 7.34.

Measurements of LC-2936 lEI: body length (inc. ch) 5.01;
car L 2.04, W 2.02, ratio 1.01; op L 2.30, W 2.02, ratio 1.14; st
L 1.14, W 0.96, ratio 1.19; lb L 0.48, W 0.34; mx L 0.68, W
0.47; ch L 0.72. Podomere lengths: Pd fe 0.71, pa 0.37, ti 0.77,
ta L 0.34, bulb L 0.66, W 0.40, total 2.59; leg 1 tr 0.21, fe 2.04,
pa 0.84, ti 2.16, mt 1.90, ta 1.01, total fe-ta 5.41; leg 2 tr 0.23,
fe 2.36, pa 0.66, ti 2.13, mt 2.16, ta 1.01, total fe-ta 5.66; leg 3
tr 0.23, fe 2.00, pa 0.58, ti 1.64, mt 1.89, ta 0.95; total fe-ta
5.34; leg 4 tr 0.25, fe 1.87, pa 0.64, ti 1.82, mt 1.51, ta 0.99,
total fe-ta 4.35.

DISCUSSION

Most fossil spiders, including Haplogynae, are known from
Cenozoic ambers. However, since haplogynes are generally
regarded as plesiomorphic among araneomorphs, they would
be expected to occur in older strata. Few Mesozoic haplogynes
are known, and almost all of them are from Cretaceous
Myanmar amber, described by Wunderlich (2008, 2012).
Among Pholcoidea, pholcids are known only from Cenozoic
ambers and Diguetidae have no fossil record, but plectreurids
have been described from the Jurassic (Selden & Huang 2010)
as well as Eocene Baltic (Wunderlich 2004) and Miocene
Dominican (Penney 2009) ambers. Some authors have
considered Plectreuridae to be among the more plesiomorphic
of the haplogyne spiders; in the introduction to his revision of
the family, Gertsch (1958, p. 1) stated: “The primitive hunters
of the family Plectreuridae are among the most generalized of
all the haplogyne ecribellate spiders”, and Krai et al. (2006), in
their study of karyotypy among basal spider clades, showed
that plectreurids exhibit the most plesiomorphic state. On the
other hand, phylogenetic analyses by Platnick et al. (1991) and
Ramirez (2000) placed plectreurids fairly high within the
haplogyne clade. A recent phylogenetic analysis, which
sampled the majority of spider families, placed plectreurids
lower down the haplogyne branch, adjacent to Hypochilidae
Marx 1888 and Filistatidae Ausserer 1867 (Agnarsson et al.
2013). Within the Plectreuridae, Montsecarachne resembles
most closely the Jurassic Eoplectreurys Selden & Huang 2010,
from which it differs in having a short embolus, lacking tibial
spurs on leg 1 of the male, and fewer macrosetae at the distal
joint of the metatarsus. In the first character, Montsecarachne
resembles the extant Plectrcinys tristis Group of Gertsch
(1958) and, in the second, the living genus Kihramoa. The lack
of a clasping spur on the tibia of leg 1 in the male, which is
present in the living Plectreurys and the Jurassic Eoplectre-
urys, but absent in the Recent Kihramoa, is replaced

20

THE JOURNAL OF ARACHNOLOGY

Figure 2. — Moiitsecarachne amiconun gen. et sp. nov. holotype counterpart LC-3780 lEI B: A. detail of carapace, chelicerae, and pedipalps; B.
explanatory drawing of A; C. tarsus of left leg 3, note pectinate paired claws; D. tarsus of right leg 2, note strong bristles dorsal to paired claws
and distal macroseta on metatarsus. Scale bars = 0.5 mm.

functionally by the strong, curved macrosetae at the distal end
of femur 1 in Montsecarachne (Figs. 1C, 3C). The femur of leg
1 of Montsecarachne is slightly curved and robust, and
approximately equal in length to the length of the carapace;
this character state resembles that in most plectreurids except
Kihramoa (femur 1 slender, much longer than carapace length;
Gertsch 1958). Interestingly, both of the Mesozoic plectreurids
have rather short carapaces in comparison with all other
plectreurid species. No phylogenetic analysis has been
performed on the Plectreuridae, so it is impossible to know
the relationships between any of the genera and species, fossil
and extant, of the family.

Biogeography. — Plectreurids are distributed today only in
the southwestern USA, Mexico, Central America and Cuba

(Alayon & Viquez 201 1), but fossils are known from Miocene
Dominican Republic amber (Penney 2009), from Eocene
Baltic amber (Wunderlich 2004) and from the Jurassic of
China (Selden «fe Huang 2010). The presence of a plectreurid in
Dominican amber is unsurprising, given their present-day
distribution; indeed, it might be expected that a living
specimen will turn up in the Recent fauna of Hispaniola
(Penney 2009). The presence of a plectreurid in middle Eocene
Baltic amber, however, suggests that the family either
migrated from Eurasia to its present area of endemism, or
that it was once more widespread and its range has contracted.
The existence of plectreurids in the early Cretaceous of Spain
and the mid-Jurassic of the North China Block (Selden &
Huang 2010) suggests that the family had a more widespread

SELDEN— SPANISH CRETACEOUS PLECTREURID

21

Figure 3. — Montsecarachne amicorum gen. et sp. nov.: A. photograph of paratype part LC-2936 lEI A; B. explanatory drawing of A;
C. photograph of paratype counterpart LC-2936 lEI B; D. explanatory drawing of C; E. explanatory drawing of F; F. detail of tarsal claws of left
leg 4. Scale bars = 1 mm.

9?

THE JOURNAL OF ARACHNOLOGY

distribution in the past; alternatively, its distribution could
have migrated from the Eastern Palearctic region to the
Western Palearctic, and then to the Western Nearctic, though
this scenario seems less likely.

Sanmartin et al. (2001) provided a concise description of the
reconstructions of the history of the Holarctic region from
the Mesozoic to the present day. By mid-Jurassic times (ca.
165 Ma), the North China Block, on which the oldest known
plectreurid, Eoplectreurys, was living by a volcanic lake
(Selden & Huang 2010), was close enough to eastern Asia for
biota to exchange between these continents (Metcalfe 2009).
The climate at the locality at this time was warm temperate
(Ren et al. 2010). However, America and Asia were widely
separated by a polar ocean in the mid-Jurassic, though some
dispersal may have been possible from Eurasia to North
America either directly, or by island-hopping via the British
Isles block (Sanmartin et al. 2001). By early Cretaceous times,
Montsecarachne was living near a lake in what is now
northern Spain, which was also part of Eurasia. This locality
was in the same warm temperate zone as occupied by
Eoplectreurys (Boucot & Scotese 2012). Dispersal of plec-
treurids had been possible from eastern to western Eurasia
through this period, though interrupted much of the time by
the presence of the central Asian Turgai Sea (Cox & Moore
2010). Also at this time, dispersal was possible from western
Eurasia to North America, via the same route through the
British Isles across the incipient Atlantic Ocean but, by the
later Cretaceous, this route became impassable. In the later
Cretaceous, a seaway developed dividing western North
America (at that time linked to Asia by the Beringian land
bridge) from eastern North America, which was becoming
more isolated from Europe as the Atlantic Ocean continued
to open northwards. By the end of the Cretaceous, the mid-
continental seaway dividing North America had regressed
and the continent became one again.

After the opening of the North Atlantic in late Cretaceous
times, terrestrial connections between Europe and North
America persisted across possible North Atlantic land bridges
until at least the early Eocene (ca. 50 Ma). The Thulean Bridge
is supposed to have connected southern Europe to Greenland
via Scotland, Iceland, and the Faeroes; Greenland was then
connected with eastern North America through the Queen
Elizabeth Islands. As the climate improved after the end-
Cretaceous extinction event and subsequent nuclear winter,
the climate warmed through the early Cenozoic, allowing an
exchange of temperate biota until the breaking of the Thulean
Bridge in the early Eocene (ca. 50 Ma). It was during the
earliest Eocene, when the combination of the availability of
the Thulean Bridge and the short-lived Paleocene-Eocene
Thermal Maximum climatic event, which saw temperatures
rise sharply, that mammals were able to interchange between
Eurasia and North America (Jones 2011), and also likely
caused a turnover in mamma! faunas (Gingerich 2006). This
was also a time when plectreurids could have migrated to
North America. Another possible North Atlantic land bridge
is the so-called de Geer Land Bridge, connecting Greenland to
Eurasia via Svalbard, though this is likely never to have been a
complete bridge, rather a stepping-stone, and may have been
too northerly and cold for thermophilic biota to have utilized
(Jones 201 1 ).

Another available land bridge between Eurasia and North
America was Beringia. Eastern Asia and western North
America became connected by land across the Bering Sea in
the mid-Cretaceous (ca. 100 Ma), and they remained joined
until the Pliocene. However, dispersal by this route appears to
have been less common than via the North Atlantic land
bridges for many plants and animals, with some exceptions
(Condamine et al. 2012). In the early Cenozoic, as the
temperature rose, boreal and, later, boreotropical forest
developed across Beringia, facilitating biotic interchange until
the Eocene-Oligocene transition event (EOT) (Hren et al.
2013), when temperatures decreased rapidly, rarely to return
to their previous levels. At the end of the Eocene, shortly after
Palaeoplectreurys was living in the Baltic amber forest
(Wunderlich 2004), the North Atlantic land bridges were no
longer available and the EOT had created a cold climate in
Beringia; so, if plectreurids had not spread to North America
before this time, then later opportunities became fewer and
less likely.

Selden & Huang (2010) discussed the possible center of
origin of the plectreurids, which was most likely on Eurasia.
Dispersal to the North American continent could have
occurred in the Jurassic, or later in the early Eocene. The
present distribution of plectreurids is most likely the result of
extinction across the large part of their range. Moreover, they
appear to have changed their habitat preference somewhat,
in that now they occur principally in arid environments.
However, although the records from Cuba (Alayon Garcia
2003) and Central America (Alayon & Viquez 2011) are not
from obvious arid habitats, the specimens were found in arid
habitats within otherwise humid environments. The Cenozoic
amber plectreurids come from humid forest environments,
though it is possible that they, too, were living in arid
situations within these environments. The Mesozoic plectreur-
ids come from perilacustrine environments, which could
include xeric habitats.

CONCLUSION

The new genus Montsecarachne can be accommodated
within the modern family Plectreuridae; it adds to the diversity
within that family and adds an additional stratigraphic and
biogeographical record. Plectreurids are restricted today to
arid habitats in the southwestern USA, Mexico, Central
America and Cuba, but Mesozoic and Cenozoic fossils show a
more widespread distribution in Eurasia. The most likely
paleobiogeographic history of the family is that it was more
widespread in the past and has suffered extinction over much
of its range, resulting in the present distribution. It is possible
that the extant plectreurids represent a living remnant of a
greater diversity of haplogynes, which were widespread during
the Mesozoic.

ACKNOWLEDGMENTS

I thank Derek Siveter, James Lamsdell and Erin Saupe
for valuable discussion; and Srs. Antonio Lacasa-Ruiz and
Albert Turull, Institut d’Estudis Ilerdencs, for arranging and
permitting the loan of the specimens for study.

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Manuscript received 24 July 2013, revised 9 October 2013.

2014. The Journal of Arachnology 42:24-33

Sex ratio bias caused by endosymbiont infection in the dwarf spider Oedothorax retusus

Bram Vanthournout'-^, Viki Vandomme' and Frederik Hendrickx^: ’Terrestrial Ecology Unit, Department of Biology,
Ghent University, Ledeganckstraat 35, 9000 Ghent, Belgium. E-mail: bram.vanthournout@hotmail.com; ^Ecology and
Genetics, Department of Bioscience, Aarhus University, Ny Munkegade 116, 8000 Aarhus, Denmark; ^Royal Belgian
Institute of Natural Sciences, Vautierstraat 29, 1000 Brussels, Belgium

Abstract. Spiders exhibit a remarkable variety of reproductive phenotypes such as induced parthenogenesis and
reproductive skew in primary sex ratio. However, observations of distorted sex ratios are mainly inferred from field catches
of adult individuals, whereas detailed information on clutch primary sex ratio and sex ratio inheritance, resulting from
multiple generations of laboratory rearing, is scarce. One of the potential causes of sex ratio variation is infection with
maternally inherited endosymbiont bacteria that alter a mother’s offspring sex ratio to increase their own fitness. Although
studies show that spiders are infected with several endosymbiont species, it was only recently discovered that endosymbiont
bacteria can cause a female sex ratio bias in this order. To explore the distribution of biased sex ratios and endosymbiont
infection patterns, we investigated sex ratio variation and bacterial presence in Oedothorax retusus Westring 1851.

Significant sex ratio variation was detected in six matrilines originating from wild-caught females, one of which consistently
showed a female bias in offspring production. Congruent with a bacterial effect, the sex ratio bias showed a clear maternal
inheritance, and treatment with antibiotics reversed the sex ratio to equal numbers of males and females. Female-biased
clutches were found to exhibit a significantly lower number of hatched spiderlings than unbiased clutches, suggesting the
occurrence of male-killing. All matrilines showed infection with the Cardinium endosymbiont, while two matrilines,
including the female biased one, were additionally infected with Wolbachia and Rickettsia. These findings indicate that
bacterial endosymbionts are responsible for the sex ratio variation in this species, and suggest that effects of endosymbiont
bacteria in the order Araneae could be more widespread than previously assumed.

Keywords: Sex ratio distortion, solitary spider, endosymbiont bacteria, male-killing, Wolbachia

Spiders display a diversity of reproductive phenotypes, such
as parthenogenesis and primary sex ratio distortion [sex ratio
at the time of fertilization (overview in Goodacre et al. 2006;
Martin & Goodacre 2009)]. Biases in offspring sex ratios have
been most extensively studied in social spider species through
both collections in the field and laboratory-based studies
(Aviles & Maddison 1991; Rowell & Main 1992; Aviles et al.
2000; Lubin & Bilde 2007). However, in solitary species,
existence of biased sex ratios is mainly inferred from
population studies where the absence or low numbers of
males could indicate either parthenogenesis and sex ratio
distortion or differential mortality of one sex (Vollrath &
Parker 1992; Baert & Jocque 1993; Levi 1996). Detailed
information on primary sex ratio distortion in solitary spiders
based on laboratory rearing is scarce (but see Gunnarsson &
Andersson 1996; Gunnarsson et al. 2004; Vanthournout et al.
2011).

One of the mechanisms that can cause a distorted sex ratio
is infection with endosymbiont bacteria. Endosymbiotic
bacteria are maternally inherited microorganisms that may
cause a variety of reproductive alterations in their hosts. Due
to their almost exclusively maternal inheritance, induction of
parthenogenesis, feminization, and male-killing, which all bias
the offspring sex ratio towards females, and the occurrence of
cytoplasmic incompatibility result in an increase of infected
females in the population (Werren & Beukeboom 1998;
Stouthamer et al. 1999; Charlat et al. 2003; Werren et al.
2008; Engelstadter & Hurst 2009). Owing to their obvious
effects on host ecology and reproductive biology, endosymbi-
ont bacteria have received increasing attention, and the
combination of screening studies and meta-analyses provides
mounting evidence that these endosymbionts are more

widespread than previously thought (Goodacre et al. 2006;
Duron et al. 2008a; Hilgenboecker et al. 2008). However,
understanding of the phenotypic effects of a large number of
such endosymbiont infections remains poorly investigated,
particularly in the Araneae.

Although several studies show that spider species exhibit a
high diversity and prevalence of endosymbiont species known
to influence their hosts’ reproductive biology (Goodacre et al.
2006; Baldo et al. 2008; Duron et al. 2008a, b; Martin &
Goodacre 2009; Yun et al. 2011), their potential effects are
verified in few cases. No effect of endosymbiont infection on
spider host reproduction could be characterized in Holocne-
mus pluchei Scopoli 1763 (Stefanini & Duron 2012). In
Pityohyphantes phrygianus C.L. Koch 1836, Gunnarsson et al.
(2009) suggested that endosymbionts play a role in influen-
cing offspring sex ratio, and recently Vanthournout et al. (2011)
showed that the endosymbiont bacterium Wolbachia is a
causative agent of a female-biased sex ratio distortion in the
dwarf spider Oedothorax gihhosus Blackwall 1841.

Despite these documented cases, it remains at present
unknown whether susceptibility to endosymbionts is confined
to only a very limited number of spider species, or whether
the effect is more widespread. To test this, we investigated the
occurrence of sex ratio variation and presence of cytoplasmic
sex ratio-distorting elements in a related species Oedothorax
relusiis Westring 1851 (Araneae: Linyphiidae: Erigoninae).
This palearctic dwarf spider is found in a variety of habitats,
usually in mosses, grasses and undergrowth, and is known to
be infected with several endosymbiont species (Goodacre
et al. 2006). Precise data on clutch sex ratio are currently
lacking, however, making this a suitable candidate for
examination.

24

VANTHOURNOUT ET AL.— SEX RATIO BIAS IN OEDOTHORAX RETUSUS

25

In this study we investigate the diversity and prevalence of the
endosymbiont community in O. retusus using endosymbiont-
specific PCR assays. To quantify the potential effects of each
endosymbiont on clutch sex ratio, we combined pedigree data
resulting from several generations of laboratory rearing and
results from a broad-spectrum antibiotic treatment. Further-
more, the phylogenetic position of the identified endosymbiont
species was determined and compared with those found in
Oedothorax gibbosus.

METHODS

1) Field collection, rearing conditions and breeding design. —
Six matrilines were set up in the laboratory by collecting adult
females by hand catches at Damvallei (Belgium) in the
summer of 2010. We placed females individually in plastic
vials of 5 cm diameter and 2.5 cm height with plaster bottoms.
Moistening the plaster with tap water kept humidity levels at
100%. A piece of moss was added to allow the construction of
a functional web. We provided fruit flies {Drosophila sp.) in
overabundance and checked food and humidity levels several
times a week. Vials were placed in a climate chamber with a
constant temperature of 20°C and a light-dark regime of 16L-
8D. Females were allowed to deposit up to three egg sacs
before being preserved in ethanol. Offspring were reared
individually as described above, except that juvenile spiders
received collembolans as a food source until the third molt.
After the final molt, we determined the sex of the spiders by
visual inspection using a stereomicroscope. This allowed
assessment of the tertiary sex ratio (number of adult male
offspring/total number of adult offspring).

Adult females were mated with unrelated males {n = 22
females, FI generation) to investigate the inheritance pattern
of the sex ratio trait and for the application of antibiotics (see

2). We reared offspring under standard conditions and again
determined tertiary sex ratio. Adult female offspring were
further mated with unrelated males to increase sample size and
to investigate the underlying mechanism (« = 19 females, F2
generation; see 4).

2) Antibiotic treatment. — It has been previously shown that
application of antibiotics is effective in eliminating endosym-
bionts in spiders (Goodacre et al. 2009; Vanthournout et al.
2011). To test whether administering antibiotics restores an
equal sex ratio (Morimoto et al. 2006; Gotoh et al. 2007), we
exposed FI fem.ales from the distorted line (Ml: Table 2) to
the broad-spectrum antibiotic, tetracycline. After reaching
adulthood, six haphazardly chosen FI females were treated by
moistening the plaster on the bottom of the vial with the
antibiotic solution (0.1%, w/v; 0.002 M). After approximately
seven days, females were allowed to copulate with first-
generation unrelated males. Offspring were reared individually
as described above with the continuous use of an antibiotic
solution. We used other FI females from the distorted
matriline as a control treatment (« = 6 females). Sex ratio
and survival of the clutches were determined and compared
between the treatments with a generalized linear mixed model
(proc GLIMMIX in SAS v. 9.1.2). To account for dependence
in sex ratio among mothers, mother ID was included as a
random effect.

3) Endosymbiont detection and phylogenetic relationship. —
We investigated the infection status of wild-caught females by

means of a PCR assay for five endosymbionts: Wolbachia,
Rickettsia, Cardinium, Spiroplasma and Arsenophonus. All
potentially cause reproductive alterations in arthropods. Since
one female died before being stored in ethanol, three of her
daughters were used in the PCR assay to determine maternal
infection status. All three daughters gave consistent results for
every endosymbiont tested.

Whole spiders were used for DNA extraction using the
Niicleospin Tissue kit (®Machery Nagel) following the
manufacturer’s recommended protocol. We used primers for
five endosymbiont bacteria: Wolbachia, Rickettsia, Cardinium,
Spiroplasma and Arsenophonus (Table 1). PCR conditions
were as follows: initial denaturation at 95°C for 2 min,
followed by 35 cycles of denaturation at 94°C for 30 sec,
annealing (for temperature, see Table 1), 30 sec, extension
(72°C, 90 sec) and a final extension at 72°C for 5 min.
Electrophoresis was performed on a 1.5% agarose gel. Gels
were stained in a solution of GELRED for approximately
15 min. Bands were visualized by UV-fiuorescence.

As Cardinium tested positive for all individuals, we were
able to confirm the reliability of the PCR detection of bacterial
infection; hence, no further positive control was necessary.
Prey items can be ruled out as a potential source of Wolbachia
contamination in the samples, as it has previously been shown
that our fruit fly and springtail breeding stocks were
uninfected (Vanthournout et al. 201 1). To test the significance
of a relationship between endosymbiont infection and sex
ratio, we used a generalized linear mixed model (Proc
GLIMMIX in SAS v.9.1.2.) with endosymbiont presence/
absence as a fixed effect. Dependence in sex ratio among
matrilines was accounted for by including matriline identity as
a random factor. The estimates obtained from the sex ratios
for infected and uninfected females were compared with an
even sex ratio using a t-test. Differences in sex ratio between
matrilines were analyzed using a chi-square test (RxC).

In order to check primer specificity and to investigate strain
diversity, we sequenced the PCR products using the BigDye
v.l.I Terminator Sequencing mix and ran them on an ABI
3710 automated sequencer. For Wolbachia, only the wsp gene
was sequenced. We used BLAST searches to identify the
closest relatives of the endosymbiont sequences obtained. The
ClustalW algorithm implemented in MEGA5 (Tamura et al.
2011) was used to align the sequences obtained with those
from other endosymbionts available in Genbank, which
mainly originate from the studies reported in Rowley et al.
(2004), Goodacre et al. (2006), Duron et al. (2008a) and Wang
et al. (2010). We then compared the phylogenetic position of
the endosymbiont wsp [Wolbachia), 16S {Cardinium) and
citrate (Rickettsia) gene sequences with those found in spiders
and other host species. For the wsp phytogeny, Wolbachia
supergroup delimitation was used as reported in Rowley et al.
(2004) and Goodacre et al. (2006). We constructed a p-
distance-based neighbor joining tree as implemented in
MEGA 5 (Tamura et al. 2011). Bootstrap percentage support
was calculated for the nodes by generating 10,000 bootstrap
values.

4) Mechanism. — Infection with male-killing endosymbionts
typically towers the clutch size to about one half, compared to
clutches produced by uninfected females. Feminization and
parthenogenesis induction do not influence clutch size. Based

26

THE JOURNAL OF ARACHNOLOGY

Table 1. — Primers used for detection of five endosymbiont genera.

Endosymbiont

Primer

Gene

Annealing temperature

Reference

Wolhachia

WSP81F

WSP691R

16Swolb99F

16Swolb99R

)vsp

16S rRNA

54°C

Braig et al. 1998

Oneill et al. 1992

Rickettsia

RICS741F

RCIT1I97R

citrate

54°C

Davis et al. 1998
Majerus et al. 2000

Cardiniimi

CLO-Fl

CLO-Rl

16S rRNA

54°C

Gotoh et al. 2007

Spiroplasuia

SP-ITS-J04

SP-ITS-N55

Spacer region between 16S
rRNA and 23S rRNA

52°C

Majerus et al. 1999

Arsenophoinis nasoniae

ArsF, ArsF3
ArsR2

16S rRNA

52°C

Duron et al. 2008a

on this difference, we can discriminate among these mecha-
nisms using two different approaches: testing for correlations
between number of offspring and sex ratio and censusing the
number of offspring as eggs and at hatching.

First, we tested for a correlation between number of adult
offspring and egg sac sex ratio among Wolhachia! Rickettsia-
infected (Ml, M2; Table 1) and among uninfected (M3-M6:
Table 1) females by means of a Pearson correlation on all
clutches, weighted for number of adult offspring. This was
further explored by investigating the relationship between the
egg sac sex ratio and the infection status of the mother, total
number of adult offspring and their interaction using a
generalized linear mixed model (Proc GLIMMIX in SAS
V. 9.1.2). Dependence in sex ratio among mothers was taken
into account by adding the identity of the mother as a random
effect. Moreover, if feminization occurs in this species, females
producing a biased clutch should produce more female
offspring than females producing offspring in equal numbers
of males and females. Therefore, we compared the number of
female offspring produced by Wolbctchial Rickettsia-'m^QciQd
and uninfected females by means of a generalized linear mixed
model (Proc GLIMMIX in SAS v. 9.1.2). To account for
dependence in sex ratio among matrilines, matriline ID was
included as a random effect.

Second, we determined the number of offspring at two
different census times: at the egg stage and at hatching from
the egg sac. Females that were used to produce the second-
generation offspring were allowed to oviposit up to three egg

sacs before being stored in ethanol. The spiderlings from the
first egg sac were allowed to emerge, and offspring were reared
to adulthood to determine the total number of emerged
spiderlings and tertiary sex ratio. The second and third egg
sacs were stored in ethanol six days after oviposition to allow
sufficient development of the eggs. Afterward, the proportion
of fertile eggs to the total number of eggs produced was
determined. Using a generalized linear mixed model (proc
GLIMMIX in SAS V.9.L2), we compared the total number of
emerged spiderlings and number of eggs produced for the
Wolhachia! Rickettsia infection status of the mother, census
time (before hatching versus after hatching), and their two-
way interaction. Identity of the mother was included as a
random effect to correct for dependence between clutches.

RESULTS

1) Sex ratio variation among maternal lines. — A highly
significant difference in average clutch sex ratio was detected
among matrilines (Table 2: df — 5, X~ = 65.6, P < 0.0001).
This difference was primarily attributed to a single matriline,
in which the wild-caught female and the daughter offspring of
at least two subsequent generations produced significantly
female-biased offspring. In the other five matrilines, equal
numbers of males and females were produced, even in
subsequent generations (M2-6: Table 2). Given that daughters
of the Ml line were in many cases crossed with males from the
other lines, persistence of the sex ratio distortion over several
generations strongly suggests a maternal inheritance (Ml:

Table 2. — Sex ratio data and endosymbiont infection status grouped by matriline.

Endosymbiont infection status

t Number ot Number ot

Matriline

Cardiniuni

Rickettsia

Wolhachia

crosses

males

Total number'

Sex ratio

F-value-

Ml

+

+

+

16

123

434

0.29

<0.0001

M2

+

+

+

5

62

135

0.46

0.38

M3

+

-

-

6

102

208

0.49

0.8

M4

+

-

-

5

73

165

0.44

0.16

M5

-h

-

-

4

88

156

0.56

0.13

M6

+

-

-

5

123

225

0.55

0.18

Tetracycline treatment

Ml

6

71

165

0.43

0.09

' Denotes the sum of the number of adult males and females.

“ Denotes the probability value of difference from an even sex ratio as calculated by a binomial test.

VANTHOURNOUT ET AL.— SEX RATIO BIAS IN OEDOTHORAX RETUSUS

27

Table 2). Conversely, three females from the undistorted lines
mated with three male offspring of Ml produced sex ratios
that were not significantly different from 0.5 (mean ± SE: 0.54
± 0.05, P = 0.22). Therefore, since no effect of males was
observed in the reciprocal crosses, this demonstrates that the
sex ratio distortion is not heritable through males and
confirms the exclusive maternal inheritance.

2) Antibiotic treatment. — Treatment of female offspring of
the distorted matriline with antibiotics significantly affected
the tertiary sex ratio (F/, /o = 6.46, P — 0.03). Untreated
females produced a significantly female-biased sex ratio (mean
± SE; 0.21 ± 0.05, Ao = -4,47, P = 0.0012), while
tetracycline treatment returned the sex ratio to an equal
proportion of males and females (mean ± SE: 0.43 ± 0.07,
I9.92 = -0.97, P = 0.4). Applying antibiotics did not influence
offspring survival, as no difference was found (Fm == 2.6, P =
0.4) in survival between tetracycline-treated (mean ± SE: 0.93
± 0.02) and control offspring (mean ± SE: 0.97 ± 0.01).

3) Endosymbiont detection and phylogenetic relationship. —
Screening of six individual females showed infection with up
to three different endosymibionts known to cause reproductive
alterations in arthropods. All females were infected with
Cardinium, while two females were infected with both
Wolbachia and Rickettsia. Over all generations, Wolbachial
Rickettsia infection status had a significant effect on the sex
ratio produced by a female (Pus = 4.62; P = 0.04), with
females infected with Wolbachia and Rickettsia producing a
significantly more distorted sex ratio than uninfected females
(mean ± SE: 0.35 ± 0.06, hs = -2.46, P = 0.02; 0.51 ± 0.04,
^35 = 0.25, P = 0.8, respectively).

For the two females testing positive for Wolbachia infection,
both the wsp and fToftflc/i/a-specific 16S rDNA primer gave
consistent results. Sequencing of the wsp primer revealed no
individual variation. BLAST searches revealed high similarity
with available Wolbachia sequences (E-values < le-199). The
wsp sequence [Genbank: JN889706] was most similar, with
sequences from the spiders Cybaeus penedentatus Bennet 2009
[Genbank: GQ480746], Araneus diadematus Clerck 1757
[Genbank: DQ231505] and Pityohyphantes phrygianus C.L.
Koch 1836 [Genbank; DQ231504], and clustered with high
support within supergroup B (Fig. 1: neighbor joining tree of
Wolbachia sequences).

The females with Wolbachia infection also tested positive
for Rickettsia. BLAST searches showed homology with
previously published Rickettsia sequences (E-values < le-
199). The Rickettsia sequence [Genbank: JN889707] showed
high similarity with the sequences of the spiders Oedothorax
gibbosus [Genbank; HQ286289], Hylyphantes graminicola
Sundevall 1830 [Genbank: DQ231487] and a Theridiidae sp.
[Genbank: DQ231486] (Fig. 2: neighbor joining tree of
Rickettsia sequences).

The Cardinium endosymbiont was found in all of the
females tested. Alignment of the sequences obtained revealed
no individual variation, and BLAST searches yielded high
similarity with available Cardinium sequences (E-values < le-
199). Sequences [Genbank: JN889705] were closely related to
the Cardinium. sequence of the spider Holocnemus pluchei
Scopoli 1763 [Genbank: EU333930] and clustered with high
support together with the sequence of the spider Oedothorax
gibbosus [Genbank: HQ286292] (Fig. 3: neighbor joining tree

of Cardinium sequences). We detected bands for Arsenophonits
in the two females infected with Wolbachia and Rickettsia.
However, sequencing and BLAST searches revealed that these
were amplifications of Rickettsia and thus constituted false
positives.

4) Mechanism. — We found a significant relationship between
the number of adult offspring and egg sac sex ratio in
Wolbachial Rickettsia infected females, with a significantly
lower proportion of males in smaller egg sacs (weighted
Pearson correlation: r = 0.57, P = 0.005: Fig. 4). For
uninfected females, no such correlation could be detected
(weighted Pearson correlation: r = 0.02, P = 0.95: Fig. 4).
There was a significant effect found for the total number of
adult offspring (Fiji = 7.05, P = 0.02) and Wolbachial
Fictoto'a infection (Fiji = 13.02, F = 0.004) on the egg-sac sex
ratio. Moreover, when Wolbachial Pdckettsia-irAcclcA mothers
produced a high number of offspring, the egg-sac sex ratio was
not biased; if a lower number was produced, the egg sac sex
ratio became significantly female biased, suggesting the
occurrence of male-killing. This was not observed in Wolba-
chial Rickettsia uninfected mothers (Fi ji = 6.60, P = 0.03).

There was no significant effect of infection status of the
female on the total number of female offspring (Fi ,3.3 = 0.02,
P — 0.9, mean ± SE: 11.9 ± 1.7 and mean ± SE: 12.2 ± 1.3
for infected and uninfected females, respectively). The total
number of spiderlings was smaller than the total number of
eggs produced, irrespective of the Wolbachial Rickettsia
infection status of the mother (F^i = 47.58, P < 0.0001).
However, significantly fewer spiderlings emerged when the
female was infected with Wolbachia and Rickettsia (mean ±
SE: 19.4 ± 1.8) than did uninfected mothers (mean ± SE:
32.8 ± 3.2, Fiji = 15.32, P = 0.0005: Fig. 5). Wolbachial
Rickettsia infection status significantly lowered the effect over
the total number of spiderlings and number of eggs (Fj^ji =
6.76, P = 0.02). Again, for this subset of mothers (F2
generation, see 1) a significant effect was found for Wolbachial
Rickettsia infection status on the offspring sex ratio (Fjn =
11.67, P = 0.005). Wolbachia and Picket tsia-mkcitA mothers
produced a significant female-biased sex ratio (mean ± SE:
0.30 ± 0.04, to = -5.08, P = 0.0002), while uninfected
mothers produced an even sex ratio (mean ± SE: 0.48 ± 0.04,
16.78 — —0.65, P — 0.54).

DISCUSSION

In this study, we report the presence of a maternally
inherited, sex-ratio-distorting bacterium in the solitary dwarf
spider Oedothorax retusus. This is deduced from several lines
of evidence: 1) one matriline produced significantly female-
biased offspring sex ratios, and several generations of
outcrossed laboratory rearing did not diminish the biased
sex ratio, 2) administering antibiotics to females of this
distorted matriline resulted in equalized sex ratios and 3) three
endosymbionts known to cause sex-ratio biases in their hosts
were found: Wolbachia, Rickettsia and Cardinium. Differential
mortality of the sexes during juvenile development is unlikely
to contribute to the sex ratio distortion, as average juvenile
survival is generally high (92%), and some highly distorted
clutches had almost 100% juvenile survival. All females were
infected with Cardinium, while only two females were infected
with Wolbachia and Rickettsia. Since 4 of the 5 uiidistorted

28

THE JOURNAL OF ARACHNOLOGY

10

Figure 1. — Phylogenetic position of Wolhachia wsp sequence of Oedotliorax retiisus [GenBank: JN889706]. Terminal taxa represent host
species. A p-distance based neighbor joining tree was constructed as implemented in MEGA 5 (Tamura et al. 2011) on a subset of Wolhachia wsp
sequences available at GenBank, with indication of the major Wolhachia supergroups (as reported in Rowley et al. 2004; Goodacre et al. 2006).
Percentage bootstrap support was calculated for the nodes. Genbank accession numbers are given in front of the taxonomic group to which the
host species belongs. Sequences that originate from spider hosts are underlined. Oedotliorax retasiis is shown in bold.

lines were not infected, and a significant relationship was
found between Wolhcichial Rickettsia infection and occurrence
of the sex ratio bias within this matriline, infection with these
endosymbionts is the most plausible causative agent of the sex
ratio distortion.

However, the relationship between bacterial presence and
sex ratio effect is not completely clear-cut. A significant
difference in sex ratio was found between Ml and M2, both
Wolhachia and Rickettsia infected matrilines (Table 1: df — 2;

- 14.51; P < 0.0002). This variable pattern of bacterial
expression of sex ratio distortion could be due to differences in
endosymbiont density (Breeuwer and Werren 1993; Hurst

et al. 2000; Bordenstein et al. 2006). Alternatively, the presence
of host suppressor genes could produce variation in the effect
on the sex ratio. Such suppressor genes are expected to evolve
in the framework of general sex ratio theory. The discovery of
such genes in butterflies and ladybirds indeed provides
empirical confirmation (Hornett et al. 2006; Majerus and
Majerus 2010a). Performing planned crosses to investigate the
precise mode of action of a proposed suppressor gene will be
necessary to demonstrate their presence in this species
(Majerus and Majerus 2010a).

The combination of these factors does not allow us to
identify the actual causal agent for sex ratio distortion in this

VANTHOURNOUT ET AL.— SEX RATIO BIAS IN OEDOTHORAX RETUSUS

29

89

EU448154 ACARI Haemaphysalis punctata
DQ081187 ACARI Haemaphysalis sulcata

EU8631 90 ACARI Haemaphysalis sulcata

FJ666771 HYMENOPTERA Pediobius rotundatus
FJ666772 COLEOPTERA Coccidula rufa

FJ666770 FIYMENOPTERA Au/ogymnus balani
93 ' FJ666769 HYMENOPTERA Au/ogymnus tritineatus

— GU131156 ACARI Amblyomma maculatum

— FJ666762 COLEOPTERA Subcoccinella vigintiquatuor

— U20242 COLEOPTERA Acfa/Za bipunctata
FJ666766 COLEOPTERA Ha/yz/a 16 guttata

— FJ666765 COLEOPTERA Ac/a//a bipunctata
FJ666768 COLEOPTERA Ada//a decempunctata
FJ666767 COLEOPTERA Calvia quatuordecimguttata

“ FJ666756 HEMIPTERA Acyrthosip/ion pisum
FJ666755 DIPTERA Bombylildae sp
- FJ666757 DIPTERA Bombylildae sp
FJ766353 HEMIPTERA Bemisia tabaci
— GU559856 HYMENOPTERA Pnigalio sp
FJ666761 LEPIDOPTERA Noctuidae sp
FJ666760 COLEOPTERA Elateridae sp
FJ666759 NEUROPTERA Chrysopid sp

85

99

89

r'

I C 1C

H

QR I E

96 ' FJ666758 NEUROPTERA Brachys tessellatus

FJ666754 COLEOPTERA Meloidae sp

■ FJ666753 COLEOPTERA Rhyzobius litura

- DQ231483 ARANEAE Meta menaei
98 D0231482 ARANEAE Meta menaei

DQ231484 AFtANEAE Gnalhonarium dentatum

99

DQ231485 ARANEAE Troxochrus scabriculus
— FM955314 COLEOPTERA Deronectes semirufus

7n [ DQ231487 ARANEAE Hvlaohantes oraminicola

DQ231486 ARANEAE Theridiidae so.
j JN889707 ARANEAE Oedothorax retusus

HQ286289 ARANEAE Oedothorax aibbosus

FM955315 COLEOPTERA Deronectes aubei
FM955313 COLEOPTERA Deronectes delarouzei
DQ231489 ARANEAE Walckenaeria cusoidata
DQ231488 ARANEAE Leotvohantes zimmermanni

DQ23 1493 ARANEAE Microneta viaria

99

jD(

I— r

FM1 77878 COLEOPTEF?A Deronectes platynotus
DQ231 492 ARANEAE Eriaone denlioalois

DQ231491 ARNAEAE Pitvohvohantes ohrvaianus

DO231490 AFiANEAE Araneus diadematus
• D0231481 ARANEAE Pitvohvohantes phrygianus

I 1

10

Figure 2. — Phylogenetic position of the Rickettsia (partial citrate sequence) endosymbiont of Oedothorax rettisiis [GenBank: JN889707].
Terminal taxa represent host species. A p-distance based neighbor joining tree was constructed as implemented in MEGA 5 (Tamura et al. 201 1 )
on a subset of Rickettsia sequences available at GenBank. Percentage bootstrap support was calculated for the nodes. Genbank accession
numbers are given in front of the taxonomic group to which the host species belongs. Sequences that originate from spider hosts are underlined.
Oedothorax retusus is shown in bold.

spider. A first indication of the identity of the sex ratio
distorter as well as a possible explanation for the apparently
variable sex ratio effect can be obtained by analyzing the
different densities of Wolhachia and Rickettsia, using quanti-
tative PCR (Goto et al. 2006). Obtaining females singly
infected with either endosymbiont could lead to more
conclusive evidence on the exact roles of each endosymbiont
and their possible interactions. This might be realized by
increasing the number of field-caught females if natural

variation is present between females infected with either
Wolhachia or Rickettsia. Extending the current study by
increasing the sample size of collected females and by
including multiple populations would equally allow for an
accurate assessment of the occurrence of the sex ratio bias in
natural populations.

Alternatively, treatment of doubly infected females with low
doses of antibiotics (Sasaki et al. 2005) or transfection of
endosymbionts (Sasaki et al. 2002) can establish such single

30

THE JOURNAL OF ARACHNOLOGY

95

AB506773 HOMOPTERA Harmalia sirokata
AB506773 HEMIPTERA Harmalia sirokata
AB506775 HOMOPTERA Euides speciosa
GQ455421 HEMIPTERA Lepidosaphes pinnaeformis
EU930867 ACARI Chaetodactylus sp
EU333931 ARANEAE Linvphia triangularis

r AY753170 ACARI Metaseiulus occidentalis

GQ455420 HEMIPTERA Protargionia larreae

I AB506779 DIPTERA Culicoides peregrinus

99 AB506776 DIPTERA Culicoides arakawae

I EU333930 ARANEAE Holocnemus oluchei

I — JN889705 ARANEAE Oedothorax retusus

81 HQ286292 ARANEAE Oedothorax aibbosus

AB241 132 ACARI Tetranychus urticae
EU333929 ARANEAE Pachvanatha deaeeri

p AB001 51 8 ACARI Ixodes scapularis

AM042540 HEMIPTERA Scaphoideus titanus

— DQ449047 ACARI Tetranychus cinnabarinus

GQ455428 HEMIPTERA Poliaspis media

7^ GQ455424 HEMIPTERA Palinaspis quohogiformis

_| 75 L GQ455412 HEMIPTERA Unaspis euonymi

|— GQ455437 HEMIPTERA Aspidiotus nerii

GQ455413 HEMIPTERA Howardia biclavis

- GQ455427 HEMIPTERA Asp/d/ofus nerii

GQ455426 HEMIPTERA Chionaspis heterophyllae

83

r GQ455411 HEMIPTERA Abgra//asp/s degenerate
^AY327472 HYMENOPTERA P/ag/omerus diaspidis
DQ854713 HYMENOPTERA Encarsia hispida
EU567084 HEMIPTERA Sem/s/a tabaci
I EU333927 ARANEAE Alooecosa pulverulenta

^ EU333926 ARANEAE Cvciosa conica

EU333928 ARANEAE Evarcha falcate

FJ388307 citrus pathogen

c,:

5

Figure 3. — Phylogenetic position of the Cardiniuw (16S rRNA gene) endosymbiont of Oedothorax retusus [GenBank; JN889705]. Terminal
taxa represent host species. A p-distance based neighbor joining tree was constructed as implemented in MEGA 5 (Tamura et al. 2011) on a
subset of Cardinium sequences available at GenBank. Percentage bootstrap support was calculated for the nodes. Genbank accession numbers
are given in front of the taxonomic group to which the host species belongs. Sequences that originate from spider hosts are underlined.
Oedothorax retusus is shown in bold.

infections. Also, we cannot exclude the possibility that the
observed sex ratio bias is caused by an as yet unidentified
endosymbiont distorter. A next generation sequencing ap-
proach would allow us to obtain a broad assessment of the
endosymbiont diversity in this spider species (Andreotti et al.
2011; Hirsch et al. 2012).

The strong correlation between number of adult offspring
and egg-sac sex ratio supports the hypothesis that the killing
of males is the most plausible mechanism of sex ratio
distortion. Feminization is highly unlikely, as infected and
uninfected females produced equal numbers of female
offspring. The occurrence of male-killing is supported by
comparing the total number of spiderlings with the total

number of eggs produced. This is the most favorable approach
for directly linking the number of emerged spiderlings, egg
number, and corresponding offspring sex ratio of one female,
since development of eggs within the egg sac hampers visual
inspection of egg development and hatching rates. Moreover,
hatching from the egg and first molt of the spiderlings occur
inside the egg sac. This causes a time lag between hatching of
the eggs and emergence of the spiderlings from the egg sac,
resulting in the inability to single out any undeveloped eggs.
As expected, spiderling number is significantly smaller than
egg number for both infected and uninfected mothers. This
can be caused by mortality during egg hatching and early
juvenile cannibalism occurring inside the egg sac. However,

VANTHOURNOUT ET AL.— SEX RATIO BIAS IN OEDOTHORAX RETUSUS

31

• Wolbachia/Rickettsia uninfected mother
o Wolbachia/Rickettsia infected mother

Figure 4. — Relationship between number of adult offspring and
proportion of male offspring in the egg sac. Open circles: WoWachia
and R/cA'c/5^/fl-infected females, filled circles: Wolhachia and Ricli-
c/55/a-uninfected females. The solid line visualizes the linear
correlation.

significantly fewer spiderlings emerge from egg sacs produced
by Wolhachia and Rickettsia-xnkciQd females. The reduction
in offspring being produced correlates with the bias toward
female offspring in these clutches, suggesting that the offspring
that do not emerge from the egg sac are predominantly males.
This is again strong evidence for the occurrence of male-
killing. Because almost all eggs showed signs of embryonic

50

after hatching before hatching

Census time

Wolhachia / Rickettsia infected mother
I I Wolhachia / Rickettsia uniniecled mother

Figure 5. — Clutch size number produced by Wolhachia and
Rickettsia infected (black bars) and uninfected (grey bars) females
in first egg sacs (after hatching, number of spiderlings) and second
and third egg sacs (before hatching, number of eggs). Bars with
the same letter annotation indicate values that are not sig-
nificantly different.

differentiation (95.7%, n = 349) in the egg sacs of infected
mothers, male-killing is most likely occurring late in embry-
onic development or during hatching.

As the sister species O. gihhosus is infected with similar
genera of endosymbionts, establishing the phylogenetic
position of the 16S sequences could present valid information
on the relatedness of the endosymbionts. This reveals a close
relatedness between the Cardimiim endosymbionts infecting
both species. This is also the case for the citrate gene sequence
in Rickettsia, which is closely related to the sequence of the
Rickettsia endosymbiont of O. gihhosus. In contrast, a clear
dissimilarity is found between the nsp sequences of Wolhachia.
The O. retiisus wsp sequence clusters within supergroup
B, while the wsp sequence of O. gibbosiis clusters within
supergroup G. Therefore, these data suggest that Cardiniwu
and Rickettsia infection predates the divergence of these
species, followed by independent invasions of different strains
of Wolhachia in the two species. However, to gain more insight
into the routes of infection in the different species and the
relatedness between endosymbionts in the genus Oedothorax,
a more detailed analysis by applying a multilocus comparison
(Baldo et al. 2006) would be most suitable.

Although our research is based on a small sample size, the
similarity of these results to a prior study of endosymbionts of
Oedothorax gibhosus is striking (Vanthournout et al. 2011).
For the populations investigated, both species seem to be fixed
for the Cardiniwu endosymbiont, while the Wolhachia
infection shows a more variable pattern with approximately
half of the individuals infected. The infection pattern of the
Rickettsia endosymbiont is different for the two species; O.
gihhosus seems to be fixed, while O. retiisus shows infection for
half of the individuals.

The effect of male-killing endosymbionts in several species
of ladybirds shows high variation in the production of
offspring sex ratios, ranging from all-female broods to the
production of significant numbers of males, (Hurst et al. 1992;
Majerus & Majerus 2010b). This is similar to the male-killing
effect in both O. retiisus and O. gihhosus, with infected females
showing a high variation in sex ratio among clutches, which is
of the same order of magnitude [O. gihhosus: 0.36 ± 0.04
(Vanthournout et al. 2011); O. retusus: 0.35 ± 0.06].

In contrast, for the butterfiy species Hypolinmas holina and
Acraea encedoii, infection with male-killers exhibits a higher
level of penetrance with the production of only all female
broods (Jiggins et al. 2001; Dyson et al. 2002). The
evolutionary significance of this difference in pattern of
expression of sex ratio distortion remains to be investigated.

Our findings suggest that the phenotypic effects of
endosymbiont bacteria on reproductive characteristics could
be more widespread in the Araneae order. This confirms the
use of a bacterial model as one possible mechanism of
different reproductive phenotypes found in many spider
species. Further studies into the effects in other spider taxa
are necessary to determine their general susceptibility to
endosymbiont bacteria and the effects on their hosts’ ecology
and evolution.

ACKNOWLEDGMENTS

We thank Lucien Mushimirwa for maintenance of the
collembolan and fruit fly breeding stocks and help in rearing

32

THE JOURNAL OF ARACHNOLOGY

the spiders in the laboratory. Andy Vierstraete performed
part of the sequencing analysis. We thank Martijn Vandege-
huchte for useful discussions on the Martin sample size. Bram
Vanthournout holds a PhD scholarship grant from the agency
for Innovation by Science and Technology (IWT grant no.
73344). Additional financial support was received from the
Belgian Science Policy (BELSPO, research project MO/36/025).

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2014. The Journal of Arachnology 42:34-43

Vertical stratification of spider assemblages in two conifer plantations in central Japan

Hiroki Oguri', Tomohiro Yoshida-^, Akihiro Nakamura^, Masashi Soga"’ and Naoki Hijii': 'Graduate School of

Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; ^Field Science Center, Faculty of Agriculture,
Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan; ^Key Laboratory of Tropical Forest
Ecology, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan
666303, P. R. China; ‘‘Department of Forest Science, Graduate School of Agriculture, Hokkaido University,

Sapporo 080-8589, Japan

Abstract. We compared the structure of spider assemblages between the upper and lower canopy layers, and between the
canopy and forest door, in plantations of evergreen cedar (Cryptomeria japonica) and deciduous larch (Larix kaempferi).

The estimated number of species was similar between the upper and lower canopy layers (49.0 vs 45.1) in C. japonica, but
was noticeably smaller in the upper canopy layer (11.3) than in the lower layer (36.9) in L. kaempferi. Arboreal spider
assemblages in the canopy differed significantly between the upper and lower layers in both C. japonica and L. kaempferi
stands, based on an abundance-based measure. However, based on an incidence-based measure, they only differed
significantly between layers in the L. kaempjeri stand. The spider assemblages also differed distinctly between the canopy
and the forest floor in both stands. Wandering spiders and orb-web builders were dominant in the canopy, while space-web
builders dominated the forest floor in the C. japonica stand. In the L. kaempferi stand, wandering spiders dominated both
the canopy and the forest floor. Our results suggested that spider assemblages in conifer plantations were distinctive among
strata because of differences in such factors as resource quality (i.e., living or dead foliage) and association with adjacent
layers along the vertical gradient of the forests.

Keywords: Community composition, forest canopy, forest floor, functional groups

Vertical stratification in forests both above ground and at
ground level is attributed to the variability of the three-
dimensional spatial arrangement of trees and other structural
elements (Ishii et al. 2004). Forest canopies provide various
food resources such as leaves, fruits, and seeds and diversified
microhabitats based on the structural complexity of foliage and
twigs, resulting in a high abundance and diversity of arthropods
(Lawton 1983; Basset et al. 2003). The forest floor also contains
a mixture of organic resources such as leaf litter, fungi, and
dead wood, with a continuous stratum packed into a thin layer
(Lavelle & Spain 2005). Spiders (Arachnida: Araneae) are one
of the most prevalent groups of predatory arthropods in species
diversity and biomass, both in the canopy and on the forest
floor (Moulder & Reichle 1972; Basset 1991; Wise 1993). These
groups occupy a highly diversified set of habitats, ranging from
various plants to the soil itself, construct a variety of web
structures (or no web for many forest-floor species) and exhibit
broad feeding behaviors.

The canopy and forest floor have different architectures
derived from the substrates that exist in each stratum, which
could be a determining factor of the structure of spider
assemblages. Previous studies have revealed that the foliage
structural complexity of the canopy and vegetation, such as
foliage density and number of leaves and branchlets, affected
spider species composition (Gunnarsson 1988, 1990; Sundberg
& Gunnarsson 1994; Halaj et al. 2000; De Souza & Martins
2005; CorcLiera et al. 2008). Likewise, the structural complex-
ity of forest-floor litter and understory vegetation, such as
litter depth, litter shape, interstitial space/volume, and ground
cover by plants, can influence spider assemblages on the forest
floor (e.g., Uetz 1975, 1979; Bultman & Uetz 1982; Docherty

^Corresponding author. E-mail: yoshitom(gcc. tuat.ac.jp

& Leather 1997; Bultman & Dewitt 2008). The canopy and
forest-floor strata can provide different microhabitats for
arthropods, presumably leading to different spider assemblag-
es among the strata.

Even-aged and monoculture forest plantations usually have
simple architecture compared to natural forests, and thus they
are good model systems for examining the effects of the vertical
structure of forests on biological communities. Japanese cedar
Cryptomeria japonica D. Don and Japanese larch Larix
kaempferi [Lamb.] Carriere, two endemic coniferous species in
Japan, are general tree plantation species that provide different
microhabitats for forest arthropods. For example, the seasonal
stability of microhabitats differs between the two species: C.
japonica is an evergreen species, whereas L. kaempferi is
deciduous. Cryptomeria japonica trees usually have a large
amount of dead foliage attached to their trunks in the lower
part of the canopy (Yoshida & Hijii 2006), whereas in L.
kaempferi forests, most of the foliage is alive in both the upper
and lower layers until the period of leaf fall in late autumn (from
October to November: Miyaura & Hozumi 1988). The
structural complexity of foliage also differs greatly; thicker,
harder, and more complex foliage forming needle-like leaves in
C. japonica, compared to the soft and clumped needles of L.
kaempferi. These differences in spatial and temporal traits
between habitats and between tree species should affect the
composition of spider assemblages in the canopy and on the
forest floor.

In the present study, we investigated the community
structures of arboreal and ground-dwelling spiders in C.
japonica and L. kaempferi plantations to test two hypotheses:
1 ) compositions of arboreal spider species differ between the
upper (living foliage) and lower (dead foliage) layers of the C.
japonica canopy, but not of L. kaempferi due to its similarities

34

OGURI ET AL.~VERTICAL STRATIFICATION OF SPIDERS

between upper and lower layers; and 2) spiders have different
community structures in the canopy and on the forest floor
due to the difference of habitat resources, such as elongate
foliage and accumulated litter.

METHODS

Study site. — The study was carried out in a 38-year-old (as
of 2008) C. japonica plantation and a 15-year-old L. kaempferi
plantation in the Experimental Forest of Nagoya University,
in central Japan (35°11'N, 137°33'E; 980 to 1000 m a.s.L).
Annual rainfall at this site averages 2100 mm, and the mean
annual air temperature is 9.7 °C (2008). Both stands are
embedded in a forested area and are more than 1000 m from
each other. Tree height and height at the lower edge of the
canopy are 24 m and 7 m in the C. japonica stand, and 10 m
and 2 m in the L. kaempferi stand, respectively. In the C.
japonica stand, the canopy is almost closed due to more
densely packed and elongated, thickened branches in the
upper layer and large numbers of dead leaves and branches
remaining attached to the trunk of each tree in the lower layer
(Yoshida & Hijii 2006). Thus the lower layer is similar in
overall architecture to the upper layer. In the L. kaempferi
stand, the canopy is more open due to less crowding of
branches mixed in with some young broadleaf trees in the
understory. The canopy of each stand was divided into upper
and lower layers at the following points: in C. japonica at the
uppermost height of dead leaves and branches attached to the
tree stems (ca. 15 m above the ground), and in L. kaempferi at
half the length of the canopy (ca. 6 m above the ground). The
vertical lengths of the upper and lower layers of the canopy are
9 m and 8 m in C. japonica, 4 m and 4 m in L. kaempferi,
respectively. Five trees of each species were selected for
sampling of the spider assemblages. The sampled C. japonica
trees were located near a 20-m tower and had a mean height
(± SD) of 22.6 ± 0.4 m and a mean diameter at breast height
of 23.9 ± 1.8 cm. The L. kaempferi trees sampled have a mean
height of 9.1 ± 0.9 m and mean diameter of 10. 1 ± 1.4 cm.
The average thickness of the litter layer on the forest lloor is
less in the C. japonica stand (0.9 ± 0.4 cm) than in the L.
kaempferi stand (3.4 ± 0.8 cm). However, many dead branches
with foliage had accumulated on the ground in some parts
of the C. japonica stand, increasing the local thickness of the
litter layer (~ 6.8 cm) and thus causing a greater habitat
heterogeneity on the forest floor than in the L. kaempferi stand
(T. Yoshida, unpubl. data).

Spider sampling. — Spiders were collected from three habi-
tats (upper and lower layers of canopies, and forest lloor) in
each tree stand at one-month intervals from 10 July to 19
December 2008. We accessed the canopies by using a 20-m
tower in the C. japonica stand and by climbing on a
connectable tube ladder on the trunks of L. kaempferi trees.
Three branches in each layer were randomly selected for spider
collection. Spiders were dislodged by beating the branches
with a 1.8-m bamboo stick and were trapped with a fine net
(0.2-mm mesh size; 60 cm in aperture diameter of a round
frame). The spiders were quickly collected with a vacuum
sampler and preserved in 70% ethanol. Spiders on the forest
floor were collected using pitfall traps, which consisted of 400-
cm^ plastic cups with openings 7.5 cm in diameter. Each trap
contained 100 ml of water, with small amounts of detergent to

35

prevent the animals from fioating, and one to two grams of
sorbic acid for preservation. Ten traps were set at least 5 m
apart from each other in a transect on the forest floor of each
stand, the openings level with the ground surface (not with the
top of the litter layer), and collected after a week.

Spiders were first sorted to genus, and then morphospecies
or identified to described species according to the keys and
descriptions of Chikuni (1989) and Ono (2009). We recorded
the number of individuals in each species for each habitat,
each forest stand, and each month. Voucher specimens were
deposited in the Laboratory of Forest Protection, Nagoya
University, Japan. Using the information in Shinkai (2006)
and Ono (2009), we divided these species into four functional
groups, which included the guilds reported by Halaj et al.
(1998, 2000), Hatley & MacMahon (1980), Uetz et al. (1999)
and Cardoso et al. (2011): 1) space-web builders, including
hackled-band weavers (Dictynidae), sheet-web weavers (Cy-
baeidae, Agelenidae, and Linyphiidae) and cobweb spiders
(Theridiidae); 2) orb-web weavers (Uloboridae, Araneidae
and Tetragnathidae); 3) wandering spiders, including jump-
ing spiders (Salticidae), ambushers (Thomisidae) and run-
ning spiders (Philodromidae and Lycosidae), nocturnal
hunters (Clubionidae, Anyphaenidae and Gnaphosidae),
and a part of Theridiidae and Araneidae that have wander-
ing foraging strategies (Shinkai 2006); and 4) edaphic spiders
(Antrodiaetidae).

Data analysis. — We excluded juveniles and unidentified
individuals prior to the analyses. Spiders collected from each
habitat (i.e., upper canopy, lower canopy and forest floor from
both tree species) were pooled for each month; hence, all
analyses were based on six monthly samples within each
habitat. We quantified the diversity of spider assemblages in
each habitat using Estimates 8.2 (Colwell 2009). With a
bootstrap estimator, we randomized the data 100 times and
calculated the estimated number of species {S^sd- Using
Estimates, we also calculated 95% confidence intervals of
the observed species richness (using MaoTau function).

We used permutational multivariate analysis of variance
(PERMANOVA: Anderson et al. 2008) to assess the effects of
forest stand (C. japonica and L. kaempferi), layer (upper and
lower canopy), sampling month and their interactions on
canopy spider assemblages. Likewise, we investigated the
effects of stand and month on the forest floor spider
assemblages. The design of the analysis is analogous to a
repeated measures ANOVA, where we treated the effect of
monthly variation as a random effect factor and the
differences in stand and layer as fixed factors. Although
PERMANOVA was developed primarily for multivariate
analysis, univariate analysis is possible using Euclidean
distances, which yield Fisher’s traditional univariate F statistic
(Anderson et al. 2008). Type III sums of squares were used to
calculate F statistics (pseudo-F statistics: Anderson et al.
2008). P values were calculated using 4999 permutations of
residuals under a reduced model. Post-hoc pair-wise analyses
were conducted for some of the variables by calculating t
statistics and P values using 4999 permutations of the data
(available within PERMANOVA routine: Anderson et al.
2008). We also used non-metric multidimensional scaling
(NMDS) ordinations, available in PRIMER6 (Clarke &
Gorley 2006), to visually represent the species compositions

36

THE JOURNAL OF ARACHNOLOGY

Table 1. — Species richness of spiders in the upper (UC) and lower
canopies (LC) and on the forest floors (FF) of the Cryptomeria
Japonica (CJ) and Larix kaempferi (LK) stands.

CJ

LK

UC

LC

FF

UC

LC

FF

Number of individuals’

n

832

889

39

662

888

299

Number of observed

species

‘S'obs

43

40

16

11

34

27

Number of estimated

species

‘S'est

49

45.1

19.5

11.3

36.9

31.5

‘ Excluding juvenile and unidentified individuals.

of canopy spiders in the upper and lower layers of the C.
japonica and L. kaempferi stands. We did not use NMDS
ordinations for the forest floor spiders because we collected
very few individuals in the C. japonica stand (Table 1).
Ordinations were conducted based on the abundance-based
(Bray-Curtis index) and incidence-based (Sorensen index)
similarity measures, with 25 restarts.

RESULTS

Spider assemblages in the canopy. — In total, we collected
3,609 individuals (excluding 51 juveniles and unidentified
individuals), representing 100 species and morphospecies from
both the canopy and forest floor during the study period
(Appendix 1). We collected 43 (with 95% confidence interval
of ± 8.7) and 40 ± 8.8 spider species in the upper and lower
canopy layers of the C. japonica stand and 1 1 ± 0 and 34 ± 3.9
species in the L. kaempferi stand, respectively (Table 1). The
estimated number of arboreal species was similar between in

the upper (49.0 species) and lower canopy layers (45.1 species)
in C. japonica, but was noticeably smaller in the upper canopy
layer (11.3 species) than in the lower layer (36.9 species) in L.
kaempferi (Table 1). With the exception of the upper layer of
L. kaempferi, the estimated species richness fell within 95%
confidence intervals of the observed number of species.

Species richness of arboreal spiders was significantly
influenced by stand and layer, although their interaction effect
was also significant (Table 2). Post-hoc pair-wise comparisons
showed that species richness was significantly greater in the
lower than in the upper layer in L. kaempferi {t = 0.24, P <
0.05), but not C. japonica {t = 0.24, P = 0.81). Unlike species
richness, monthly variation was the only (but highly significant)
factor influencing spider abundance (Table 2). The densities of
spiders in both canopy layers peaked from August to October in
both stands and then tended to decrease toward December
(Fig. la, b).

Among the functional groups, wandering spiders were a
significantly more abundant and species-rich group than orb-
web weavers and space-web builders in the canopies of both
the C. japonica and L. kaempferi stands (PERMANOVA,
pseudo-F = 12.8, P < 0.001 for abundance; pseudo-F = 12.6,
P < 0.001 for species richness: see Fig. 2a, b). Orb-web
weavers were the second most abundant group in the C.
japonica canopy, whereas space-web builders were much more
abundant than orb-web weavers in the L. kaempferi canopy. The
proportions of orb-web weavers were lower in abundance but
higher in species richness in the lower canopy of the L. kaempferi
stand (Fig. 2a, b). Statistical tests showed that the stand had a
significant influence on proportional abundances of space-web
builders and orb-web weavers, whereas the layer was only

Table 2. — Summary results of PERMANOVA, showing pseudo-F values and degrees of freedom {df) of stand, layer, month and their
interaction effects on spiders collected from the canopy. Spiders were analyzed using species composition (assemblage), total abundance, species
richness and three major functional groups, based on the abundance-based (upper half of the table) and incidence-based (lower) data. Functional
groups were analyzed using proportional abundance (upper) or species richness (lower) per site.

Assemblage Prop, abund. Prop, abund. Prop, abund.

df

( abundance-based)

Abundance

SW

OW

WS

Stand

1

7.33

**

0.87

n.s.

13.01

15.73

327

n.s.

Layer

1

8.09

**

6.25

n.s.

1.82

n.s.

35.89

0.58

n.s.

Month

5

8

35.96

32.08

IM

17.22

Stand X Layer

1

5.86

1.71

n.s.

1.24

n.s.

9.28

*

0.08

n.s.

Stand X Month

5

5.68

0.25

n.s.

16.43

14.09

11.91

Layer X Month

5

1.06

n.s.

0.77

n.s.

2.1

n.s.

1.22

n.s.

1.73

n.s.

Residual

5

Assemblage

Prop, species

Prop, species

Prop, species

df

(incident-based)

Species richness

SW

OW

WS

Stand

1

7.86

99.46

0.79

n.s.

2.76

n.s.

0.06

n.s.

Layer

1

6.3

14.43

0.002

n.s.

18.1

*

19.16

Month

5

6.08

**

8.39

*

22.18

1.74

n.s.

15.67

Stand X Layer

1

9.35

*

7.35

*

1.93

n.s.

7.38

17.54

Stand X Month

5

5.45

0.23

n.s.

14.42

**

2.22

n.s.

14.76

Layer X Month

5

1.51

n.s.

0,63

n.s.

2.43

n.s.

0.92

n.s.

0.87

n.s.

Residual 5

*■. P < 0.05.

**: P < 0.01.

***: P < 0.001.
n.s.: P > 0.05.

SW: Space-web builders, OW: Orb-web weavers, WS: Wandering spiders.

OGURI ET AL.— VERTICAL STRATIFICATION OF SPIDERS

37

(a) Upper canopy

C. japonic a

(b) Lower canopy

(c) Forest floor

C. japonic a

L. kaempferi

Figure 1 . — Seasonal changes in the densities of spiders in the a) upper and b) lower layers of the canopy and c) on the forest floors in the
Cryptomeria japonka and Larix kaempferi stands. Values represent mean ± standard error.

significant with respect to orb-web weavers (Table 2). However,
as suggested by the significant interaction effect of stand
and month, proportional abundances of space-web builders
were higher in L. kaempferi than in C. japonka, but marked
differences were observed in winter only (viz. November and
December: Table 2, Fig. 2a). Likewise, proportional abundanc-
es of orb-web weavers were generally greater in C. japonka, but
the differences were much greater in lower layers in early
summer (July). Monthly variation was significant in abundances
of all three functional groups; however, the differences were
more pronounced within the L. kaempferi canopy than within C.
japonka (Table 2, Fig. 2a). A significantly greater proportional
species richness of wandering spiders was observed within the
upper than the lower canopy layers in L. kaempferi, but similar
trends were not observed in C. japonka, presumably due to the
interaction effect between stand and layer (Table 2, Fig. 2b).
Likewise, a greater proportional species richness of orb-web
spiders was observed in the lower than in the upper canopy
layers of L. kaempferi, but not in C. japonka (Table 2, Fig. 2b).

Significant monthly variations were suggested for wandering
spiders; however, due to the presence of interaction effects, such
a variation was observed only in L. kaempferi, where the species
richness declined to zero in winter. As opposed to wandering
spiders, proportional species richness of space-web spiders
increased in winter in the L. kaempferi stand (Table 2, Fig. 2b).

The community compositions of arboreal spider species
according to both the abundance-based and incidence-based
measures differed significantly between stands and between
layers; however, there was also an interaction effect be-
tween these two factors (Table 2). NMDS ordinations and
post-hoc pair-wise comparisons showed that all four treat-
ments significantly separated species assemblages when using
abundance-based Bray-Curtis measures (Fig. 3). When we
used incidence-based Sorensen measures, however, spider
assemblages did not differ significantly between the upper
and lower canopies of the C. japonica.

Spider assemblages on the forest floor. — We sampled 39
individuals of 16 (with 95% confidence interval of ± 6.6)

38

THE JOURNAL OF ARACHNOLOGY

(a) No. individuals

CJ - Lower canopy

100ir-n „ r

Jill Aug Sep Oct Nov Dec

LK - Lower canopy

Jul Aug Sep Oct Nov Dec

100

80

60

40

20

(b) No. species

CJ “ Upper canopy

LK - Upper canopy

ilUIUI

100

80

60

40

20

CJ " Lower canopy

n

LK “ Lower canopy

:1m luM

Jul Aug Sep Oct Nov Dec

Jul Aug Sep Oct Nov Dec

Wandering spiders ^ Space-web builders □ Orb-web weavers

Figure 2. — Seasonal changes in the percentages {%) of a) individuals and b) species richness of functional groups in the upper and lower layers
of the canopy in the Cryptomeria japonica (CJ) and Larix kaempferi (LK) stands.

species (excluding juveniles and unidentified individuals) and
299 individuals of 27 ± 5.3 species on the forest floors of the
C. japonica and L. kaempferi stands, respectively (Table 1).
Only three spider species were found both in the canopy and on

the forest floor: Octonoba sybotides (Bosenberg & Strand 1906),
Tetragnatha yesoensis S. Saito 1934 and Pseudomicrargus
latitegulatus (Oi 1960) (Appendix I). The estimated number of
forest-floor species (19.5 species) was smaller than those in the

OGURI ET AL.— VERTICAL STRATIFICATION OF SPIDERS

39

(a) Abundance-based (Bray-Curtis) stress: o 1 1

9 UpperCJ
O LowerCJ
A UpperLK
LowerLK

Figure 3. — Nonmetric multidimensional scaling ordination plots
for spider assemblages in the upper and lower layers of the canopy in
the Cryptomeria japonica (CJ) and Larix kaempferi (LK) stands
according to a) abundance-based (Bray-Curtis index) and b)
incidence-based (Sorensen index) similarity measures. J: July, A:
August, S: September, O: October, N: November, D: December.

upper (49.0 species) and lower canopy layers (45. 1 species) of C
japonica, whereas the value for the forest floor in L. kaempferi
(31.5 species) was larger than that in the upper canopy layer
(1 1.3 species). The active density of ground-dwelling spiders on
the forest floor of the C. japonica stand was relatively constant
across the study period, whereas the density showed a peak in
July and tended to decrease toward December on the forest
floor of the L. kaempferi stand (Fig. Ic).

The abundance of space-web builders accounted for a
greater proportion than that of wandering spiders in the C.
japonica stand, whereas the opposite was found within the L.
kaempferi stand (Fig. 4). Few orb-web weavers and edaphic
spiders were collected throughout the study period.

DISCUSSION

Our results showed that arboreal spider assemblages assessed
by the abundance-based measure differed significantly between
the upper and lower layers of the C. japonica and L. kaempferi
stands, but those assessed by the incidence-based measure
differed significantly between layers of the L. kaempferi stand
only. This result may partly support the first hypothesis that
different spider assemblages would be established between
the upper and lower layers of C. japonica trees because of
differences in potential resources for spider habitats between the

layers (i.e., living foliage versus dead foliage). Two possible
factors may be responsible for the existence of different spider
assemblages within the canopy of C. japonica and L. kaempferi
stands. First, arboreal spiders might prey on phytophagous
arthropods in the upper canopy layer and on detritivorous
microarthropods in the lower layer of C. japonica trees. The
different composition of spider assemblages within the canopy
might not depend on the physical structure of the habitats
because the structural complexity was not so different between
the upper layer (mainly living foliage) and lower layer (dead
foliage) of the canopy in C. japonica. Shimazaki & Miyashita
(2005) suggested that on the forest floor in C. japonica stands,
smaller web-building spiders depend more on the prey derived
from the detrital food web than do larger spiders. Although we
did not perfonn a quantitative comparison, the evidence that
detrital microarthropods (e.g., Collembola) were abundant
specifically on the dead foliage of C. japonica (Yoshida & Hijii
2005) supports the dominance of smaller spiders in the lower
layer of the canopy.

Second, the difference in spider assemblages between the
canopy layers in the L. kaempferi stand was attributed to a large
number of less abundant species (mainly orb-web weavers) in
the lower layer (these species were largely absent in the upper
layer). The less abundant species might colonize from
understory vegetation that is next to the lower canopy layer.
Although we did not collect spiders from this layer, some
studies have shown that understory vegetation shared some
spider species with those found on the canopy (Sorensen 2003;
Larrivee & Buddie 2009; Aikens & Buddie 2012; Pinzon et al.
2013). Turnbull (1960) reported that in general spider species
were stratified across the vertical structure of forests, but that
they also frequently extended their distributions beyond each of
their preferred strata. Pinzon et al. (2013) showed a species
turnover along the vertical gradient (forest floor, understory
and lower canopy) of white spruce stands. Pinzon et al. (2013)
predicted that the community composition in the upper canopy
was also different from other strata, and Aikens & Buddie
(2012) and our result support their prediction.

Several studies have shown that the community composi-
tion of spiders differed between the canopy and forest floor,
but that some spider species shared strata in coniferous
(Pinzon et al. 2013), deciduous (Turnbull 1960), and montane
(Sorensen 2003) forests. Pinzon et al. (2011) showed that
spiders on the forest floor are more similar to those in the
canopy than to those on the understory vegetation, suggesting
that the two habitats could be linked by spiders moving along
tree trunks. Our findings, however, indicated that the canopy
habitats shared few spider species with the forest floor in the
C. japonica and L. kaempferi stands. The reason is unknown,
but may be attributed to the microenvironment of tree trunks
(e.g., bark structure) and understory vegetation (e.g., biomass
and/or architecture), which can serve as a ‘habitat filter’
between the canopy and forest floor.

The proportions of functional groups also differed between
the canopy and forest floor. Wandering spiders were dominant
in both layers in the L. kaempferi stand, whereas wandering
spiders and orb-web builders were dominant in the canopy
and space-web builders on the forest floor in the C. japonica
stand. Although we need to be cautious about differences in
sampling methods, this difference between the layers could be

40

THE JOURNAL OF ARACHNOLOGY

(a) No. individuals

CJ - Forest floor

LK - Forest floor

5b 20

o

o 100

CO

(D

bO

cd

<D

P 40

}— I

(D

Jul Aug Sep Oct Nov Dec

(b) No. species

CJ “ Forest floor

Jul Aug Sep Oct Nov Dec

LK “ Forest floor

iimi

Jul Aug Sep Oct Nov Dec

Jul Aug Sep Oct Nov Dec

I Wandering spiders ^ Space-web builders □ Orb-web weavers
□ Edaphic spiders

Figure 4. — Seasonal changes in the percentages (%) of a) individuals and b) species richness of functional groups on the forest floors in the
Cryptomeria japonica (CJ) and Larix kaempferi (FK) stands.

related to the differences in structural complexity of the
habitat substrates. Field manipulations of foliage density by
Hatley & MacMahon (1980) and Halaj et al. (2000) showed
that wandering spiders decreased with the removal of foliage,
but increased when branches were tied up, as opposed to web-
building spiders, which showed weaker responses to foliage
manipulations. In our study, the relative abundances of
wandering spiders in the L. kaempferi canopy substantially
decreased in November and were almost absent in December.
This would be due to the decrease in structural complexity of
foliage associated with the seasonal leaf fall of L. kaempferi in
late autumn (Miyaura & Hozumi 1988). Thus, both the
canopies of C. japonica and L. kaempferi trees would provide
dense foliage structures more favorable for wandering spiders
than for web builders. On the forest floor of the C. japonica
stand, branches with dead foliage made a structurally hetero-
geneous litter layer with much interstitial space. The structural
complexity of the accumulated litter layer allowed a greater
abundance of web-building spiders (Bultman & Uetz 1982), and
space-web builders are known to build webs in narrow spaces,
such as those formed between the needles of conifer trees
(Stratton et al. 1979). Accordingly, the space-web builders might
have dominated the forest floor of the C. japonica stand.

In conclusion, our analyses in the C. japonica and L.
kaempjeri stands suggest that distinctive spider assemblages

were established between vertical strata, reflecting the differ-
ences in factors, such as resource quality (i.e., living- or dead
foliage, accumulated litter) and association with adjacent
layers, along the vertical gradient of the forests. Basset et al.
(2003) noted that arthropod stratification in forests could be
determined by four types of factors: abiotic factors, forest
physiognomy and tree architecture, resource availability and
arthropod behavior. Further quantitative approaches related
to these factors are required for a comprehensive under-
standing of the vertical stratification and horizontal spacing
of spider assemblages in forest ecosystems.

ACKNOWLEDGMENTS

We thank Mr. Y. Imaizumi, Mr. N. Yamaguchi, Mr. N.
Takabe and all the members of the Laboratory of Forest
Protection, Nagoya University, for their assistance with
fieldwork. This study was funded by the Japan Society for the
Promotion of Science KAKENHI program (grant
numbers 17380091, 21380093 and 24780144).

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Manuscript received 10 May 2013, revised 16 December 2013.

42

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Numbers of individuals of spiders collected from upper (UC) and lower layers (LC) of the canopy and forest floor (FF) in the
CryptODien'a japonica (CJ) and Larix kaeinpferi (LK) stands. Juveniles and unidentified spiders (denoted by asterisks) were not included in the
analyses.

Functional group

CJ

LK

family

species

UC

LC

FF

UC

LC

FF

Edaphic spiders

0

0

1

0

0

0

Antrodiaetidae

AiUrocliaetiis roretzi (L. Koch 1878)

-

-

1

-

-

-

Space-web builders

131

89

30

279

336

99

Agelenidae

AIIoclubio)toides sp.

-

-

-

-

-

1

Coeloles personatiis Nishikawa 1973

-

-

4

-

-

2

Coelotes decolor Nishikawa 1973

-

-

1

-

-

2

Coeloles gifiiensis Nishikawa 2009

-

-

-

-

-

3

Coelotes spp.

-

-

-

-

-

25

Orumcekki satoi (Nishikawa 2003), n. comb.

-

-

3

-

-

12

Tegecoelotes corasides (Bosenberg & Strand 1906)

-

-

1

-

-

-

Agelenidae juvenile *

2

8

8

-

-

-

Cybaeidae

Cybtieiis nipponicus (Uyemura 1938)

-

-

2

-

-

-

Cyhaeiis kirigammensis Komatsu 1963

-

-

4

-

-

-

Cvhaeus tottoriensis lhara 1994

-

-

1

-

-

-

Cyhaeiis sp.l

-

-

8

-

-

-

Cyhaeiis spp.

-

-

-

-

-

3

Dictynidae

Latliys iiiacidosa (Karsch 1879)

62

38

-

-

-

-

Latlivs sexocidata Seo & Sohn 1984

1

15

-

-

-

-

Hahniidae

Halinia corlicicola Bosenberg & Strand 1906

-

-

-

-

-

1

Aprifroiitalia mascidii (Karsch 1879)

-

-

-

-

-

1

Ceratinopsis setoensis (Oi 1960)

-

-

-

-

-

4

Linyphiidae

Floroiiia exonuila (L. Koch 1878)

-

-

-

-

9

-

Gonaliiiiii japoiiiciini Simon 1984

-

-

-

-

-

1

Neolinypliia fusca Oi 1960

-

-

-

-

4

-

Neriene hrongersniai (van Helsdingen 1969)

-

-

5

-

-

-

Neriene spp.

-

-

-

-

-

3

Nippoiioiieta ohliqua (Oi 1960)

-

-

1

-

-

35

Ponhoninia spp.

-

-

-

-

-

2

ProUnyphia linihatiiiella (Bosenberg & Strand 1906)

4

11

-

-

-

-

Pseudoiiiicnirgus lalitegidatus (Oi 1960)

-

1

-

-

-

4

Straiidella yagiiniiiiai H. Saito 1982

-

1

-

-

-

-

Tiiriiiypliia yiiiiolianieiisis (Bosenberg & Strand 1906)

8

6

-

-

11

-

Theridiidae

Aiielosinnis crassipes (Bosenberg et Strand 1906)

1

-

-

-

-

-

Cliikiiiiia alhipes (S. Saito 1935)

-

-

-

-

3

-

Chrysso foliata (L. Koch 1878)

-

-

-

-

3

-

Coleosoimi octoiiiacidatiini (Bosenberg & Strand 1906)

1

-

-

-

-

-

Eiioplogiiatiui ahriipta (Karsch 1879)

1

-

-

-

-

-

Eiioplogiiatlia caricis (Fickert 1876)

-

-

-

-

1

-

Episiiiiis aj finis Bosenberg et Strand 1906

2

3

-

2

4

-

Eiiryopis flavoniacidata (C. L. Koch 1836)

7

-

-

-

-

-

Parasteatoda japonica (Bosenberg & Strand 1906)

1

1

-

-

-

-

Phoroncidia altiventris Yoshida 1985

-

1

-

-

-

-

Takariis chikiinii (Yaginuma 1960)

5

1

-

-

-

-

Takavus takavensis (S. Saito 1939)

37

11

-

277

273

-

Yiino/ianiel/a lyrica (Waickenaer 1842)

1

-

-

-

28

-

Orb-web weavers

147

281

3

53

92

1

Araneidae

Alenatea fiiscocoloratiis (Bosenberg & Strand 1906)

1

1

-

-

1

-

Araneiis acusisetiis Zhu & Song 1994

21

23

-

2

13

-

Araneiis niacaciis Uyemura 1961

-

1

-

-

-

-

Araneiis rotimdicornis Yaginuma 1972

-

-

-

-

1

-

Araneiis Stella (Karsch 1879)

1

-

-

-

-

-

Araneiis iivennirai Yaginuma 1960

1

-

-

-

-

-

Araneiis viridiventris Yaginuma 1969

-

1

-

-

-

-

Araneiis spp.

6

12

-

-

-

-

Araniella displicata (Hentz 1847)

6

-

-

-

-

-

Araniella yaginiiniai Tanikawa 1995

-

-

-

-

6

-

Cyclosa ginmiga Yaginuma 1959

-

-

-

-

2

-

Eriopliora sachalinensis (S. Saito 1934)

3

4

1

“

9

“

OGURI ET AL.— VERTICAL STRATIFICATION OF SPIDERS

43

Appendix 1. — Continued.

Functional group

CJ

LK

family

species

UC

LC

FF

UC

LC

FF

Neoscona punctigera (Doleschall 1857)

1

-

-

-

-

-

Neoscona scylla (Karsch 1879)

1

-

-

-

2

-

Neoscona suhpullaia (Rosenberg & Strand 1906)

-

1

-

-

1

-

Parazygiella disper (Kulczynski 1885)

-

2

-

-

9

-

Yaginumia sia (Strand 1906)

3

2

-

-

-

-

Araneidae juvenile *

1

-

-

-

-

-

Tetragnathidae

Leucauge subblanda Rosenberg & Strand 1906

-

-

1

-

-

-

Leucauge sp.

-

-

-

-

1

-

Tetragnatha shinanoensis Okuma & Chikuni 1978

11

21

-

-

2

-

Tetragnatha yesoensis S. Saito 1934

79

99

-

51

45

1

Uloboridae

Octonoba sybotides (Rosenberg & Strand 1906)

13

114

1

-

-

-

Wandering spiders

554

519

5

330

460

199

Anyphaenidae

Anyphaena pugil Karsch 1879

51

46

-

8

3

-

Araneidae

Chorizopes nipponicus Yaginuma 1963

1

1

-

-

-

-

Clubionidae

Clubiona jucunda (Karsch 1879)

69

22

-

-

-

-

Clubiona kurosawai Ono 1986

2

5

-

-

5

-

Clubiona lena Rosenberg & Strand 1906

-

-

-

-

-

1

Clubiona spp.

46

30

1

11

29

1

Corinnidae

Otacilia komurai (Yaginuma 1952)

-

-

-

-

-

1

Gnaphosidae

Drassyllus sliaanxiensis Platnick & Song 1986

-

-

-

-

-

9

Drassyllus sasakawai Kamura 1987

-

-

-

-

-

2

Drassyllus spp.

-

-

-

-

-

3

Gnaphosa akagiensis Hayashi 1994

-

-

-

-

-

1

Lycosidae

Pardosa laura Karsch 1879

-

-

-

-

-

170

Pirata clercki (Rosenberg et Strand 1906)

-

-

-

-

-

8

Pirata yagimmai Tanaka 1974

-

-

4

-

-

-

Philodromidae

Philodromus subaureolus Rosenberg & Strand 1906

60

8

-

25

3

-

Salticidae

Evarcha albaria (L. Koch 1878)

-

-

-

-

3

.

Evarcba sp.

-

-

-

-

1

-

Plexippoides armulipedis (S. Saito 1939)

3

1

-

-

-

-

Plexippoides doenitzi (Karsch 1879)

-

-

-

13

21

-

Rhene atrata (Karsch 1881)

1

-

-

-

-

-

Sibianor kochiensis (Rohdanowicz & Proszynski 1987)

-

-

-

-

-

1

Sibianor spp.

-

-

-

-

-

2

Sitticus spp.

-

-

-

-

5

-

Stertinius kumadai Logunov, Ikeda & Ono 1997

6

34

-

-

-

-

Yaginumaella striatipes (Grube 1861)

15

20

-

-

-

-

Salticidae juvenile *

-

1

-

-

-

-

Theridiidae

Argyrodes cylindratus Thorell 1898

-

5

-

-

-

-

Ariamnes cylindrogaster Simon 1888

-

1

-

-

-

-

Keijia sterninotata (Rosenberg et Strand 1906)

81

212

-

-

-

-

Phycosoma amamiense (Yoshida 1985)

1

-

-

-

8

-

Phycosoma nmstelimim (Simon 1888)

45

11

-

-

-

-

Rhomphaea sagana (Donitz et Strand 1906)

-

1

-

-

-

-

Thomisidae

Diaea subdola O. Pickard-Cambridge 1885

128

13

-

74

71

-

Lysiteles coronatus (Grube 1861)

32

15

-

160

241

-

Synaema chikunii Ono 1983

12

94

-

39

61

-

Tmarus rimosus Paik 1973

1

-

-

-

-

-

Xysticus spp.

-

-

-

-

9

-

Unidentified *

13

9

3

4

2

Total

848

907

50

666

890

299

2014. The Journal of Arachnoiogy 42:44-51

Assessing spider diversity on the forest floor: expert knowledge beats systematic design

Elvira Sereda', Theo Blick^, Wolfgang H. O. Dorow^, Volkniar Welters' and Klaus Birkhofer'-^: 'Department of Animal
Ecology, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. E-mail:
Elvira.Sereda@allzooi.bio.uni-giessen.de; -Senckenberg Gesellschaft fur Naturforschung, Senckenberganlage 25, 60325
Frankfurt am Main, Germany; ^Department of Biology, Biodiversity and Conservation Science, Lund University,
Solvegatan 37, 223 62 Lund, Sweden

Abstract. The design of sampling schemes affects the results of biodiversity inventories. As an approach for quantifying
the implications of such effects, we compared data on spider communities sampled in a beech-dominated forest floor
habitat by 1) a regular grid of pitfall traps (systematic design) and 2) an expert-based distribution of traps (stratified
design). We tested whether the two designs would lead to similar conclusions about the diversity and composition of
ground-dwelling spider communities. Estimates of species richness, rarefied species richness and activity density calculated
per trap were significantly higher in the stratified than in the systematic design. The community composition based on the
presence or absence of sampled species or based on log-transformed activity densities differed significantly. Most of the
dissimilarity between the community estimates of the two designs was attributable to three species, with Pardosa saltans
Topfer-Hofmann 2000 being more common in traps of the stratified design and Tenuiphantes zimmermanni (Bertkau 1890)
and Walckenaeria cuspidata Blackwall 1833 being more frequently observed in traps of the systematic design. Our study
suggests that a stratified sampling design is better suited for inventory surveys of spider communities of forest-floor
habitats, as trap locations of this design reflect specific habitat needs. It is important to note that inventories are a major
field for the application of such designs and that greater care is needed for the application of inferential statistics. For
example, the non-randomness that is caused by expert selection of sampling sites may violate fundamental assumptions of
simple linear models.

Keywords; Araneae, biodiversity, inventory, expert-based sampling, regular sampling, sampling design

Biodiversity research provides crucial information for the
development of conservation strategies (Brooks et al. 2004).
Strict inventories that generate comprehensive taxonomic lists
for a discrete spatiotemporal unit are thus prerequisites for
protecting species richness (Longino & Colwell 1997). Moreover,
reliable estimates of species composition are needed to enable
researchers to monitor biodiversity changes successfully (Dorow
et al. 1992; Colwell & Coddington 1994; Buckley & Rough-
garden 2004). As a contribution to this issue, we compared
estimates of diversity and species composition of ground-
dwelling spiders in a forest-floor habitat of a beech forest with
two different sampling designs (systematic vs. stratified; e.g.,
Southwood & Henderson 2000).

Systematic designs that are based on a regular distribution of
sampling locations in a study area (Woodcock 2005) are a
common approach in diversity surveys (e.g., sampling transects
for flower-visiting insects: Rundlof et al. 2008). However, such a
design depends on a priori decisions on the distance between
sampling points in relation to the scale of environmental
heterogeneity and the mobility of the focal taxa. A regular
placement of sampling locations further assumes that environ-
mental gradients which affect the analysed taxa are constant
over the study area and do not vary over different spatial scales
(Quinn & Keough 2002). Systematic designs may therefore be
most appropriate for homogeneous habitats with weak or very
simple environmental gradients. Dorow et al. (2007) suggested
that stratified sampling of pre-defmed subpopulations provides
an appropriate alternative for biodiversity inventories, since it
may improve precision by taking account of specific habitat
types (see also Hayek & Buzas 1997). In stratified designs,
specific microhabitats can be selected based on expert
knowledge, and this approach may thus provide a more precise

estimate of diversity in heterogeneous study regions than
random sampling (Southwood & Henderson 2000). In general,
subjective selection of sampling locations biases analyses of
ecological data by preconceptions of the investigator (Hirzel &
Guisan 2002). However, subjectivity may be necessary and valid
for certain research questions (McCune & Grace 2002). A strict
inventory of species richness in heterogeneous habitats, for
example, may only be reliable if the sampling design is biased by
expert knowledge toward locations that support rare species
and habitat specialists. An important assumption for using data
from stratified designs is that information about the stratum is
included as a predictor in statistical models (Quinn and Keough
2002). Comparative studies on the trade-offs between system-
atic and stratified designs are generally rare (Hirzel & Guisan
2002) and not available for invertebrate communities in
temperate forests.

Our study focused on spiders, because this taxon forms a
diverse group in temperate forests, and species are sensitive to
environmental heterogeneity (Wunderlich & Blick 2006;
Ziesche & Roth 2008; Birkhofer et al. 2010). Data were
collected with pitfall traps in a 34.8 ha area for 16 months. The
spatial arrangement of traps either followed a systematic
design (regular grid) or a stratified design (expert-based
selection of 14 pre-defined habitat structures). We hypothesize
that the design based on expert knowledge would provide a
more complete estimate of spider diversity than the systematic
design based on a regular grid.

METHODS

Study site and sampling. — The study was conducted in the
strict forest reserve “Locheiche” located in the National Park
Kelierwald-Edersee in the northern part of Hesse, Germany

44

SEREDA ET AL.— EXPERT KNOWLEDGE BEATS SYSTEMATIC DESIGN

45

Figure 1. — Trap locations (points) in the 34.8 ha study area in the
Kellerwald strict forest reserve “Locheiche” with the systematic
(circles, one trap per point) and the stratified design (triangles, three
traps per point).

(480-555 m a.s.l.; 5r08'30.45"N, 08°59'21.82"E) as a part of
the long term studies in the forest reserves of Hesse (Dorow et
al. 2010). The forest has not been managed since 1988, and
beech trees (Fcigus sylvatica L.) of an age of 81-120 years grow
on the north and west exposed slopes of the study area.
Additional tree species are Quercus petraea (Mattuschka)
Liebl., Larix decidua Mill., Acer pseudoplatanus L. and Picea
abies (L.) H. Karst. The annual mean temperature is 7.6°C,
and the average annual precipitation is 765 mm (www.
naturwaelder.de). The soil type is a cambisol with a pH of
5.1 in the uppermost horizon (Harmonized World Soil
Database 2009).

In total, 77 funnel pitfall traps (diameter 10 cm, filled with
approximately 200 ml of 70% ethanol and 99.5% glycerin at a

ratio of 2:1) were placed on the forest lloor (for details see
Dorow et al. 1992). Thirty-five traps were arranged in a
regular grid with an inter-trap distance of 100 m (systematic
design, referred to as SYS below: Fig. 1), and 14 triplets of
traps (42 traps in total) were placed at pre-defmed locations
with a distance of 5 m between traps in a triplet (stratified
design, referred to as STR below: Fig. 1, Table 1). We account
for these differences in inter-trap distances within and between
designs in our analyses (see statistical analysis). Forest
inventory points at 100X100 m grid intersections were
established on the forest floor, and pitfall traps of the
systematic design were placed next to these standardized
locations. Locations of the traps in the stratified design were
defined based on an inspection of the study area and
structures outlined in Table 1. Traps were open for 16 months
(29 October 2008 to 23 March 2010) and were emptied every
4 weeks. In winter, traps were not emptied before spring due to
snow cover from 1 1 December 2008 to 25 March 2009 and
from 25 November 2009 to 23 March 2010. Spiders were
determined using standard keys (Roberts 1987, 1995; Nentwig
et al. 2013), and the nomenclature followed Platnick (2013).
Juveniles were only identified to the family level and were not
included in the analysis.

Statistical analysis. — Before analyses commenced diversity
metrics were corrected for differences in sampling effort
between designs (systematic: 35 traps vs. stratified: 42 traps)
by using the following approach. Traditional diversity metrics,
such as species richness (including species richness that was
rarefied to a minimum of 24 individuals observed in one trap),
activity density or the inverse Simpson index were calculated
as means per trap over the 16 month study period and are
presented as average values per trap. As the capture
probability of pitfall traps varies with both activity and
density of the species, the term activity density should be used
(Heydemann 1957). To make the results more intuitive we
used the inverse of the Simpson index instead of its original
formulation, as an increase in the inverse index reflects an
increase in diversity (Magurran 1988).

Table 1. — Description of trap locations in the stratified (1-14) and systematic design (201-235). Note that each location of the stratified design
was sampled with three pitfall traps.

Trap ID

Description

1

2

3

4

5

6

7

8

9

10
11
12

13

14

201-206, 208-224, 226,
227, 230-232, 235
225, 228, 229, 233, 234
207

Beech-spruce-larch forest with needle and leaf litter

Border of forest-driveway with Avenella Jlexuosa

Border of forest-driveway with grasses and Urtica dioicci

Woodrush beech forest, underlayer without herb layer

Woodrush beech forest, stony hilltop

Dense beech young stands with maple

Charcoal pile with Cardamine bulbifera

Edge of the forest with several shrub species

Charcoal pile with grass and young beech stands

Glade with grass and young beech stands

Young spruce plantation

Mixed beech-oak-larch forest

Border of forest-driveway, stony, poor herb layer

Border of forest-driveway, with young stands of beech and larch

Forest floor covered with beech litter, without herb layer

Forest floor covered with beech and needle litter, without herb layer

Forest floor with grass and young stands of beech

46

THE JOURNAL OF ARACHNOLOGY

Q.

CD

m

m

0)

c

o

'u-

m

®

o

CD

CL

CO

® 10

CO

0)

o

m

Q.

m

X5

<4—

fl3

cr

25

20

15

10

14

- C)

13

a,

CO

CO

CO

CD

C

12

11

9 -

7 -

Q.

CO

CO

c

CD

■o

>4

tj

<

Cu

CO

t_

X

■O

c.

c

0

CO

CL

1

CO

CD

>

c

Figure 2. — Median, 75 and 95% quartiles and outliers for a) species richness, b) activity density, c) rarefied species richness (n = 24) and d)
inverse Simpson index per pitfall trap for spider assemblages sampled in a systematic (SYS, gray) or stratified (STR, white) design.

Diversity and abundance metrics were compared between
designs by one-way permutational analysis of variance with
permutation of residuals under a reduced model and design
(systematic vs. stratified) as fixed factor (PERMANOVA:
Anderson 2001). We included X and Y coordinates of all trap
locations as co-variables in our models to account for the fact
that some traps within, but also between, designs were located
more closely to each other. All univariate tests were based on
Euclidean distances and 10,000 permutations. The univariate
PERMANOVA based on Euclidean distances is analogous to a
traditional one-way ANOVA, but P-values are obtained from
permutations (Anderson and Millar 2004). We thus avoid
the assumption of normality in our statistical models (e.g.,
Anderson et al. 2008) and show all results using box and
whisker plots as recommended by Dytham (2003) for such data.

To assess the differences in community composition between
sampling designs, we calculated resemblance matrices based on

Sorensen (presence or absence of species) or Bray-Curtis (log
x-hl -transformed activity densities) distances between traps
in both designs. We log-transformed activity density data to
weigh down the contribution of abundant species to differences
between the two designs and to emphasize the importance of
rare species (Clarke et al. 2006). We used principal coordinate
analysis (PCO) based on Bray-Curtis distances to visualize the
dissimilarity of communities between traps from both designs
(Clarke & Warwick 2001). To explore the individual contribu-
tion of species to dissimilarities between the two designs, we used
similarity percentage analysis (SIMPER; Clarke & Warwick
2001). We further tested for homogeneity of multivariate
dispersion by comparing the distances of communities per traps
to group centroids between both designs (PERMDISP routine).
All analyses were performed using PRIMER version 6.1.13
with the PERMANOVA + add-on version 1.0.3 (PRIMER-E,
Plymouth, UK: Anderson et al. 2008).

SEREDA ET AL.— EXPERT KNOWLEDGE BEATS SYSTEMATIC DESIGN

47

Figure 3. — Principal coordinates analysis based on Bray-Curtis
similarities of log-transformed activity density data from all traps.
The size of the bubbles corresponds to the number of individuals
sampled in each pitfall trap in the systematic (SYS, gray) or stratified
(STR, white) design for a) Pardosa saltans (bubble size range: 1-173
individuals), b) Tenuiphantes zimmermanni (1-62 individuals) and c)
Walckenaeria cuspidata (1-39 individuals). Letters stand for traps that
did not contain any individuals from the species in the stratified
(expert-based, E) or systematic (S) sampling. Note that bubbles
may overlap.

RESULTS

In total, 8012 adult spiders were sampled from 96 species in
14 families (see Appendix 1). Traps in the STR design
contained 90 species, of which 42 were exclusively found in
the STR design. Traps of the SYS design contained 54 species,
of which 6 were exclusively found in the SYS design. Species

richness (pseudo-Fij4 = 31.59, P < 0.001) and activity density
(pseudo-Fij4 = 13.99, P < 0.001) per trap were significantly
lower in the SYS than in the STR design (Figs. 2a, b). Rarefied
species richness was also significantly lower in the SYS design
(Fig. 2c; pseudo-Fi_74 = 17.18, R < 0.001). The inverse
Simpson index did not differ significantly between designs
(Fig. 2d; pseudo-Fi,74 = 1.10, F = 0.301).

Community composition based on the presence or absence
of species in traps (Sorensen similarity, pseudo-Fi,74 = 7.04, P
< 0.001) or based on log-transformed activity densities
(Fig. 3, Bray-Curtis similarity, pseudo-Fijs = 8.02, P <
0.001) differed significantly between the two designs. Al-
though both designs shared 47 out of 96 species, similarity
percentage analyses indicated that three common species
contributed most to the significant dissimilarity between
communities (Fig. 3). Pardosa saltans Topfer-Hofmann 2000
was more common in traps of the STR design (mean
abundance of 51 individuals across all traps) and almost
absent from the SYS design (only two individuals were
collected in one trap of the systematic design). In contrast,
Tenuiphantes zimmermanni (Bertkau 1890) and Walckenaeria
cuspidata Blackwall 1833 were more frequently observed in
traps of the SYS design. In general, the multivariate dispersion
of community composition was significantly smaller in the
SYS design, indicating that community composition varied
less between traps than in the STR design (PERMDISP; F[ 75
= 49.18, F < 0.001).

DISCUSSION

Our study suggests that the stratified design provides a more
representative estimate of diversity and a more comprehensive
summary of community composition in the study area than a
systematic design. Species richness was higher in the stratified
design, and the number of exclusive species only sampled with
this design was almost an order of magnitude higher than for
the systematic design. However, expert knowledge is needed to
select sample locations in stratified designs in order to sample
all relevant microhabitats. In contrast, systematic designs do
not require such knowledge, but decisions about the extent of
the sampling area, the number of sample points and the inter-
point distances also require a priori assumptions.

It has been previously suggested that systematic designs may
not adequately represent the composition of communities, since
environmental gradients that acted on the mammal species
studied were not covered (Read et al. 1988; Pearson & Ruggiero
2003). The effectiveness of stratified methods to sample rare
species in heterogeneous habitats was also highlighted for plant
communities in coastal wetlands (Croft & Chow-Fraser 2009).
In our study, the number of unique spider species was seven
times higher in the stratified design, even though the same
sampling technique was used and the survey lasted over the
same period (16 months). Differences between designs were
attributed to some common spider species; for example, F.
scdtans was predominantly collected by traps in the stratified
design. This pattern highlights preferences of F. scdtans for
particular forest habitats (e.g., Hendrickx et al. 2001 ) that were
only sampled in the stratified design. This observation also
demonstrates the danger of missing specific habitat types if trap
locations are arranged in a regular grid that is related to the
number, size and distribution of habitats in the study area.

48

THE JOURNAL OF ARACHNOLOGY

Tenuiphantes zimmermanni and W. cuspidata were more
frequently observed in traps of the systematic design, but both
species were also present at particular locations of the stratified
design. This pattern reflects the rather broad habitat prefer-
ences of these two sheet-web weavers.

Community composition of spiders in individual traps was
significantly more homogeneous in the systematic design than
in the stratified design, reflecting a more diverse range of
microhabitats sampled in the stratified design. The vast
majority of Central European beech forests consist of a
relatively uniform stand of dense beech trees without a shrub
and herb layer (Standovar & Kenderes 2003; Galhidy et al.
2006). These areas are interspersed by small patches of
different structure (e.g., wayside herbs, seepage springs,
glades, rocks). To cover such elements in a systematic design
requires an enormous effort and resources that may not be
available for biodiversity inventories. Although the study
presented here clearly illustrates that a stratified sam.pling
design is more efficient than a systematic design, we
acknowledge that the observed differences may be limited to
the study location. Thus additional studies are needed to
confirm our results for other habitats in general.

To conclude, our results suggest that forest surveys aiming
at strict inventories of ground-active arthropods should not be
based on systematic designs even in moderately heterogeneous
study areas. That approach is more expensive and provides a
less precise estimate of diversity and community composition.
We propose, instead, that stratified designs should be used for
strict inventories in European forests if expert knowledge is
available and that the use of systematic designs should be
reserved for spatial analyses (e.g., Birkhofer et al. 2011; Sereda
et al. 2012) or surveys in more homogeneous habitats (e.g.,
Diekotter et al. 2010). It is important to note that inventories
are a major field for the application of such designs and that
greater care is needed for the application of inferential
statistics. For example, the non-randomness that is caused
by expert selection of sampling sites may violate fundamental
assumptions of simple linear models.

ACKNOWLEDGMENTS

We thank two anonymous reviewers and the subject editor
for comments on a previous version of the manuscript. This
research was supported by a DAAD grant and a grant of the
National Park Kellerwald-Edersee to E.S. Research was
conducted in cooperation with and financially supported by
“Landesbetrieb Hessen-Forst”.

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Manuscript received 22 February 2013, revised August 6 2013,

Appendix 1. — Species and number of individuals (juveniles excluded) sampled by the systematic (SYS) and the stratified (STR) approach.

Family/Species

SYS

STR

Agelenidae

Coelotes terrestris (Wider 1834)

507

479

Histopona torpida (C. L. Koch 1837)

115

87

Inerinocoelotes inermis (L. Koch 1855)

129

213

Malthonica silvestris (L. Koch 1872)

21

18

Amaurobildae

Amaurobius fenestralis (Strom 1768)

26

29

Clubionidae

Clubiona comta C. L. Koch 1839

0

1

C reclusa O. P. -Cambridge 1863

0

1

C. Westring 1851

1

6

Dictynidae

Cicurina cicur (Fabricius 1793)

128

211

Dysderidae

Dysdera erythrina (Walckenaer 1802)

0

1

Harpactea hombergi (Scopoli 1763)

0

8

Gnaphosidae

Drassodex lesserti (Schenkel 1936)

0

2

Haplodrassus signifer (C. L. Koch 1839)

0

1

H. silvestris (Blackwall 1833)

1

16

H. umbratilis (L. Koch 1866)

0

1

Micaria pulicaria (Sundevall 1831)

0

2

Zelotes clivicola (L. Koch 1870)

0

17

Z. ereheus (IYiotqW 1871)

0

3

Z. subterraneus (C. L. Koch 1833)

0

128

Hahniidae

Hahnia helveola Simon 1875

3

7

H. pusilla C. L. Koch 1841

0

17

Linyphiidae

Agyneta conigera (O. P. -Cambridge 1863)

0

1

Asthenargus paganus (Simon 1884)

0

2

Bathyphantes gracilis (Blackwall 1841)

0

1

B. nigrinus (Westring 1851)

0

1

Bolyphantes alticeps (Sundevall 1833)

0

5

Centromerus brevivulvatus Dahl 1912

1

0

C. cavernarum (L. Koch 1872)

2

66

C. dilutus (O. P. -Cambridge 1875)

54

167

C. pabulator (O. P.-Cambridge 1875)

1

48

C. sylvaticus (Blackwall 1841)

21

134

50

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Continued.

Family/Species

SYS

STR

CeratineUa brevis (Wider 1834)

1

35

Linyphiidae

Dicvmbium (Blackwall 1836)

28

35

Diplocephalus crista tus (Blackwall 1833)

0

1

D. latifrons (O. P.-Cambridge 1863)

1

13

D. picinus (Blackwall 1841)

51

122

Diplostyla concolor (Wider 1834)

1

65

Drapetisca socialis (Sundevall 1833)

3

0

Eiitelecara erythropiis (Westring 1851)

0

1

Erigone atra Blackwall 1833

0

1

Formiphantes lephthyphantiformis (Strand 1907)

0

1

Goncitium rubellimi (Blackwall 1841)

26

40

Helopbora iitsigiiis (Blackwall 1841)

0

42

Jacksoiiella falconeri (Jackson 1908)

8

0

Lepthyphantes niimitiis (Blackwall 1833)

0

1

L. twdifer Simon 1884

0

1

Linyphia hortensis Sundevall 1830

0

3

Macrargus rufiis (Wider 1834)

36

34

Maso simdevalli (Westring 1851)

0

2

Micrargus herbigradus (Blackwall 1854)

26

130

Microneta viaria (Blackwall 1841)

36

26

Monocephalus fuscipes (Blackwall 1836)

0

1

Neriene clathratci (Sundevall 1830)

1

1

N. emphana (Walckenaer 1841)

1

0

Nusoncits nasutiis (Schenkel 1925)

1

1

Obscuriphantes obscuriis (Blackwall 1841)

0

1

Pallidiiphantes pallidus (O. P.-Cambridge 1871)

1

3

Pocadicnemis pumila (Blackwall 1841)

0

1

Porrhomma campbeUi F. 0. P.-Cambridge 1894

4

1

P. pallidum Jackson 1913

7

13

Pseiidocarorita thaleri (Saaristo 1971)

4

3

Saloca diceros (O. P.-Cambridge 1871)

26

64

Tapinocyba insec fa (L. Koch 1869)

327

256

T. pallens (O. P.-Cambridge 1872)

64

161

T. praecox (O. P.-Cambridge 1873)

0

1

Temiiphantes alacris (Blackwall 1853)

4

2

T. cristaliis (Menge 1866)

1

15

T. flavipes (Blackwall 1854)

10

73

T. mengei (Kulczynski 1887)

4

12

T. tenebricola (Wider 1834)

1

34

T. tenuis (Blackwall 1852)

0

7

T. zimmernumni (Bertkau 1890)

443

487

Tliyreosthenius parasiticus (Westring 1851)

1

0

Wcdckenaeria acuminata Blackwall 1833

0

11

W. corniculans (O. P.-Cambridge 1875)

24

45

Linyphiidae

IV. cucullata (C. L. Koch 1836)

47

111

W. cuspidata Blackwall 1833

566

407

W. dysderoides (Wider 1834)

8

7

IV. mitrata (Menge 1868)

0

1

W. ohtusa Blackwall 1836

4

14

Liocranidae

Agroeca brunnea (Blackwall 1833)

0

4

Apostenus fuscus Westring 1851

0

2

Lycosidae

Alopecosa pulverulentu (Clerck 1757)

0

12

Pardosa amentata (Clerck 1757)

0

1

P. pullata (Clerck 1757)

0

1

P. (Walckenaer 1802)

0

45

P. saltans Topfer-Hofmann 2000

2

1018

SEREDA ET AL.— EXPERT KNOWLEDGE BEATS SYSTEMATIC DESIGN

51

Appendix 1. — Continued.

Family/Species

SYS

STR

Trocbosa terricola Thorell 1856

2

75

Xerolycosa nemonilis {Westring 1861)

0

1

Salticidae

Euophrys frontalis (Walckenaer 1802)

0

3

Neon reticulatus (Blackwall 1853)

9

9

Segestriidae

Segestria senoculata (Linnaeus 1758)

0

3

Tetragnathidae

Metellina segmentatci (Clerck 1757)

1

0

Pachygnatha degeeri Sundevall 1830

3

1

Theridiidae

Robertas lividus (Blackwall 1836)

33

41

R. Jackson 1914

7

4

Total:

2833

5179

2014. The Journal of Arachnology 42:52-73

The conservation value of secondary forests in the southern Brazilian Mata Atlantica from a

spider perspective

Florian Raub', Hubert Hofer'-'^, Ludger Scheuermann^ and Roland BrandP: ’Staatliches Museum fiir Naturkunde

Karlsruhe, Erbpriiizenstr. 13, D-76133 Karlsruhe, Germany; “LenzstraBe 6, D-76137 Karlsruhe, Germany; ^Department
of Animal Ecology, Philipps-Universitat Marburg, Karl-von-Frisch-Str. 3, D-35032 Marburg, Germany

Abstract. In many tropical areas of the world, pristine forests have become rare. Nevertheless, due to shifts in the human
population the area covered by secondary forests is increasing. These forests may harbor a rich flora and fauna and are
considered to be main refuges for species of primary forests. However, this issue is far from clear. To assess the
conservation value of secondary forests in the Atlantic Forest of Brazil, we compared the diversity of spiders in differently
aged secondary forests with old-growth forests. Within a larger project treating several invertebrate taxa, we sampled
spiders using a standard protocol in 24 sites of three successional stages (5-8, 15-20, 30-50 years old) and old-growth
forests (>100 years untouched) in two nature reserves. We describe the diversity and structure of the assemblages using
morphospecies and genera and analyze richness at the genus level. Generic richness and diversity showed no differences
between successional stages; i.e., did not increase from the youngest to older forests, but guild diversity did increase. The
youngest stage showed the highest variability in generic composition, and the turnover of genera and species was strong
between the younger forests (5-20 years old) and forests older than 30 years. High alpha diversity, high turnover among
sites and the lack of differences in richness between stages support the value of secondary forests for species conservation in
the region studied.

Keywords: Araneae, diversity, guild structure, Atlantic Forests, Brazil

The Brazilian Atlantic forest (Mata Atlantica) is one of the
“hottest hotspots” of biodiversity (Laurance 2009), due to its
exceptional species richness and high number of endemic taxa
in the various forest types (Forzza et al. 2012). Flowever, the
coastal region of Brazil has experienced an exceptionally high
degree of forest conversion and deforestation (Myers et al.
2000; Ribeiro et al. 2009) for more than 500 years. In contrast
to the more strongly deforested areas of the Atlantic coast, in
the state of Parana in southern Brazil large remnants of
Atlantic forests still exist, forming a mosaic of patches of old-
growth forests (sensu Clark 1996; also see Wirth et al. 2009)
and secondary forests in various stages of succession. These
secondary forests originate mainly from abandoned buffalo
pastures. Recently the issue of the importance of these
secondary forests for the conservation of biodiversity initiated
a controversial discussion (see Bihn et al. 2008b).

Conservation strategies and management in the tropics are
often based on large, exotic and beautiful or rare, endangered
vertebrate species. However, the overwhelming part of biodi-
versity consists of invertebrates. Furthermore, invertebrates are
involved in numerous important ecosystem functions (e.g.,
nutrient cycling or pollination). The analyses of invertebrate
diversity for conservation are usually restricted to species
numbers or lists of species of selected taxa. Although the
number of species is not a quality measure per se, richness and
diversity measures, which include the relative abundance of
species, are valuable approximations to biodiversity and the
conservation value of a habitat (Gaston 1996; Gotelli & Colwell
2001; Brose et al. 2003; Magurran 2004). This is especially true
when autecological data are lacking; i.e., when knowledge of the
distribution, natural history traits and habitat preferences for
most of the species is sparse. However, to evaluate the richness
of an assemblage a reference is needed. Comparing species

‘‘Corresponding author. E-mail: hubert.hoefer@smnk.de

numbers of assemblages in secondary vegetation with the
original (primary) vegetation seems to be a meaningful
approach to estimate degradation, to recognize the loss of
functional diversity (Bihn et al. 2008b, 2010) and to classify
areas with regard to their conservation value (Dunn 2004),
although there is some evidence of functional redundancy
(Lawton et al. 1998; Loreau et al. 2001).

The Brazilian-German cooperative project SOLOBIOMA
(Hofer et al. 2007, 2011) studied the biogeochemistry and, in a
multi-taxon approach, the diversity of earthworms (Rombke et
al. 2009), enchytraeids (Schmelz et al. 2009, 201 1 ), ants (Bihn et
al. 2008a,b), beetles (Hopp et al. 2010, 2011; Ottermanns et al.
2011) and spiders in order to evaluate the conservation value of
secondary forests in the Mata Atlantica. The overall aim of this
project was to check the possibility of classifying secondary
forest stages by their soil fauna and comparing that with the
“traditional” classification by age and vegetation. In the
absence of true primary vegetation in this region, we had to
rely on “old-growth” forests as a reference.

Spiders are a species-rich taxon in the tropics. In Brazil the
taxonomy is comparatively well studied (Brescovit et al. 2011),
and meaningful faunistic inventories are available (Hofer 1990,
1997; Silva 1996; Silva & Coddington 1996; H5fer & Brescovit
2001; Rego et al. 2007; Venticinque et al. 2008; Bonaldo et al.
2009). However, in these studies there is a strong bias toward
the Amazonian region. During the most recent years and based
on taxonomic advances and faunistic knowledge, several studies
in the Mata Atlantica, focusing on spiders, have approached
ecological questions (effects of disturbance, fragmentation and
vegetation type; Benati et al. 2005; Candiani et al. 2005;
Oliveira-AIves et al. 2005; Podgaiski et al. 2007). However,
studies with well-replicated designs are still rare (Dias et al.
2005; Bonaldo et al. 2007; Lo-Man-Hung et al. 2008; Pinto-
Leite et al. 2008; Ricetti & Bonaldo 2008). To assess the
conservation value of secondary forests, we sampled spiders on

52

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

53

Atlantic

Ocean

Curitiba

Bala

Nature

Ca-A-2

O

Ca-M-2

O

Ca-H-2

O

Ca-M-3

, Graphic; S. Scharf, Karlsruhe

Figure 1. — Location of the study region in Parana state, Brazil and the sampling sites in the two Nature Reserves Rio Cachoeira (Ca) and
Itaqui (It); successional stages: H - herbaceous, A - arboreal, M - medium, F - old-growth forest.

the ground and in the lower vegetation in three different stages
of secondary and old-growth forests, appraising the changes in
richness and composition of genera across the successional
gradient.

METHODS

Study area. — This study was conducted in the coastal region
of Parana State in southeastern Brazil. Originally the region
was covered by dense ombrophilous lowland and submontane
forests (IBGE 1992), but these ecosystems suffered massive
exploitation and were largely converted to buffalo pastures
(IPARDES 1995). Today, the landscape is characterized by a
mosaic of open land, secondary forests and few relatively large
patches of old-growth forests. The regional climate is humid
subtropical (Koppen’s Cfa: Strahler & Strahler 2005), with
mean temperatures between 16.2 °C in July and 24.5 °C in
February (IPARDES 2001). Average precipitation ranges
between 2000 and 3000 mm year”' (Roderjan & Kunyoshi
1988). Rainfall is more or less evenly distributed throughout
the year, although with some seasonality (low rainfall from
April to August).

The areas studied are part of an ecological restoration
program (Feretti & Britez 2006). Sampled sites were located in
two private nature reserves (RPPN) “Reserva Natural do Rio
Cachoeira” and “Reserva Natural Serra do Itaqui” (Fig. 1).

Both are owned and managed by the Brazilian NGO “Society
for wildlife research and environmental education” (SPVS)
and are part of the Environmental Protection Area (EPA) of
Guaraquegaba and also the Mata Atlantica Biosphere Reserve.
Within their areas of 12,000 and 6,700 ha, respectively, ranging
from sea level to elevations of 700 m a.s.l., different successional
stages from pasture to forest were categorized a priori by the
SPVS using age and vegetational structure, based on ortho-
photos from 1952, 1980 and 2002 and knowledge of the
residents on historical use.

Study design. — In both reserves (subsequently called locali-
ties), which are located approximately 25 km apart (Cachoeira;
25.3142°S, 48.6958°W; Itaqui: 25.2733°S, 48.4872°W), we
sampled spiders along a chronosequence of four forest stages:
5-8 years old (H - herbaceous stage), 10-15 years old (A -
arboreal stage), 35-50 years old (M - medium stage) and >
100 years old (F - old-growth); the latter was used as a reference
stage. In each stage we sampled three spatially separated
replicate sites of 30 X 50 m^ each. In total, 12 sites (3 replicates
X 4 stages) were studied in both localities (Fig. 1 ) during several
days of sampling (see below) in springtime (October/November)
of 2005 (Cachoeira) and 2007 (Itaqui). The springtime period
provides a high degree of sampling completeness without the
necessity of resampling throughout the year (Baldissera et al.
2003; Rodrigues 2005; Podgaiski et al. 2007).

54

THE JOURNAL OF ARACHNOLOGY

Table 1. — Absolute and relative abundance and richness of the spider families captured on lower vegetation (by beating and looking up). N =
number of individuals, G = number of genera, S = number of morphospecies.

Family

N

% N

G

% G

S

% S

Theridiidae

1010

37.4

29

19.6

96

30.2

Linyphiidae

374

13.8

9

6.1

23

7.2

Salticidae

370

13.7

29

19.6

, 52

16.4

Araneidae

234

8.7

21

14.2

53

16.7

Anyphaenidae

148

5.5

8

5.4

10

3.1

Thomisidae

119

4.4

7

4.7

10

3.1

Pholcidae

98

3.6

3

2.0

12

3.8

Uloboridae

69

2.6

3

2.0

5

1.6

Tetragiiathidae

60

2.2

5

3.4

9

2.8

Dictynidae

58

2.2

1

0.7

1

0.3

Mimetidae

33

1.2

3

2.0

4

1.3

Scytodidae

32

1.2

1

0.7

2

0.6

Oonopidae

24

0.9

5

3.4

5

1.6

Theridiosomatidae

24

0.9

5

3.4

12

3.8

Corinnidae

10

0.4

4

2.7

7

2.2

Oxyopidae

9

0.3

3

2.0

4

1.3

Hahniidae

8

0.3

1

0.7

1

0.3

Zoridae

5

0.2

1

0.7

2

0.6

Miturgidae

4

0.2

2

1.4

2

0.6

Lycosidae

3

0.1

1

0.7

1

0.3

Deinopidae

2

0.1

1

0.7

1

0.3

Hersiliidae

2

0.1

1

0.7

1

0.3

Sparassidae

2

0.1

1

0.7

1

0.3

Amaurobiidae

1

0.0

1

0.7

1

0.3

Ctenidae

1

0.0

1

0.7

1

0.3

Philodromidae

1

0.0

1

0.7

1

0.3

Synotaxidae

Sum: 27

1

2702

0.0

1

148

0.7

1

318

0.3

Sampling methods and identification. — A structured sam-
pling, following a widely accepted standard protocol (Cod-
dington et al. 1991), was applied to sample spider diversity in
these forests:

a) Ground hand sampling (“looking down” of Coddington
et al. 1991): two experienced persons sampled for one
hour at night with headlights, exploring all structures
below knee level, resulting in one sample per person, two
samples per site.

b) Aerial hand sampling (“looking up” of Coddington et al.
1991): one person sampled for one hour at night,
exploring all structures from knee height upwards to
overhead arm’s reach; i.e., lower vegetation, resulting in 1
sample per site.

c) Beating: Three persons striking vegetation at any
reachable level (i.e., lower vegetation) with a stick,
collecting the spiders falling on a 50 X 50-cm tray held
below, for one hour. Twenty beating points made one
sample. Depending on the person sampling, a different
number of samples per site (3-9) resulted.'

d) Pitfall trapping: Ten traps per site were installed to
capture active ground spiders for one week, usually
resulting in 10 samples per site, with a few failures. Traps
were 330 ml PE cups with an opening diameter of 7.5 cm,
filled with 100 ml of 4% formaldehyde solution and
protected against rain by transparent plastic plates.

The spiders sampled were stored in 75% ethanol. All adult
spiders were determined to morphospecies or to morphogenera

if possible, using a conservative approach to delimit morpho-
species and morphogenera. All analyses are based on adult
spiders. Notwithstanding the progress in spider taxonomy in
the Neotropics, severe shortcomings in the analyses of the
diversity of tropical faunas remains a prime difficulty in
identifying specimens to the species level or to sort all adult
specimens to the level of morphospecies. This is due to the high
number of inadequately described species and the lack of
identification keys (Uehara-Prado et al. 2009). We therefore
used genera as a surrogate for the comparison of species
richness and diversity, which has been shown to be a successful
strategy even at local scales (Andersen & Hauge 1995; Balmford
et al. 1996; Baldissera et al. 2008; Bihn et al. 2008b).

Identifications were made by the first and third authors,
with help from Brazilian experts at Butantan Institute, Sao
Paulo (IBSP) and Museu de Ciencias Naturais da Fundagao
Zoobotanica, Porto Alegre (MCN). Morphospecies numbers
(in the appendix) were assigned according to IBSP and MCN
numeration to assure future comparability. Voucher material
is deposited at the entomological department of Universidade
Federal do Parana in Curitiba (UFPR), at IBSP and MCN.

Data analysis. — We pooled the complementary captures
from the different methods and strata for all analyses.
Richness and diversity of the spider assemblages per site
(alpha diversity) were described by the numbers of genera (G)
observed, the ratio of genera/individuals (G/N), the Shannon
index (H), the Shannon evenness measure (E) and log series a
(Magurran 2004). We used rarefaction (Hurlbert 1971; Cole-
man 1982; Gotelli & Entsminger 2004; Magurran 2004) for the

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

55

Table 2. — Absolute and relative abundance and richness of the spider families captured on the ground (by pitfall traps and looking down).
N = number of individuals, G = number of genera, S = number of morphospecies.

Family

N

% N

G

% G

S

% S

Zoridae

855

47.7

1

0.9

8

3.6

Theridiidae

210

11.7

25

21.6

48

21.6

Linyphiidae

126

7.0

12

10.3

29

13.1

Ctenidae

121

6.8

2

1.7

6

2.7

Pholcidae

69

3.9

5

4.3

13

5.9

Lycosidae

62

3.5

5

4.3

9

4.1

Pisauridae

54

3.0

1

0.9

2

0.9

Araneidae

49

2.7

10

8.6

22

9.9

Mysmenidae

40

2.2

3

2.6

4

1.8

Hahniidae

26

1.5

1

0.9

7

3.2

Salticidae

25

1.4

8

6.9

14

6.3

Corinnidae

18

1.0

3

2.6

6

2.7

Amaurobiidae

17

1.0

1

0.9

3

1.4

Oonopidae

16

0.9

4

3.5

5

2.3

Ochyroceratidae

14

0.8

1

0.9

3

1.4

Theridiosomatidae

12

0.7

2

1.7

4

1.8

Tetragnathidae

11

0.6

5

4.3

7

3.2

Thomisidae

11

0.6

3

2.6

6

2.7

Anyphaenidae

10

0.6

3

2.6

3

1.4

Scytodidae

10

0.6

1

0.9

2

0.9

Nemesiidae

8

0.5

2

1.7

2

0.9

Titanoecidae

7

0.4

1

0.9

1

0.5

Mimetidae

4

0.2

1

0.9

2

0.9

Gnaphosidae

2

0.1

1

0.9

1

0.5

Palpimanidae

2

0.1

2

1.7

2

0.9

Prodidomidae

2

0.1

1

0.9

1

0.5

Anapidae

1

0.1

1

0.9

1

0.5

Caponiidae

1

0.1

1

0.9

1

0.5

Deinopidae

1

0.1

1

0.9

1

0.5

Dipluridae

1

0.1

1

0.9

1

0.5

Liocranidae

1

0.1

1

0.9

1

0.5

Miturgidae

1

0.1

1

0.9

1

0.5

Nesticidae

1

0.1

1

0.9

1

0.5

Symphytognathidae

1

0.1

1

0.9

1

0.5

Synotaxidae

1

0.1

1

0.9

1

0.5

Trechaleidae

1

0.1

1

0.9

1

0.5

Uloboridae

1

0.1

1

0.9

1

0.5

Zodariidae

1

0.1

1

0.9

1

0.5

Sum: 38

1793

116

222

direct comparison of generic richness between the single sites.
It was calculated with R version 2.10.2 (R Development Core
Team 2009), using the rarefy function of the package VEGAN
1.17-2 (Oksanen et al. 2009). To evaluate the proportion of
rare genera at the single site, we calculated the relative
abundance of singletons (proportion of genera with one
individual from the total genera number per site: Magurran
2004). We calculated the nonparametric sample-based estima-
tors Chao 2 and ICE (Magurran 2004) with Estimates 8.0
(Colwell 2005). A coverage measure was calculated for each
site using the number of observed genera as a percent of the
estimated richness.

Similarity across stages (beta diversity) was analyzed with
qualitative presence/absence (Sorensen index) and quantitative
(abundance) data for assemblage structure (NESS = Normal-
ized Expected Species Shared: Grassle & Smith 1976). In
contrast to the Sorensen Index, NESS is a quantitative
similarity measure, which accounts for the individual numbers
of shared species in the sites compared or assemblages (as in

the Renkonen or Bray-Curtis qualitative index), but weights
the rare species with ascending values for the sample size.
Therefore it seems to be a good measure for tropical
communities, where rare species account for a considerable
part of the recorded species (Chazdon et al. 1998; Novotny &
Basset 2000). We calculated NESS with the program BIODIV
97 for Excel.

To visualize differences in spider assemblages of the forest
stages and localities, we used a three-dimensional ordination
based on a non-metric multidimensional scaling (nMDS)
analysis, calculated on Bray-Curtis similarities of square-root
transformed abundances of genera using Winkyst 1.0 (100
random perturbations) and Canoco for Windows 4.53 (Ter
Braak 2002). The similarity matrix was tested for spatial
autocorrelation using the mantel function of the R package
ECODIST. The spatial distribution of the study sites had no
effect on the patterns of beta diversity {P = 0.98).

To complete the comparison of the forest stages, we used
available guild classifications for the Neotropical spider fauna

56

THE JOURNAL OF ARACHNOLOGY

Table 3. — Alpha diversity of spiders in the Cachoeira sites (genus based, samples from all methods pooled). Site codes; Ca Hl-3 = Cachoeira
sites of herbaceous stage, Ca Al-3 of arboreal stage, Ca Ml-3 of medium stage, Ca Fl-3 of old-growth. N = number of individuals, G =
number of genera, H = Shannon Index, E = evenness, oc = Fishers’s alpha index, Ra = rarefied genera number, Sg = portion of singletons,
Chao 2 = estimated generic richness, ICE = sample-based richness estimate. Coverage = number of observed genera as a percentage of Chao 2-
estimated richness, SD = standard deviation, CV = coefficient of variation.

Site

N

G

G/N

H

E

A

Ra (SD)

Sg

Chao 2 (SD)

ICE

Coverage

Ca HI

165

51

0.31

3.5

0.66

25.3

44.0 (2.1)

0.39

61.6 (6.2)

73.5

82.8

Ca H2

139

40

0.29

3.1

0.57

18.8

36.4 (1.6)

0.55

69.0 (16.1)

86.8

58

Ca H3

194

51

0.26

3.3

0.52

22.5

40.5 (2.4)

0.45

79.0 (14.8)

83.0

64.6

Ca Al

169

37

0.22

2.6

0.36

14.6

30.7 (2.0)

0.49

53.3 (10.2)

62.1

69.4

Ca A2

134

50

0.37

3.4

0.59

28.9

46.4 (1.6)

0.54

94.4 (22.0)

119.7

53

Ca A3

185

52

0.28

3.4

0.58

24.0

41.5 (2.4)

0.46

100.6 (25.4)

103.7

51.7

Ca Ml

137

49

0.36

3.2

0.51

27.3

44.5 (1.7)

0.61

86.9 (18.8)

115.0

56.4

Ca M2

155

46

0.30

3.4

0.56

22.1

39.8 (2.0)

0.52

94.1 (26.2)

93.1

48.9

Ca M3

169

44

0.26

3.0

0.45

19.3

36.4 (2.1)

0.50

70.3 (14.5)

78.1

62.6

Ca FI

216

54

0.25

3.3

0.50

23.1

40.6 (2.5)

0.46

95.7 (22.5)

88.4

56.4

Ca F2

212

49

0.23

3.2

0.51

20.0

36.7 (2.5)

0.47

83.7 (18.6)

89.5

58.5

Ca F3

241

57

0.24

3.3

0.48

23.6

39.9 (2.7)

0.46

79.6 (11.3)

92.8

71.6

Total

2116

157

220.4 (25.2)

200.0

71.3

Mean

176.3

49.3

0.28

3.2

0.52

22.5

39.8

0.49

80.7

90.5

61,2

CV

19%

12%

17%

8%

15%

17%

11%

12%

18%

18%

16%

(Hofer & Brescovit 2001; Dias et al. 2010). We assigned the
specimens to 16 distinct guilds. The assignment of a species to
a guild is usually based on the family, in some cases on the
genus, which was possible for almost all specimens in our
samples. In a few cases we had to apply personal knowledge of
the biology of a taxon based on our own observations in the
field, the sampling method and information in the literature
(Silva & Coddington 1996; Alvares et al. 2004). Only the
Amaurobiidae (18 individuals) were not assigned to a guild
due to the unclear taxonomic status and lack of ecological
information for Neotropical species. For the comparison of
guild structure in the different stages, data from the two
localities were pooled.

The rarefied genera numbers, the estimated richness and the
alpha diversity values were tested for significant effects of the
stage (four levels) and the locality (two levels) with two-way

ANOVAs using Statistica 8.0 (StatSoft 2007). Permutational
multivariate analysis of variance (Permanova, Version 1.6:
Anderson 2001, 2005) was used to analyze the generic
turnover in the spider assemblage of different forest stages
and to underpin the ordination with a statistical analysis. We
tested the main factors of the residuals and their interaction
terms with 9999 permutations using Bray-Curtis dissimilarities
between the study sites.

Indicator analysis was done with R, version 2.10.1 (R
Development Core Team 2009) and the packages MASS
(Venables & Ripley 2002) and labdsv (Roberts 2007). Because
indicators of single stages were weak, we pooled the beating tray
data of the two younger and the two older stages to one group
each [stages H and A = young (Y), stages M and F = old (O)] in
order to achieve a distinctive separation with indicator genera of
high indicator values for younger and older forests, respectively.

Table 4. — Alpha diversity of spiders in the Itaqui sites (genera based, samples from all methods pooled). Site codes: It Hl-3 = Itaqui sites of
herbaceous stage. It Al-3 of arboreal stage, It Ml-3 of medium stage. It Fl-3 of old growth. N = number of individuals, G = number of genera,
H = Shannon index, E = evenness, oe = Fishers’s alpha index, Ra = rarefied genera number, Sg = portion of singletons, Chao 2 = estimated
generic richness, ICE = sample-based richness estimate. Coverage = number of observed genera as a percentage of Chao 2-estimated richness,
SD = standard deviation, CV = coefficient of variation.

Site

N

G

G/N

H

E

a

Ra (SD)

Sg

Chao2 (SD)

ICE

Coverage

It HI

121

38

0.31

3.2

0.64

19.0

37.5 (0.7)

0.42

51.6 (8.3)

64.7

73.6

It H2

253

54

0.21

3.2

0.45

21.0

37.1 (2.7)

0.48

97.2 (22.2)

103.3

55.6

It H3

125

34

0.28

2.4

0.31

15.4

32.5 (1.1)

0.68

71.7 (21.4)

128.6

47.4

It Al

150

44

0.29

3.2

0.53

21.0

38.6 (1.9)

0.52

75.7 (17.3)

90.6

58.1

It A2

230

50

0.22

2.5

0.26

18.5

32.7 (2.6)

0.54

117.3 (37.4)

101.8

42.6

It A3

160

42

0.26

3.1

0.54

17.8

35.4 (1.9)

0.46

61.1 (12.0)

71.1

68.7

It Ml

205

43

0.21

3.0

0.49

16.6

33.4 (2.3)

0.44

70.7 (16.1)

79.7

60.8

It M2

277

54

0.20

3.2

0.46

20.0

37.3 (2.7)

0.37

78.2 (13.6)

77.3

69.1

It M3

281

57

0.20

3.3

0.46

21.6

38.7 (2.8)

0.37

73.5 (8.9)

85.2

77.6

It FI

123

33

0.27

3.1

0.69

14.8

32.4 (0.7)

0.36

47.3 (9.9)

51.3

69.8

It F2

274

60

0.22

3.4

0.47

23.7

39.4 (2.9)

0.45

109.2 (24.7)

105.5

54.9

It F3

180

44

0.24

3.2

0.58

18.6

37.2 (2.0)

0.36

60.4 (9.7)

67.0

72.8

Total

2379

154

196.8 (17.3)

191.5

78.3

Mean

198.3

46.1

0.24

3.1

0.49

19.0

36.0

0.45

76.2

85.5

62.6

CV

32%

19%

16%

10%

25%

14%

7%

21%

29%

25%

18%

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

57

Table 5. — Sampling effort, generic richness (observed and estimated) and diversity per stage (means and standard deviations from three
replicates, all samples pooled). N = number of individuals, G = number of genera, a = Fishers’s alpha index, Ra = rarefied genera number, Sg
= portion of singletons (pooled data for the three replicates), Chao 2 = estimated generic richness, ICE = sample-based richness estimate.
Coverage = number of observed genera as a percentage of Chao 2-estimated richness. SD = standard deviation. Abbreviations for stage as in
Tables 3 and 4.

Stage

Samples

Total N

Mean N (SD)

Total G

Ra (SD)

Sg

a (SD)

Chao 2 (SD)

ICE

Coverage

Ca H

60

498

166 (28)

89

47.6 (3.4)

0.34

31.6 (2.3)

1 16.6 (12.5)

125.2

76.3

Ca A

69

488

163 (26)

85

43.7 (3.4)

0.34

29.8 (2.2)

111.3 (11.9)

126.2

76.3

Ca M

70

461

153 (16)

83

42.9 (3.3)

0.41

29.5 (2.3)

115.8 (14.3)

135.5

71.7

Ca F

72

669

223 (16)

84

40.9 (3.2)

0.36

25.4 (1.7)

119.3 (17.0)

113.6

70.4

It H

69

499

166 (75)

86

44.9 (3.4)

0.34

30.0 (2.2)

102.5 (8.0)

115.8

83.9

It A

65

540

180 (44)

82

39.3 (3.3)

0.43

26.9 (2.0)

116.5 (15.2)

132.2

70.4

It M

72

763

254 (43)

89

40.6 (3.3)

0.33

26.1 (1.7)

117.6 (13.9)

118.6

75.7

It F

62

577

192 (76)

81

41.4 (3.2)

0.40

25.7 (1.8)

123.5 (19.7)

127.4

65.6

Total

539

4495

187 (51)

192

49.8 (3.8)

0.23

40.7 (1.4)

248.8 (22.3)

229.3

77.2

RESULTS

the

vegetation.

Very few mygalomorphs

(i.e..

Nemesiidae,

A total of 11,293 individuals were collected from 539
samples, of which only the 4,495 (39.8%) adults were identified
and sorted to 43 families, 192 genera and 440 morphospecies
(Appendix 1). We were able to identify and name 155 species
according to the available literature. Although the two
localities were sampled in different years, similar numbers of
spiders were collected; 2,1 16 individuals of 33 families and 157
genera in Cachoeira (2005) and 2,379 individuals of 37 families
and 154 genera in Itaqui (2007). The ratios of females/males
(0.941, 0.948) and adults/juveniles (0.673, 0.669) were also
similar.

Overall, Theridiidae ranked first in abundance, accounting
for 27% of all adults, and also in species richness with 117
morphospecies in 34 genera. The theridiid genera Dipoemi ( 1 9
morphospecies), Theridion (16), Crvptachaea (13) and Thy-
moites (10) showed the highest species richness. Only the
araneid genus Mcmgora was represented by a comparably high
number of morphospecies (10). Zoridae ranked second with
19% of the individuals, but only eight morphospecies. The
spider assemblages in Cachoeira and Itaqui showed a similar
ranking (Spearman r = 0.36) of family abundance values, but
Theridiidae and Linyphiidae were nearly twice as abundant in
Itaqui as in Cachoeira. The Araneidae (58 morphospecies/2I
genera), Salticidae (55/29) and Linyphiidae (43/15) accounted
together for more than 35% of all species and 34% of all
genera collected.

As expected, sampling in different strata (groimd/vegeta-
tion) yielded strongly complementary sets of lineages. In the
vegetation 74% of all spiders captured were web-builders.
Theridiidae and Linyphiidae alone accounted for more than
50% (Table 1), with more than 100 species. The only abundant
hunting spiders in the vegetation were Salticidae (55 morpho-
species) and Anyphaenidae (10 species). There was no
dominant (10% criterion) species or genus in the vegetation,
and the 316 morphospecies (148 genera) collected showed that
this stratum houses a large part of the total diversity. In strong
contrast, half of all spiders captured on the ground belong to
one genus of small hunting zorids, and 70% of all were
hunting spiders (Table 2). All abundant hunting-spider
families (Zoridae, Ctenidae, Lycosidae, Pisauridae) were
represented by few genera and species and thus overall
richness (216 morphospecies, 116 genera) was lower than in

Dipluridae) were collected.

Alpha diversity. — The number of individuals ranged from
134 to 241, representing 37 to 57 genera, in Cachoeira and
from 121 to 277, representing 33 to 60 genera, in Itaqui.
Means of all generic richness values were very close, and the
coefficient of variation rarely exceeded 20% (Tables 3, 4). The
same applied to the diversity indices. Typical for nonrecurring
sampling of tropical habitats, nearly half of the morphospecies
or genera were represented by only one adult specimen per site
of a stage (singletons: Tables 3, 4, Appendix 1 ). Both estimators
produced very similar values (mean of 81 genera in Cachoeira,
76 in Itaqui), corresponding to a coverage of over 60%. The
richness of genera (total, mean rarefied, estimated) was very
similar across the stages of forest succession (see means in
Table 5).

After correcting for the sampling effort (number of samples,
individuals), no differences between the stages were found.
None of the statistical tests (two-way ANOVAs with stage and
locality as factors and rarefied and estimated generic richness
and the two diversity indices as dependent variables) showed a
significant effect of stage or locality. The spider assemblages in
younger stages were as rich in genera and as diverse as in the
old-growth forests. At the stage level the portion of singletons
was 33% or higher, the estimated number of genera, based on
the Chao 2 and ICE estimators, was mostly less than twice the
number of observed genera and, consequently, coverage was
higher than 66% (Table 5). At the morphospecies level the
portion of singletons in the stages was even higher.

Beta diversity. — Qualitative similarity (Sorensen index) of
the different stages at each locality ranged from 0.53 (youngest
stage with older) to around 0.7 (between older stages)
(Tables 6, 7), reflecting a turnover of genera (and species)

Table 6. — Qualitative (Sorensen index, upper right) and quantita-
tive (NESS index, lower left, ni = 228) similarities between the forest
stages in Cachoeira reserve, based on genera data, all methods
pooled. Abbreviations for stage as in Table 3.

Ca H

Ca A

Ca M

Ca F

Ca H

0.58

0.56

0.53

Ca A

0.76

0.70

0.67

Ca M

0.72

0.93

0.71

Ca F

0.70

0.92

0.92

58

THE JOURNAL OF ARACHNOLOGY

Table 7. — Qualitative (S0reiisen index, upper right) and quantita-
tive (NESS index, lower left, m = 248) similarities between the forest
stages in Itaqui reserve, based on genera data, all methods pooled.
Abbreviations for stage as in Table 4.

It H

It A

It M

It F

It H

0.59

0.54

0.53

It A

0.82

0.65

0.58

It M

0.72

0.85

0.66

It F

0.67

0.76

0.91

along the successional gradient. Furthermore, within-stage
similarities were not higher, ranging from 0.4 (stage H) to 0.7
(stage F) in Cachoeira and from 0.3 (H) to 0.6 (M) in Itaqui.
Similarities of the same stages from the two reserves were
usually higher (Tables 8, 9) than of the different stages within
the same locality (Tables 6, 7). Similarities between stage H
and other stages were always lowest; the spider assemblage of
the herbaceous stage differed strongly from the older stages.

Quantitative similarity (NESS) is generally higher than
qualitative similarity (Tables 6-9), indicating that the domi-
nant genera (respectively, species) were abundant in all stages.
This is also obvious in the list of the ten most abundant genera
(respectively, species) of the two localities, representing 49%
and 50%, respectively, of all adults (Tables 10, 1 1). One zorid
genus clearly dominated in all stages, and the positions of
many abundant genera in the list are also very similar.
Abundant spider species reflecting the turnover between
younger (H, A) and older forests (M, F) are the linyphiids
of the genus Anodoration and several theriidid genera
(Spinthariis, Theridion, Thwaitesia) in Itaqui and the dictynid
Tballumetus and pholcids of the genus Mesabolivar in
Cachoeira. In Itaqui the latter was also found exclusively in
the two older stages, but was not among the ten most
abundant genera (see also indicator analysis).

Multivariate analysis, — The ordination (Stress = 0.12:
Fig. 2) shows the stages of both localities arranged along the
first axis. The younger stages (H, A) are especially well
separated from each other and from the older stages, with the
exception of one herbaceous site in Cachoeira. A much higher
variability of the youngest (H) stage is obvious. Sites of the two
older stages (M, F) ordinate close to each other. Sites at Itaqui
and Cachoeira separate along the second and third axes.
Although the nMDS is based on Bray-Curtis distances, which
are more biased to dominant species than the NESS measures,
the ordination visualizes the same differences between sites as
the NESS values (Tables 6, 7, 9). Several genera (mainly orb-
and sheet-weavers, some anyphaenids) characterized the
youngest herbaceous stage, whereas the older stages grouped
apart from the younger by pholcids (Mesabolivar spp.), the

Table 8. — Qualitative similarity (Sorensen) between the forest
stages in both reserves, based on genera data, all methods pooled.
Abbreviations for stage as in Tables 3 and 4.

It H

It A

It M

It F

Ca H

0.65

0.56

0.49

0.51

Ca A

0.58

0.62

0.65

0.66

Ca M

0.56

0.66

0.69

0.69

Ca F

0.50

0.64

0.68

0.68

Table 9. — Quantitative similarity (NESS, m = 228) between the
forest stages in both reserves, based on genera data, all methods
pooled. Abbreviations for stage as in Tables 3 and 4.

It H

It A

It M

It F

Ca H

0.82

0.75

0.65

0.65

Ca A

0.76

0.85

0.91

0.85

Ca M

0.68

0.80

0.89

0.86

Ca F

0.57

0.80

0.91

0.92

anyphaenid genus Patrera, the uloborid genus Miagrammopes
and the theridiid genus Spintharus. The nMDS ordination was
confirmed by a Permanova analysis. The four stages showed
highly significant differences concerning their composition of
spider assemblages {F = 2.34; P = 0.0007).

Functional diversity, — Weavers were more abundant than
hunting spiders in all stages (62/38%-56/44%), with the
exception of the young arboreal stage (49/51%). Most spiders
(40%) belonged to the diurnal space-web weavers, and these
were more abundant in the herbaceous stage than in the older
ones. Twenty-one percent were ground runners, most abun-
dant in the young arboreal stage and less in the herbaceous.
Spiders known to be diurnal dominated the collections with
44% of all individuals, while nocturnal spiders accounted for
20%. The portion of diurnal spiders decreased with the age of
the stages from 53% to 41%. In older forests distinctly more
orb weavers (e.g., near the ground), sedentary sheet-web
weavers and nocturnal ground ambushers (i.e., ctenids) were
caught than in the younger stages. Ground runners were most
abundant in the more open young arboreal stage (due to a
higher proportion of lycosids). The number of guilds in the
stages was nearly equal, but the diversity of guilds appeared to
increase from the young herbaceous to the old forest stages
(Table 12).

Indicator analysis. — Indicators of single stages were weak,
so the two younger (H -i- A) and the two older (M -h F) stages
were pooled to show a clear separation by genera (Table 13).
Spintharus and Miagrammopes showed high indicator values
for the older forest stages, whereas Anodoration and Titidius
were indicatory taxa for the younger forests. The same genera
fitted best to the nMDS ordination space, but species arrows
are not shown in Fig. 2 to maintain legibility.

DISCUSSION

Given the project’s approach, we put time and effort into
the use of replicates to allow for a statistical analysis of
biodiversity patterns of spiders in secondary forests, rather
than to attempt to inventory the entire spider assemblage.
We therefore did not undertake a special effort to sample
cryptic, specialized or rare species, but rather used an
accepted and widely used protocol to sample the spider
assemblage on the ground and lower vegetation. By doing so
we also made our samples per site comparable within our
study and to other studies in the Neotropics. Due to
difficulties in identifying the species and to avoid a biased
result by wrong morphospecification of the partly unde-
scribed tropical species, we based our richness measures and
estimates on genera. According to other studies, genera serve
as a reliable base for evaluating species richness (Baldissera
et al. 2008; Bihn et ai. 2010).

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

59

Table 10. — Assemblage structure (relative abundance of the ten most abundant genera) and total number of individuals (Ind.) in the four
forest stages in Cachoeira reserve, pooled from all methods in all sites Abbreviations for stage as in Table 3.

Family

Genus

Ca H %

Ca A %

Ca M %

Ca F %

Ca total %

Ca Ind.

Zoridae

gen. 1

15.0

16.1

22.3

29.7

20.3

429

Salticidae

Tariomi

5.1

6.7

3.8

4.1

5.1

108

Theridiidae

Dipoena

5.3

3.9

5.6

5.4

5.0

105

Linyphiidae

Sphecozone

6.4

3.3

0.4

7.6

4.3

90

Theridiidae

Theridion

3.1

2.4

0.8

6.7

3.1

66

Pholcidae

Mesabolivar

0.2

1.8

1.2

9.1

2.9

61

Araneidae

Mangora

2.0

0.9

2.8

6.5

2.8

60

Anyphaenidae

Pat r era

0.0

1.5

3.8

3.9

2.2

47

Dictynidae

Tlialhimetiis

0.0

0.0

4.0

5.4

2.1

45

Theridiidae

Spinthanis

0.4

1.5

1.6

5.4

2.1

45

The temporal distant sampling of the two localities had no
effect on any of the analyzed variables (total number of
individuals, genera, families; ratios of female/male and adult/
juvenile). The absence of autocorrelation in the dataset
indicates that neither the temporal distance of the two
sampling campaigns nor the spatial distance between the two
localities had significant effects on the sampled spider
assemblages.

Shortcomings in the methods, sampling protocol and
identification could have masked differences in richness
between the stages. Probably old-growth forests offer more
specific microhabitats (e.g., in bromeliads or dead wood) for
specialized, cryptically living or rare (less abundant, not widely
distributed, not active during the whole year) species. These
species cannot be assessed by either strongly vision-based
sampling or by beating the easily accessible lower vegetation
(Dias et al. 2000; Rinaldi et al. 2002). Thus, to assess and
evaluate the diversity of complex habitats such as an old-
growth forest in an unbiased way, more effort might be
necessary, using special sampling techniques for specialized
species. It is even questionable whether the old-growth sites
studied, although not strongly altered by humans, were
suitable as a reference in place of primary forests. They could
have obscured differences between stages or a directed
succession, being “old-growth successional states” in them-
selves. The lack of any native earthworm species in the
investigated sites and the high dominance of the invasive
species Pontoscolex corethrurus in all, even the oldest, forest
sites (Rombke et al. 2009) shed some light on the long history
of anthropogenic influence in the region.

Notwithstanding these possible constraints, our survey of
spiders revealed a high richness at the genus and species level
when compared to other studies in the realm of the Atlantic
Forests. Some of them, however, sampled in urban parks,
plantations or small forest fragments (Rinaldi & Ruiz 2002;
Benati et al. 2005; Candiani et al. 2005; Oliveira-Alves et al.
2005). Comparably high richness values were recorded by
Brescovit et al. (2004), Podgaiski et al. (2007) and Baldissera et
al. (2008) for Atlantic forests and Ricetti & Bonaldo (2008) for
Amazonian forests. The differences in both sampled and
estimated alpha-diversity values between sites (of all types)
and also between the two sampled reserves of our study were
low and not significant. Even the youngest successional stages
in the study area house a considerable diversity of spiders.
This is not unusual, because such habitats often show high
structural heterogeneity, prey availability and ecotone char-
acteristics, which increase species numbers (Kotze & Samways
1999; Baldissera et al. 2003; Platen 2006; Petillon & Garbutt
2008).

The high turnover of species between all sites, independent
of the stage, was interesting. Stages differ in their species
composition, not in richness. Variability within the a priori
defined stages originates from the heterogeneity of structural
and microclimatic conditions (openness, plant density), which
in all stages is based on physical and pedological heterogeneity
(exposition, inclination, soil type, groundwater level). The
higher variability within the youngest stage (visible in the
ordination) is probably caused by differences in historical
(largely unknown) land use (e.g., the use of machines,
fertilizers or pesticides), which mainly influences early

Table 11. — Assemblage structure (relative abundance of the ten most abundant genera) and total number of individuals (Ind.) in the four
forest stages in Itaqui reserve, pooled from all methods in all sites. Abbreviations for stage as in Table 4.

Family

Genus

It H %

It A %

It M %

It F %

It total %

It Ind.

Zoridae

gen. 1

1.4

30.9

18.3

15.1

18.1

431

Theridiidae

Dipoena

2.4

6.1

7.9

3.6

5.3

126

Linyphiidae

Sphecozone

5.0

2.8

6.0

5.4

4.9

117

Theridiidae

Spinthanis

0.2

1.9

9.7

5.2

4.8

115

Linyphiidae

Anodoration

16.2

2.8

0.0

0.0

4.0

96

Salticidae

Tariona

0.4

1.5

4.3

4.9

3.0

71

Theridiidae

Thwaitesia

0.4

1.3

1.6

7.1

2.6

62

Ctenidae

Isoctenus

0.8

2.4

2.9

3.8

2.6

61

Theridiidae

Episinus

6.4

1.3

2.2

0.9

2.6

61

Theridiidae

Theridion

9.2

0.2

0.1

1.9

2.5

59

60

THE JOURNAL OF ARACHNOLOGY

Figure 2. — Three-dimensional representation of a non-metric multidimensional scaling analysis (nMDS), based on Bray-Curtis distances;
generic data pooled from all methods and sites in Cachoeira and Itaqui and square-root transformed (stress = 0.12).

Table 12. — Guild structure of the spider assemblage in the four stages, data of both localities and all methods pooled. Taxa assigned to guilds
following Dias (2010) or Hofer and Brescovit (2001)’. H - herbaceous, A - arboreal, M - medium, F - old-growth forest.

Stages

Assigned families (genera)

Guild

H

A

M

F

Diurnal aerial ambushers

32

46

34

19

Thomisidae, Philodromidae

Diurnal aerial hunters

5

4

2

I

Miturgidae 2 (Radulphius), Oxyopidae

Diurnal ground runners

1

0

0

0

Liocranidae

Nocturnal aerial ambushers

2

2

0

1

Hersiliidae, Sparassidae,Trecha!eidae

Nocturnal aerial hunters

77

37

68

46

Anyphaenidae, Scytodidae, Corinnidae

Aerial runners

91

101

121

110

Salticidae, Minietidae

Nocturnal ground ambushers

13

29

39

49

Ctenidae, Nemesiidae

Nocturnal ground hunters

10

15

20

12

Salticidae 2 (Asaphobelis), Oonopidae, Palpimanidae,
Caponiidae, Zodariidae, Prodidomidae

Ground runners/Nocturnal ground hunters

34

13

0

20

Lycosidae 1, Gnaphosidae

Ground runners

111

276

251

224

Miturgidae 1 ( Teminius, Strotarchis), Zoridae

Diurnal ground orb weavers'

3

8

5

25

Mysmenidae, Symphytognathidae

Diurnal space-web weavers

487

374

460

459

Dictynidae, Linyphiidae, Synotaxidae, Theridiidae, Nesticidae

Nocturnal ground weavers'

12

2

20

12

Deinopidae, Dipluridae, Titanoecidae Anapidae, Hahniidae

Nocturnal space web weavers

1

2

4

7

Ochyroceratidae

Sedentary sheet weavers'

16

54

58

97

Pholcidae and Pisauridae 2 {Architis)

Orb weavers

103

65

138

154

Araneidae, Tetragnathidae, Theridiosomatidae, Uloboridae

Shannon Index H

1.75

1.83

1.87

1.94

Evenness E

0.64

0.68

0.69

0.72

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

61

Table 13. — Indicator analysis of the vegetation-bound spiders
(beating tray data): O = older stages (M & F); Y = younger stages
(H & A).

Cluster

Indicator value

Probability

Spinthariis

O

0.82

0.003

Miagrcumnopes

O

0.82

0.003

Patrera

o

0.76

0.002

Mangora

o

0.69

0.007

Thallumetus

o

0.67

0.003

Mesabolivar

o

0.63

0.041

Faiditus

o

0.50

0.013

Chrosiothes

0

0.42

0.043

Onoculus

0

0.42

0.043

Anodoration

Y

0.92

0.001

Titidius

Y

0.77

0.003

Hetschkia

Y

0.59

0.027

succession. During further succession, differences in biotic
(prey availability, structure) and abiotic (climate) habitat
parameters within and between the stages appear to decrease.
An experimental manipulation of food and structure in one
arboreal stage and the old-growth forest suggested food
limitation of the decomposer fauna, but also revealed no effect
of food or structure or any influence of stage on the spiders
(Raub et al. 2014). Spiders are mostly generalist predators and
seem to adapt easily to different food conditions and prey
types (Uetz 1992), as long as suitable habitat structures and
climate are provided. Baldissera et al. (2008) also found no
differences in family, generic and species composition of the
spider assemblages of natural Araucaria forest fragments and
Eucalyptus monocultures, when appropriate habitat structures
where provided.

The richness of our sites is comparable to other studies in
Atlantic forests (see above), but some studies showed a
different (family level) composition of assemblages (Rinaldi et
al. 2002; Rinaldi & Ruiz 2002) and also significant differences
in richness between young secondary and old-growth forest
sites (Pinto-Leite et al. 2008; Uehara-Prado et al. 2009). We
assume such differences to be caused by different uses of the
sampled areas; for example, the use of pesticides or heavy
machinery, and by the influence of the matrix of a forest
fragment (see above).

Studies from tropical forest regions in the Brazilian Amazon
revealed distinctly lower species richness of spiders in
anthropogenic altered landscapes with forest patches than in
a continuous forest cover (Lo-Man-Hung et al. 2011).
However, as shown by Rego et al. (2005), taxa-specific
responses can also lead to opposite responses in Neotropical
forest fragments. High spider richness in the younger
secondary sites should be regarded carefully in the context
of conservation issues and not be taken as an absolute
measure of habitat quality. Other invertebrate groups
investigated in the same area showed an increase in richness
along the successional gradient (Bihn et al. 2008b; Hopp et al.
2010).

The use of indicator taxa is becoming more and more
important in the context of the growing anthropogenic
pressure on highly diverse and threatened tropical ecosystems.
For the evaluation of the conservation potential and state of
secondary and old-growth tropical forests, precise but quick

and cheap tools such as indicators are needed (Uehara-Prado
et al. 2009). However, the use of indicator taxa in the
evaluation of ecosystems is a controversial topic, especially
because of the indirect effects in food webs (Abrams et al.
1996), together with the lack of knowledge of the interrelations
between the taxa. Therefore a multi-taxon approach with a
carefully selected set of organisms (Kotze & Samways 1999;
Cabra-Garcia et al. 2012) should be used. Nonetheless, the
results of our indicator analysis can be used for evaluations of
secondary forest areas in the southern Mata Atlantica region.
The identified genera can serve as indicator taxa for the
evaluation of priority areas for forest conservation. For future
evaluations they should be combined with the outcomes of
other arthropod studies (Bihn et al. 2008b; Hopp et al. 2011;
Ottermanns et al. 2011), and ecological traits should also be
included to establish a reliable multi-taxon approach for the
implementation of conservation strategies (Kotze & Samways
1999; Uehara-Prado et al. 2009).

Recovery of (species) richness can be relatively fast. Dunn
(2004) reported a time span of 20^0 years for ant and bird
richness recovery, which is comparable to the age of our
medium-aged secondary forests. However, the regeneration of
the original forest community often needs much more time
(Dunn 2004; Bihn et al. 2008b). Among spiders, some forest-
dwelling Lycosidae still do not seem to find adequate habitat
in the oldest secondary stage. We therefore assume that
mature secondary forests can host a highly diverse spider
community, but do not serve as surrogate habitats for all old/
primary forest dwelling genera or species. A classification of
forests by the diversity and structure of spider assemblages
would separate young (< 15 years) from median to old forests
(> 30 years), in good accordance with results on beetles (Hopp
et al. 2010), but not on ants (Bihn et al. 2008a).

Our study did not show a succession of spider diversity from
species-poor young secondary vegetation toward a species-rich
old-growth fauna, but rather a turnover of spider genera along
the successional gradient, strongest between the two young
and the two older stages; i.e., between ages of 20 to 30 years.
We interpret the high alpha diversity and turnover between
sites of the same stage as an expression of a rich regional
spider fauna, maintained by the mosaic landscape of forests of
different ages and mainly stochastic processes in the estab-
lishment of spider assemblages in early successional stages.
Our study region presents a highly diverse mosaic texture, with
large patches of old-growth forest acting as refuges for spiders
(Rodrigues et al. 2009), never far away even from the youngest
secondary stages. This variation in vegetation complexity, and
the large set of microhabitats provided, is able to host highly
diverse spider assemblages (Ricetti & Bonaldo 2008). We
assume that ideal preconditions for colonization and repop-
ulation of secondary habitats have been met in the region.
Spiders survived the deforestation and fragmentation of the
coastal forests in Parana due to the constant availability of
retreat habitats for later resettlement.

ACKNOWLEDGEMENTS

This study was conducted within the SOLOBIOMA project
and as part of the Mata Atlantica program based on a
German-Brazilian Government agreement. It was funded by
the German Federal Ministry of Education and Research

62

THE JOURNAL OF ARACHNOLOGY

(BMBF-sig.: 01LB0201) and the Brazilian National Council
for Scientific and Technological Development (CNPq). The
Society for Wildlife Research and Environmental Education
(SPVS) and the Federal University of Parana (UFPR) gave
permission to work in their sites and laboratories. We are
grateful to Rainer Fabry for his constant assistance in
organizing work and the staff of SPVS at the Cachoeira
reserve for valuable help during the fieldwork. Without the
help of our Brazilian colleagues (E.S.S. Alvarez, A.D.
Brescovit, A. A. Bonaldo, L Cizauskas, E.H. Buckup, A. A.
Lise, E.O. Machado, M.A.L. Marques, R. Ott, D. Polotow, C.
Rheims, E.N.L. Rodrigues, G.R.S. Ruiz) identifying spiders,
this work would not have been successful. We thank David
Russell for correction of our English style, and Stano Pekar
and two anonymous reviewers for very helpful comments on a
previous version of the manuscript.

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Manuscript received 9 July 2013, revised 9 January 2014.

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

65

Appendix 1 . — List of morphospecies of adult spiders recorded in the forest stages {H - herbaceous, A - arboreal, M - medium, F - o!d-growth
forest) of the two nature reserves Cachoeira (Ca) and Itaqui (It) (specimens from all methods and replicate sites pooled).

Locality and Stage

Taxon

Ca-H

Ca=A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Amaurobiidae

Amaurobiidae sp. 1

0

0

0

1

0

0

0

6

Amaurobiidae sp. 2

0

0

0

0

0

I

0

0

Amaurobiidae sp. 3

0

0

0

I

0

0

0

0

Amaurobiidae sp. 4

0

0

3

2

0

0

3

1

AnapMae

Anapidae sp.

0

0

0

0

0

0

1

0

Anyphaenidae

Amaurobioidinae sp. 1

1

0

0

0

0

0

0

0

Aysha sp. 1

11

2

0

1

7

1

I

1

Aysha sp. 2

1

0

0

0

0

0

0

0

Aysha sp. 4

0

0

0

1

0

0

0

0

Buckupiella imperatriz Brescovit 1997

4

1

0

0

4

0

0

0

gen. 1 sp. 1

16

0

0

0

0

0

0

0

Patrera cita (Keyserling 1891)

0

10

19

18

0

2

15

9

Temnida sp. 1

0

0

0

0

1

0

0

0

Wulfila sp. 1

0

0

0

0

0

1

0

0

Wulfilopsis sp. 1

2

1

1

3

8

2

10

4

Araneidae

Araneidae sp. 8

0

1

0

0

0

0

0

0

Acacesia tenella (L. Koch 1871)

1

0

0

0

7

0

0

0

Acacesia yacuiensis Glueck 1994

0

0

0

0

0

2

1

0

Alpaida biasii Levi 1988

0

0

0

0

3

0

0

0

Alpaida canoa Levi 1988

0

0

2

0

0

0

1

0

Alpaida rubeUula (Keyserling 1892)

0

0

0

1

0

0

0

0

Alpaida septemmammata (O.P.-Cambridge 1889)

0

0

0

0

0

0

0

1

Alpaida sp. 3

0

0

0

1

0

0

0

0

Alpaida sp. 5

2

0

0

0

0

0

0

0

Alpaida tijuca Levi 1988

1

0

1

0

0

1

3

0

Alpaida truncata (Keyserling 1865)

0

0

1

0

0

0

0

0

Araneus iguacu Levi 1991

0

1

0

2

0

2

3

3

Araneus tijuca Levi 1991

0

0

2

1

2

1

I

0

Araneus unanimus (Keyserling 1879)

0

0

0

1

0

0

0

0

Araneus uniformis (Keyserling 1879)

0

0

0

0

1

0

0

0

Bertrana rufostriata Simon 1 893

1

0

0

0

7

0

0

0

Bertrana sp. 1

0

0

0

0

2

0

0

0

Cyclosa fililineata Kingston 1932

1

0

4

8

1

2

1

2

Cyclosa morretes Levi 1999

0

0

0

3

0

0

0

2

Enacrosoma anomalum (Taczanowski 1873)

0

0

0

0

0

1

0

0

Eustala sp. 1

1

0

0

1

0

0

0

0

Eustala sp. 2

1

0

0

0

1

0

0

0

Eustala sp. 3

0

0

0

0

3

0

0

0

Eustala sp. 4

0

0

0

0

2

0

0

0

Eustala sp. 6

1

0

0

0

1

0

0

0

Eustala sp. 8

0

0

0

0

3

0

0

0

Hypognatha sp. !

0

0

I

0

0

0

0

0

Kaira echinus (Simon 1897)

0

1

0

0

0

0

0

0

Kapogea sellata (Simon 1895)

0

0

1

0

1

0

0

0

Mangora blumenau Levi 2007

4

0

2

14

0

4

6

2

Mangora bocaina Levi 2007

0

1

0

0

1

4

1

0

Mangora botelho Levi 2007

0

0

1

0

0

0

0

0

Mangora caparu Levi 2007

1

0

1

3

0

0

3

1

Mangora chacobo Levi 2007

0

0

0

0

0

1

0

0

Mangora manicore Levi 2007

4

3

3

3

1

0

1

5

Mangora melanocephala (Taczanowski 1874)

1

0

0

0

2

0

0

0

Mangora missa Levi 2007

0

1

0

0

0

0

1

!

Mangora sp. 1

0

1

7

10

0

2

2

0

Mangora sp. 2

0

0

0

0

0

0

1

2

66

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Metazygia manu Levi 1995

0

0

0

0

1

0

1

0

Micrathena crassispina (C.L. Koch 1836)

0

0

0

0

0

0

0

1

Micrathena excavata (C.L. Koch 1836)

0

0

1

1

0

1

0

I

Micrathena sanctispiritus Brignoli 1983

0

0

0

1

0

0

0

0

Micrathena triangularis (C.L. Koch 1836)

1

2

0

0

0

0

1

0

Micrepeira albomaculata Schenkei 1953

0

0

0

1

0

0

0

0

Parawixia audax (Blackwall 1863)

3

0

0

0

2

0

1

2

Parawixia kochi (Taczanowski 1873)

0

0

0

0

I

0

0

0

Parawixia monticola (Keyserling 1892)

0

0

0

0

0

0

3

1

Scoloderus cordatus (Taczanowski 1879)

0

0

1

0

2

0

0

4

Scoloderus gibber (O.P.-Cambridge 1898)

1

1

0

0

0

0

0

0

Testudinaria gravatai Levi 2005

0

0

0

1

0

2

0

0

Verrucosa sp. 1

0

!

1

2

0

1

1

3

Wagneriana eupalaestra (Mello-Leitao 1943)

0

0

2

1

0

0

0

0

Wagneriana heteracantha (Meilo-Leitao 1943)

0

0

2

0

0

0

0

0

Wagneriana iguape Levi 1991

0

1

1

1

0

1

2

1

Wagneriana Janeiro Levi 1991

0

1

1

6

0

1

3

1

Wagneriana taim Levi 1991

3

0

0

0

2

0

0

1

Wixia sp. 1

0

1

0

0

0

0

0

0

Caponlidae

Caponiidae sp.

0

0

0

0

0

0

1

0

Corinnidae

Castianeira sp. 1

0

0

0

0

1

0

0

0

Castkmeira sp. 2

0

1

0

0

0

0

0

0

Corinna sp. 1

0

1

0

1

0

0

1

0

Corinna sp. 2

1

1

0

0

0

0

0

0

Corinna sp. 3

0

1

0

0

0

0

0

0

Corinna sp. 4

0

0

0

0

0

0

1

0

Corinna sp. 5

I

0

0

0

2

0

1

0

Corinna sp. 6

0

0

0

0

0

1

0

0

Corinna sp. 7

0

0

0

0

0

0

1

0

landuba varia (Keyserling 1891)

3

2

0

1

1

0

0

0

Myrmecium sp. 1

0

0

1

0

0

0

0

1

Trachelas sp. 1

0

1

0

1

0

0

1

0

Trachelas sp. 2

0

0

0

0

0

0

1

0

Ctenidae

Ctenus medius Keyserling 1 89 1

1

3

4

4

0

1

3

2

Ctenus ornatus (Keyserling 1877)

1

0

0

0

0

0

0

0

Ctenus sp. 1

0

0

0

i

0

0

0

0

Isoctenus janeirus (Walckenaer 1837)

0

0

0

1

0

0

4

0

Isoctenus ordinario Polotow & Brescovit 2009

0

0

0

1

0

2

4

1

Isoctenus strandi Mello-Leitao 1936

7

11

4

16

4

11

15

21

Deinopidae

Deinopis sp. 1

0

0

1

2

0

0

0

0

Dictynidae

Thallumetus sp. 1

0

0

20

25

0

0

6

7

Dipluridae

Trechona rufa Vellard 1924

0

0

0

0

0

0

1

0

Gnaphosidae

Gnaphosidae sp.

0

0

0

0

2

0

0

0

Hahniidae

Hahniidae sp. 1

0

0

5

10

0

0

0

0

Hahniidae sp. 2

0

0

0

0

0

0

1

0

Hahniidae sp. 3

0

0

1

0

0

0

0

0

Hahniidae sp. 4

0

0

2

0

0

0

0

0

Hahniidae sp. 5

I

0

1

0

0

0

0

0

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

67

Appendix 1. — Continued.

Locality and Stage

Taxon Ca-H Ca-A Ca-M Ca-F It-H It-A It-M It-F

Hahniidae sp. 6
Hahniidae sp. 7

Hersiliidae

Ypypuera crucifera (Vellard 1924)

Linyphiidae

Linyphiidae indet. 1

Linyphiidae indet. 2

Linyphiidae sp. 1

Linyphiidae sp. 2

Linyphiidae sp. 3

Anodoration claviferum Millidge 1991
Anodoration sp. 1

Asemostera tacuapi Rodrigues 2007
Dubiaranea sp.

Dubiaranea sp. 1
Dubiaranea sp. 2

Exechopsis conspicua Millidge 1991
Exechopsis sp.

Exechopsis sp. 1
Exechopsis sp. 2
Exocora sp.

Exocora sp. 1
Labicymbium sp.

Labicymbium sp. 1
Lepthyphantes sp.

Lepthyphantes sp. 2
Linyphiinae indet. 1
Linyphiinae indet. 2
Meioneta sp.

Meioneta sp. 1
Meioneta sp. 2
Meioneta sp. 3
Meioneta sp. B

Moyosi prativaga (Keyserling 1886)
Moyosi sp.

Moyosi sp. 1
Psilocymbium sp.

Psilocymbium sp. 1
Psilocymbium sp. 2
Scolecura sp.

Sphecozone diversicolor (Keyserling 1886)
Sphecozone labiata (Keyserling 1886)
Sphecozone personata (Simon 1894)
Sphecozone sp.

Sphecozone sp. 1

Sphecozone tumidosa (Keyserling 1886)
Sphecozone venialis (Keyserling 1886)
Vesicapalpus simplex Millidge 1991

Liocranidae
Gen. 1 sp. 1
Lycosidae
Agalenocosa sp. 1
Hogna sp. 1
Hogna sp. 2

Hogna sternalis (Bertkau 1880)

Lobizon sp. 1
Lobizon sp. 2

Lycosa erythrognatha Lucas 1836

0 0 0
0 0 0

0 0 0

0 4 0

0

0

0 2 0

0 0 0 0 0

0

0

0

2

0

22

2

0

0

2

0

0

1

0

1

0

0

3

0

0

1

0

2

0

0

0

0

0

0

0

1

5

6
1
1

17

0

10

1

3

0

0

0

0

0

3

0

0

5
0
0
0
0
0
0

6
1

0

0

0

0

3

0

0

0

0

0

2

0

0

3

1

1

0

0

0

0

0

0

3

13

0

5

1

0

0

0

0

0

0

2

0

0

1

1

0

0

0

2

0

0

0

2

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

0

2

0

0

0

0

0

0 1

0 3

0 0

0 0

0 0

0 0

0 81

0 3

0 0

0 0

0 0

0 0

0 0

0 0

0 0

2 0

0 0

0 0

0 0

0 0

0 0

1 0

0 0

0 0

0 1

0 0

0 3

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 19

0 0

1 0

0 0

0 6

0 0

34 0

0 0

0 0 0

0 0 0

21 1 0

1 0 0

5 7 2

0 0 0

15 0 0

0 0 1

0 0 0

0 0 0

1 0 0

0 0 2

16 5 0

0 1 2

0 0 0

3 0 0

0 0 1

0 0 0

1 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 1

1 8 8

0 8 0

0 0 0

0 0 0

0 0 0

0 0 0

1 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 5

5 7 7

0 0 0

0 0 0

2 0 0

8 39 19

0 1 0

10 0 0 0

0 0 0

7 0 0
0 2 0
4 2 0
0 5 0
0 0 0
0 4 0
0 0 0

0 0 0 0 0

0 2 0 0 0

0 0 0 0 0

0 3 0 0 0

9 0 0 0 3

3 11 000

0 1 0 0 0

68

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Lycosa inornata Blackwall 1862

3

0

0

0

1

0

0

0

Lycosinae sp. 1

Mimetidae

0

0

0

5

0

0

0

0

Ero sp. 1

2

2

8

7

2

2

3

5

Ero sp. 2

2

0

0

0

0

1

0

0

Gelcmor sp. 1

0

0

0

1

0

0

0

1

Mimetinae sp. 1

0

0

1

0

0

0

0

0

Miturgidae

Radiilphius sp. 1

1

1

0

0

0

0

0

1

Strotarchus sp. 1

0

1

0

0

0

0

0

0

Teminius insularis (Lucas 1857)

0

0

0

0

1

0

0

0

Mysmenidae

Mysmenidae sp. 1

0

0

0

1

0

0

0

0

Itcipiia sp.

I

2

1

0

0

0

0

0

Itapua sp. 1

0

0

1

0

0

0

0

0

Microdipoena sp. 1

Nemesiidae

0

0

0

0

2

6

2

24

Acanthogonatus sp. 1

0

0

1

1

0

1

2

0

Pycnothele sp. 1

Nesticidae

0

0

2

1

0

0

0

0

Eidnuinella sp.

0

0

0

1

0

0

0

0

Ochyroceratidae

Ochvrocera sp. 1

0

0

0

0

1

1

3

1

Ochyroceni sp. 2

0

0

0

3

0

0

1

3

Ochyrocera sp. 3

0

0

0

0

0

1

0

0

Oonopidae

Gamasomorpha sp.

0

0

1

0

0

0

0

0

gen. 2 sp. 1

0

0

0

0

0

1

0

0

Neoxyphitms sp. 1

0

0

0

3

0

1

0

0

Oonops sp. 1

2

2

1

1

3

3

3

1

Oonops sp. 2

0

0

0

0

0

4

2

0

Orchestina sp. 1

0

0

0

0

0

0

4

0

Predatoroonops sp. 1

0

1

0

3

0

0

2

1

Triaeris stenaspis Simon 1891

0

0

0

0

1

0

0

0

Oxyopidae

Hamataliwa sp. 1

0

2

1

0

0

0

0

0

Hamataliwa sp. 2

0

1

0

0

2

0

1

0

Oxyopes sal ficus Hentz 1 845

1

0

0

0

0

0

0

0

Pence t ia Jlava Keyserling 1877

Palpimanidae

0

0

0

0

1

0

0

0

Palpimanidae sp.

0

0

1

0

0

0

0

0

Notiotiwps hirabeni (Zapfe 1961)

Philodromidae

0

0

0

0

0

0

0

1

Cleocnemis sp. 1

0

0

0

0

0

1

0

0

Pholcidae

Pholcidae sp. 1

11

5

3

3

0

0

0

0

Mesabolivar aff. brasiiiensis (Moenkhaus 1898)

0

0

0

1

0

0

0

0

Mesabolivcir aff. cyaneotaeniatus (Keyserling 1891)

0

0

0

3

0

0

0

0

Mesabolivar aff. guapiara Huber 2000

0

0

0

1

0

4

1

6

Mesabolivar brasiiiensis (Moenkhaus 1898)

0

1

3

11

0

0

0

0

Mesabolivar cyaneotaeniatus (Keyserling 1891)

0

0

0

2

0

0

0

0

Mesabolivar luteus (Keyserling 1891)

0

9

I

24

0

4

7

4

Mesabolivar rudilapsi Machado, Brescovit & Francisco 2007

1

0

2

0

0

6

1

2

Mesabolivar sp. 1

0

0

0

0

1

0

3

0

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

69

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Mesabolivar sp. 2

0

2

0

0

0

3

0

2

Metagonia aff. bonaldoi Huber 2000

0

I

0

1

0

0

0

0

Metagonia furcata Huber 2000

0

0

1

1

0

1

0

0

Metagonia sp. 1

0

3

2

5

0

5

4

0

Metagonia sp. 2

0

0

0

0

0

1

0

0

Nine tines sp. 1

0

0

0

0

0

0

0

2

Ninetines sp. 2

0

1

1

0

0

0

1

1

Tupigea nadleri Huber 2000

0

2

0

1

0

0

6

0

Tupigea sp. 1

0

1

3

0

0

0

0

0

Pisauridae

Architis brasiliensis (Mello-Leitao 1940)

2

2

18

20

0

3

1

7

Architis capricorna Carico 1981

1

0

0

0

0

0

0

0

Prodidomidae

gen. sp.

0

0

0

0

0

0

2

0

Salticidae

Salticidae sp. 26

1

0

0

0

0

0

0

0

Salticidae sp. 38

0

0

0

0

1

0

0

0

Salticidae sp. 45

0

0

0

0

0

1

0

0

Salticidae sp. 46

0

0

0

0

0

1

0

0

Salticidae sp. 54

0

0

0

0

0

0

3

0

Salticidae sp.

0

0

0

0

0

0

2

0

Amphidraus sp. 1

0

1

1

0

0

0

0

0

Amycinae sp.l

0

0

2

0

0

0

1

1

Amycinae sp.2

0

1

0

0

0

1

0

0

Arnoliseus graciosa Braul & Lise 2002

0

4

1

4

0

0

2

3

Asaphobelis physonychus Simon 1902

3

2

2

1

1

1

0

1

Atelurius sp. 1

1

0

0

0

0

0

1

0

Chira spinosa Mello-Leitao 1945

1

0

0

0

1

0

0

0

Chim thysbe Simon 1902

2

0

0

0

0

0

0

0

Coryphasia sp. 1

0

0

0

1

0

0

0

0

Coryphasia sp. 2

0

2

3

0

0

1

1

0

Cotinusa sp. 1

2

1

2

0

0

0

0

1

Cotinusa sp. 2

0

0

0

0

0

1

1

0

Cylistella sp. 1

0

0

1

1

0

3

2

0

Cyllodania sp. 1

0

0

0

0

1

0

0

0

Dendryphantinae sp.l

1

0

0

0

0

0

0

0

Dendryphantinae sp.2

0

0

0

0

1

0

0

0

Euophryinae sp. 2

0

0

0

2

0

0

I

0

Euophryinae sp. 3

2

0

0

0

0

0

0

0

Euophryinae sp. 4

0

0

0

2

0

0

0

0

Euophryinae sp. 5

1

0

0

0

0

0

0

0

Euophryinae sp. 6

0

3

0

0

1

1

0

0

Euophryinae sp. 7

0

0

1

0

0

1

1

2

Euophryinae sp. 8

1

0

0

0

1

0

0

0

Euophryinae sp. 9

1

0

0

1

0

0

0

0

Euophryinae sp. 10

0

0

1

0

0

0

1

2

Euophryinae sp. 1 1

0

0

0

0

0

0

0

1

Euophryinae sp. 12

0

0

0

0

0

0

1

0

Euophryinae sp. 13

0

0

0

0

0

1

0

0

Euophryinae sp. 14

0

0

0

0

0

1

0

0

gen. n. sp. 1

0

0

1

0

0

0

0

0

Hyetussinae sp. 1

0

0

1

3

0

0

0

2

Ilargus sp. 1

0

0

0

0

1

0

0

0

Itata sp. 1

0

0

0

1

0

0

0

0

Lyssomanes sp. 1

0

0

0

0

0

0

0

1

Lyssomanes sp. 2

0

0

0

0

0

0

1

0

Maeota dichrura Simon 1901

0

1

0

0

5

1

0

1

Noegus sp. 1

8

9

11

7

5

4

7

10

Ramboia sp. 1

3

0

0

0

2

0

4

0

70

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Romitia sp. 1

2

0

0

0

1

1

0

0

SemnoUus sp. 1

0

1

0

0

1

0

0

1

Semora napaea Peckham & Peckham 1892

0

0

0

0

4

0

0

0

Synemosvna lauretta Peckham & Peckham 1892

1

0

0

0

3

0

0

0

Synemosyninae sp. 1

0

0

0

1

0

2

3

0

Tanybelus aeneiceps Simon 1 902

0

0

0

0

0

0

0

1

Tariona sp. 1

18

38

19

19

1

7

33

28

Tariona sp. 3

0

3

0

0

0

0

0

0

Tariona sp. 4

5

4

0

0

0

0

0

0

Tariona sp. 5

2

0

0

0

0

0

0

0

Tariona sp. 6

0

0

0

0

1

1

0

0

Vinnius imcatus Simon 1902

3

0

0

0

0

0

0

0

Scytodidae

Scytodes antonina Rheims & Brescovit 2009

6

2

9

0

7

6

3

3

Scytodes globida 1849

0

0

0

1

0

0

0

0

Scytodes sp. 1

0

0

2

1

0

1

1

0

Sparassidae

Olios sp. 1

1

0

0

1

0

0

0

0

Symphytognathidae

Anapistula sp. 1

0

0

1

0

0

0

0

0

Synotaxidae

Synotaxus sp. 1

0

0

0

0

0

1

0

1

Tetragnathidae

Azilia histrio Simon 1 895

1

0

0

0

0

0

0

1

Chrysometa boraceia Levi 1986

0

0

0

0

2

0

2

0

Chrysometa ludibunda (Keyserling 1893)

0

2

1

2

1

1

13

2

Chrysometa sp.

0

0

0

1

0

0

3

0

Cyrtognatha sp. 1

9

3

1

0

4

0

2

0

Glenognatha sp. 1

1

0

0

0

0

0

0

0

Leucauge sp.

0

0

0

0

0

1

0

1

Leucauge sp. 1

0

1

1

0

0

2

0

5

Leucauge sp. 2

0

0

1

0

0

0

5

0

Tetragnatha sp.

0

2

0

0

0

0

0

0

Theridiidae

Theridiidae sp. 1

0

0

0

0

0

0

0

1

Achaearanea sp. 1

0

0

0

0

0

1

0

0

Achaearanea sp. 18

0

0

0

1

0

0

0

0

Achaearanea tingo Levi 1963

0

1

1

0

0

1

0

0

Ameridion imanimum (Keyserling 1891)

0

0

0

0

4

0

0

0

Anelosimus ethicus (Keyserling 1884)

0

0

0

0

1

0

0

0

Anelosimus nigrescens (Keyserling 1884)

1

0

0

0

0

0

0

0

Argyrodes elevatus Taczanowski 1873

0

0

0

1

0

0

0

0

Argyrodes sp. 1

0

0

0

0

0

1

0

1

Ariamnes attemiatus (O.P. -Cambridge 1881)

0

0

2

1

0

0

1

2

Ariamnes longissimus Keyserling 1891

1

1

0

0

1

1

0

0

Ariamnes sp. 2

0

0

0

0

0

0

1

0

Chrosiothes niteroi Levi 1964

0

0

0

3

13

4

7

9

Chrosiothes perfidus Marques & Buckup 1997

0

1

1

3

2

3

0

5

Chrosiothes sp. 1

0

0

0

2

0

0

0

0

Chrysso gounellei Levi 1962

1

13

0

0

0

1

0

0

Chrysso nigrosterna Keyserling 1891

0

0

0

0

0

3

3

0

Chrysso ruhrovittata (Keyserling 1884)

0

0

0

0

1

0

0

0

Chrysso sp. 3

1

2

0

1

0

0

0

6

Chrysso sp. 32

0

0

0

0

0

0

0

1

Coleosoma sp. 1

0

0

0

0

2

0

0

0

Cryptachaea altiventer (Keyserling 1884)

2

1

0

0

1

0

0

0

Cryptachaea digitus (Buckup & Marques 2006)

0

0

0

0

0

0

0

3

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

71

Appendix 1. — Continued.

Taxon

Locality and Stage

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Cryptachaea cinnabarina (Levi 1963)

0

0

0

0

0

0

1

2

Cryptachaea hirta (Taczanowski 1873)

0

0

0

0

8

0

0

0

Cryptachaea isana (Levi 1963)

3

0

0

0

0

0

0

0

Cryptachaea jequirituba (Levi 1963)

0

0

0

0

0

2

0

0

Cryptachaea migrans (Keyserling 1884)

0

0

0

3

0

0

0

0

Cryptachaea passiva (Keyserling 1891)

2

2

3

6

2

5

2

7

Cryptachaea rioensis (Levi 1963)

0

0

0

0

0

0

1

0

Cryptachaea sicki (Levi 1963)

0

0

0

0

0

0

0

1

Cryptachaea sp. 1

0

0

0

0

0

0

1

0

Cryptachaea taim (Buckup & Marques 2006)

1

0

I

1

0

2

4

3

Cryptachaea trigut tata (Keyserling 1891)

0

4

3

2

1

3

3

0

Dipoena atlantica Chickering 1943

0

0

0

0

1

1

0

1

Dipoena bryantae Chickering 1943

0

0

0

0

0

1

0

0

Dipoena cordiformis Keyserling 1886

0

0

1

0

0

0

0

0

Dipoena duodecimpunctata Chickering 1943

0

0

0

1

0

0

0

0

Dipoena ira Levi 1963

4

0

0

1

1

0

1

1

Dipoena keyserlingi Levi 1963

1

0

0

0

0

0

2

0

Dipoena pumicata (Keyserling 1886)

1

4

3

2

3

0

1

I

Dipoena pusilla (Keyserling 1886)

0

0

4

5

0

2

0

1

Dipoena santacatarinae Levi 1963

5

1

4

0

0

0

0

0

Dipoena sp.

1

0

0

0

0

0

1

2

Dipoena sp. 2

8

10

11

3

1

20

42

4

Dipoena sp. 3

0

0

0

0

0

0

1

0

Dipoena sp. 6

0

0

0

0

0

3

0

0

Dipoena sp. 8

1

0

0

0

3

0

0

0

Dipoena sp. 12

5

9

5

12

3

4

11

9

Dipoena sp. 21

0

1

0

0

0

0

0

0

Dipoena sp. 22

0

0

0

0

0

0

1

1

Dipoena sp. 58

0

0

0

0

0

2

0

0

Dipoena variabilis (Keyserling 1886)

0

1

0

1

0

0

0

1

Emertonella taczanowskii (Keyserling 1886)

0

0

0

1

1

1

0

0

Episinus sp.

0

0

0

0

0

1

0

0

Episinus sp. 1

3

2

6

4

9

6

17

5

Episinus sp. 2

0

0

0

0

21

0

0

0

Episinus teresopolis Levi 1964

1

0

0

0

2

0

0

0

Euryopis sp.

1

0

0

0

0

0

0

0

Euryopis sp. 1

2

0

0

0

2

1

0

0

Exalbidion sp. 1

6

1

0

3

I

1

0

0

Exalbidion sp. 2

0

1

0

0

0

0

0

0

Faiditus acuminatus (Keyserling 1891)

0

0

0

0

0

2

0

0

Faiditus sp. 1

0

0

1

1

0

0

0

0

Faiditus sp. 2

0

1

0

1

0

0

1

1

Faiditus sp. 3

0

0

0

3

0

0

1

0

gen. 2 sp. 2

0

0

0

0

0

0

1

0

Guaraniella mahnerti Baert 1984

0

0

0

0

4

15

31

5

Guaraniella sp.

0

0

0

0

0

0

1

0

Guaraniella sp. 1

3

0

0

5

0

0

0

0

Hadrotarsinae sp. 1

0

0

0

0

0

0

1

0

Hadrotarsinae sp. 2

0

0

0

0

3

0

0

0

Hadrotarsinae sp. 3

0

0

0

3

0

0

0

0

Helvibis sp. 1

2

3

0

0

0

0

1

0

Hetschkia gracilis Keyserling 1886

14

5

2

0

4

0

1

0

Janula bicorniger (Simon 1894)

0

1

13

6

0

2

21

32

Neopisinus cognatus (O.P. -Cambridge 1893)

0

1

0

0

0

0

0

0

Phycosotna altum (Keyserling 1886)

4

6

2

0

13

2

0

0

Rhomphaea sp. 1

1

0

1

1

0

0

0

0

Rhomphaea sp. 2

0

0

1

0

0

0

0

1

Spintharus gracilis Keyserling 1886

2

10

8

25

1

10

74

30

Stemmops sp. 2

1

0

0

0

0

0

0

0

Styposis sells Levi 1 964

0

0

0

0

1

1

0

1

Tekellina erica Marques & Buckup 1993

1

0

0

0

0

0

2

0

72

THE JOURNAL OF ARACHNOLOGY

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Tekellina sp. 3

0

0

0

1

0

0

0

0

Tekellina sp. 4

0

0

0

0

0

0

0

1

Theridion biezankoi Levi 1963

1

0

0

0

0

0

1

0

Theridion mimitissimum Keyserling 1884

0

1

0

0

0

0

0

0

Theridion opolon Levi 1963

0

0

1

0

0

0

0

0

Theridion plaumanni Levi 1963

11

2

0

0

43

0

0

1

Theridion quadripartitum Keyserling 1891

0

0

1

3

0

0

0

0

Theridion sp. 1

1

0

0

0

1

0

0

0

Theridion sp. 2

0

0

0

1

0

0

0

0

Theridion sp. 1 1

0

0

0

0

0

1

0

0

Theridion sp. 28

0

0

0

0

0

0

0

1

Theridion sp. 32

1

12

1

23

0

0

0

8

Theridion sp. 36

0

0

0

1

0

0

0

0

Theridion sp. 40

0

1

0

0

0

0

0

0

Theridion sp. 47

0

0

1

1

0

0

0

0

Theridion sp. 63

0

0

0

1

0

0

0

0

Theridion sp. 68

0

0

0

1

1

0

0

0

Theridion teresae Levi 1963

1

0

0

0

1

0

0

1

Theridula gonygaster (Simon 1873)

0

0

0

0

1

0

0

0

Thwailesia affinis O.P. -Cambridge 1882

7

9

7

1

2

7

12

41

Thwaitesia sp. 1

0

0

1

1

0

0

0

0

Thvmoites aniens Levi 1964

8

2

0

0

4

0

0

0

Thymoites melloleitaoni (Bristowe 1938)

0

0

0

1

0

15

1

1

Thymoites sp.

1

0

0

0

0

1

0

0

Thymoites sp. ?

0

0

0

0

0

2

0

0

Thymoites sp. 1

4

1

2

4

1

0

1

0

Thvmoites sp. 2

5

0

1

3

0

0

0

0

Thymoites sp. 4

1

0

0

0

0

0

1

2

Thvmoites sp. 5

0

0

1

0

0

0

0

0

Thymoites sp. 7

0

0

2

3

0

7

1

0

Thymoites sp. 12

1

0

0

0

0

0

0

0

Wamha crispulus (Simon 1895)

4

1

0

0

0

0

0

0

Wirada sp. 1

0

0

0

0

0

0

1

0

Theridiosomatidae

gen. indet. 1

0

1

0

0

1

0

0

0

gen. indet. 3

1

0

1

5

0

0

0

0

gen. indet. 4

0

0

0

1

0

0

0

1

gen. sp. 1

0

0

1

0

0

0

0

1

Chthonos sp.

1

0

0

0

0

0

0

0

Chthonos sp. i

0

0

0

0

0

1

3

0

Chthonos sp.2

0

0

0

0

0

0

1

0

Naatio sp.

0

0

2

0

0

2

0

1

Naatlo sp. 1

0

0

0

0

0

0

1

0

Plato sp. 1

0

0

0

0

0

0

0

I

Theridiosoma sp. 1

5

0

0

1

0

0

0

0

Theridiosoma sp. 3

0

0

0

2

0

0

0

0

Theridiosoma sp. 4

0

0

0

1

0

0

0

1

Thomisidae

Acentroscelus sp. 1

0

2

1

0

0

0

1

1

Aphantochilus taurifrons (O.P. -Cambridge 1881)

0

0

1

1

0

1

0

0

Deltoclita sp. 1

1

0

1

1

2

1

2

0

Epicadimis sp. 1

0

1

0

2

0

0

2

1

Misnmenops sp. 1

1

0

0

0

0

0

0

0

Onocohis sp. 1

0

0

7

1

0

0

0

1

Titidius sp. 1

8

10

1

0

2

5

0

0

Tmarus sp. 1

3

12

4

3

9

3

9

5

Tmariis sp. 2

4

7

2

0

2

2

2

1

Tmarus sp. 3

0

0

1

1

0

0

0

0

Tmarus sp. 4

0

0

0

1

0

0

0

0

Tmarus sp. 5

0

1

0

0

0

0

0

0

RAUB ET AL.— CONSERVATION OF SECONDARY FORESTS FOR SPIDERS

73

Appendix 1. — Continued.

Locality and Stage

Taxon

Ca-H

Ca-A

Ca-M

Ca-F

It-H

It-A

It-M

It-F

Titanoecidae

Goeldia sp. 1

0

0

0

0

7

0

0

0

Trechaleidae

Neoctenus comosus Simon 1897

0

0

0

0

1

0

0

0

Uloboridae

Conifaber sp. 1

0

1

0

0

0

0

0

0

Miagrammopes sp. 1

0

1

8

6

0

2

2

3

Miagrammopes sp. 2

4

0

8

8

0

3

9

9

Uloborus sp.

0

0

0

0

0

0

0

1

Uloborus sp. 1

0

0

0

1

0

0

1

3

Zodariidae

Zodariidae sp.

0

0

0

0

0

0

1

0

Zoridae

gen. 1 sp. 14

8

0

0

0

0

0

0

0

gen. 1 sp. A

30

74

88

103

21

110

107

64

gen. 1 sp. B

0

2

I

0

0

5

1

1

gen. 1 sp. C

20

28

2

21

16

50

31

20

gen. 1 sp. D

14

0

20

12

0

0

0

0

gen. 1 sp. E

1

0

0

0

0

0

0

0

gen. 1 sp. F

0

1

0

0

0

0

0

0

gen. 1 sp. G

0

3

0

1

0

2

1

2

* Nomenclature (order) of the morphospecies originates from the system of the experts/hosting institutions: IBSP - Instituto Butantan, Sao
Paulo; MCN: Museu de Ciencias Naturals da Fundapao Zoobotanica, Porto Alegre.

2014. The Journal of Arachiiology 42:74-78

Trophic niche and predatory behavior of the goblin spider Triaevis stenaspis (Oonopidae):

a springtail specialist?

Stanislav Korenko', Katerina Hainouzova' and Stano Pekar-: 'Department of Agroecology and Biometeorology, Faculty
of Agrobiology, Food and Natural Resources, Czech University of Life Sciences, Kamycka 129, 165 21 Prague 6,
Suchdol, Czech Republic. E-mail: korenko.stanislav@yahoo.com; -Department of Botany and Zoology, Faculty of
Sciences, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic

Abstract. Triacris stciuispis Simon 1891 is a parthenogenetic goblin spider that has been introduced into greenhouses all
over Europe. Here we investigated its trophic niche and predatory behavior. Potential prey in the greenhouses included
predominantly springtails, aphids, and other spiders. Out often potential prey types offered in the laboratory, T. steiiapsis
captured only three types, the primary one being springtails. The spider rarely caught the other two types, termites and
crickets, and completely rejected beetles, ants, aphids, thrips. Hies, spiders and mites. Triaevis stenaspis did not build webs
for prey capture, but instead used the grasp-and-hold tactic. Prey-capture efficiency decreased with springtail body size, the
spider using more than three bites to capture large springtails. Large springtails defended themselves by saltation with the
spiders still attached to their backs. Our study supports the hypothesis that T. stenaspis is a specialized predator of
springtails, being effective in the capture of this type of prey.

Keywords: Stenophagy, prey acceptance, capture behavior, prey

Spiders are mostly known as euryphagous predators,
consuming a wide variety of prey (Nentwig 1987), but several
spider species are prey specialists. Known examples of trophic
specialists include myrmecophagous spiders of the genus
Zodariou (Zodariidae) (e.g., Pekar 2004), oniscophagous
spiders of the genus Dysclera (Dysderidae) (Rezac et al.
2008), araneophagoLis spiders of the genus Portia (Salticidae)
and Palpiauimis (Palpimanidae) (Harland & Jackson 2001;
Pekar et al. 2011), lepidopterophagous spiders of the genus
Mastopliora (Araneidae) (Yeargan 1988) and termitophagous
spiders of the genus Anwioxemts (Ammoxenidae) (Dippenaar-
Schoeman et al. 1996).

Trophic specialists possess various kinds of specific adap-
tations that are effective for their preferred prey. Pekar et al.
(2011) documented specific behavioral and morphological
adaptations in araneophagous Palphuamis spiders. Harland &
Jackson (2001) identified a specific behavioral adaptation,
“cryptic stalking”, in araneophagous Portia fiiiihriata Do-
leschall 1859, which prevents prey dangerous to spiders from
identifying Portia as a predator. Researchers observed unique
physiological adaptations in ant-eating spiders of genus
Zodariou (Pekar et al. 2008; Pekar & Toft 2009), one being
that they cannot metabolize alternative prey at all, or at most
to a much more limited degree.

Goblin spiders (Oonopidae) occur throughout the temper-
ate and tropical regions of the world, in habitats as diverse as
deserts, savannahs, mangroves and rainforest (e.g., Joeque &
Dippenaar-Schoeman 2006). To date, arachnologists have
described more than one thousand species, making goblin
spiders one of the largest families among the Haplogynae
(Platnick 2012). Yet very little is known about the trophic
niche of oonopid spiders. Scientists have found oonopids in a
variety of underground microhabitats such as ant nests
(Jacobson 1933, Weber 1957), termite mounds (Benoit 1964)
and caves (Harvey & Edward 2007), but also on the soil
surface (Ubick 2005) and on tree bark and in the canopy (e.g..

Fannes et al. 2008). All oonopid species appear to be active
cursorial hunters, as they do not build prey-capture webs.
They have been observed to feed on springtails, mites,
firebrats, psocids, and other spiders (Bristowe 1948; Knoflach
et al. 2009; Korenko et al. 2009; Hansen 1992). Researchers
have even detected some oonopids scavenging insect remains
on webs of larger spiders (Bristowe 1948; Knoflach et al.
2009). To date, rigorous analysis of the trophic niche of
oonopids is lacking.

Triacris stenaspis Simon 1891 seems to be indigenous to
West Africa (Platnick et al. 2012) and, according to Platnick
(2012), its range stretches from Central to South America,
including Antilles and Europe. It has become a resident in
Europe and is successfully surviving in heated greenhouses
(Korenko et al. 2007). It is known to be parthenogenic in
Europe (Korenko et al. 2009).

Since greenhouses are rather poor in arthropod species
diversity (Mahr et al. 2001), we expect T. stenaspis to be
trophically specialized on an abundant prey. Based on
knowledge from our previous study when T. stenaspis was
successfully reared on a monotypic diet including only
springtails (Korenko et al. 2009), we hypothesized that T.
stenaspis prefers, or at least is adapted to, springtail prey. Such
trophic specialization is either fixed across populations or is a
plastic response of a population to locally abundant prey, as
was recently found for Oecohius spiders (Liznarova et al. 2013).
In the former case, specialized traits used for capture and
utilization of prey occur in all populations and are leading to
the evolution of species-specific adaptations for particular prey
types. In the latter case, a predator does not possess specialized
traits, but it can be locally specialized on profitable prey to
enhance its versatility at a particular time and place. Our aim in
this study was to investigate the trophic niche of T. stenaspis,
namely the prey spectrum and the predatory behavior of this
species in order to test our hypothesis on prey-specialization
and to investigate whether it is effective in the capture of prey.

74

KORENKO ET \L.—TRIAERIS STENASPIS: A SPRINGTAIL SPECIALIST?

75

METHODS

On eight sampling dates during the autumn and winter
2006-2008, we collected all ground-dwelling arthropods
occurring in the microhabitat of T. stenaspis' occurrence in
the botanical garden of the Masaryk University in Brno
(Czech Republic) to estimate the potential prey spectrum. Our
team members hand-collected arthropods by lifting stones and
rotten pieces of wood, inspecting the ground and plant roots
underneath using a pooter, and then put the arthropods in
tubes with ethanol and identified them to order in the
laboratory. We only considered specimens of a total body
size less than six mm as potential prey.

Altogether, we collected 60 adult females of T. stenaspis in
the greenhouse in order to perform observations in the
laboratory. The body length of spiders was very similar,
ranging between 1.6 and 1.7 mm. We placed spiders singly in
cylindrical containers (diameter 35 mm, height 40 mm) with a
layer of plaster of Paris at the bottom, and kept them at room
temperature, 22 ± 3.5°C, to replicate conditions in the
greenhouse. The plaster was moistened with a few drops of
water to retain sufficient humidity. Spiders were fed springtails
Sinella curviseta (Brook 1882) at 3^ day intervals.

We tested the following 1 1 prey types of ground-living
arthropods for acceptance: beetle imagoes (Curculionidae)
(average body length = 1.93 mm, SD = 0.20), first instars of
crickets Acheta domestica (Linnaeus 1758) (Gryllidae)
(2.81 mm, SD = 0.46), aphids (Aphididae) (1.81 mm, SD =
0.25), thrips (Thysanoptera) (1.41 mm, SD = 0.15), mites
(Trombidiidae) (0.87 mm, SD = 0.10), early instars of crab
spiders of genus Xysticus (Thomisidae) (1.4 mm, SD = 0.38),
ants Tetramorium caespitum (Linnaeus 1758) (Formicidae)
(3.14 mm, SD = 0.20) and a mixture of springtail species
(Collembola). Our team collected all of these prey from the
soil in the greenhouse (1.06 mm, SD = 0.41). Termites
Reticulitermes sp. (Isoptera), (3.54 mm, SD = 0.30), larvae
and imagoes of Drosophila mekmogaster Meigen 1830 (Diptera)
(3.8 mm, SD = 0.54 for larvae and 2.4 mm, SD = 0.50 for
imagoes) and springtails Sinella curviseta (Brook 1882)
(1.45 mm, SD = 0.12) (Entomobryidae) came from laboratory
cultures. The body length of prey was measured using an ocular
ruler in the stereomicroscope before each trial.

Altogether we performed 150 trials over a period of 60 days
to test prey acceptance. For each prey type, 12 individuals of
adult female T. stenaspis were used. The order of tested prey
was based on the availability of particular prey. Each tested
spider was kept singly in a dish (diameter 35 mm, height
45 mm), which was marked by number, 1-60, in the order in
which the individuals were collected. Twelve individuals from
the set of 60 specimens were randomly (without replacement)
selected for each prey type. When all individuals were used in
the first round of tests, another 12 individuals were randomly
(without replacement) selected from those that had already
been used. Each individual was tested a maximum of two or
three times. Each individual was used in the trial five days
after being satiated with springtails. Individuals that did not
feed during the day of satiation were not used in the next trial.
At least six days elapsed between the repeated uses of the same
individual.

Before each trial, spiders were placed singly in an
experimental dish (diameter 35 mm, height 15 mm) with a

Table 1. — Relative incidence of potential prey types found on the
soil in the greenhouse (n = 148).

Potential prey

Relative incidence

Isopoda

0.12

Myriapoda

0.02

Araneae (other than Triaeris)

0.09

Schizomida

0.07

Collembola

0.61

Ensifera

0.02

Formicidae

0.03

Coleoptera larvae

0.02

Other larvae

0.02

thin (2-3 mm) layer of plaster of Paris at the bottom. After
five minutes of acclimatization, the potential prey was released
into the experimental dish, and the occurrence of capture
(incidence) was recorded. If the spider accepted the prey, or
did not catch the prey within 120 minutes following encounter
with prey, we terminated the trial.

The capture efficiency was studied in detail using S.
curviseta springtails as prey. Thirty adult female spiders,
selected randomly (without replacement) from the set of 60,
were starved for five days and placed in arenas as described
above. After five minutes of spider acclimatization in the dish,
a springtail S. curviseta of body length between 0.5 mm-2 mm
(prey-predator body length ratio = 0.3-1. 2) was released.
Spiders were selected randomly from the same experimental
group as in the previous experiment. We recorded the attack
latency, the capture behavior and the number of attempts
required to capture springtails. The attack latency was the
time between when the spider oriented itself toward the
springtail and successfully completed the attack, (i.e., when
prey was held in the chelicerae). We measured the body length
of springtails before each experimental trial.

Statistical analyses were conducted within the R environ-
ment (R Development Core Team 2011). The relationship
between prey size and attack latency, springtail size and
number of attacks were analyzed using Generalized Linear
Models with Gamma (GLM-g) or Poisson (GLM-p) error
structure, respectively (Pekar & Brabec 2009). We used
Generalized Estimating Equations with binomial error struc-
ture (GEE-b) to compare the incidence of captured prey
assessed in a binary form. GEE is an extension of GLM used
for repeated measurements by specifying correlation among
measurements (Pekar & Brabec 2012). GEE was used since
measurements were not independent due to repeated use of the
same individual spiders.

RESULTS

The most abundant potential prey arthropods in the
greenhouse (n - 148) were springtails (Entomobryidae)
followed by aphids, other spiders, schizomids, ants, myria-
pods, coleopteran larvae, other larvae and crickets (Table 1).
From 1 1 potential prey types tested in the laboratory, T.
stenaspis accepted only springtails at high incidence rates
(83%, u — 42, Fig. 1). Crickets and termites were rarely
accepted (8%, n = 12, both prey types). The prey-predator
body-length ratio of accepted prey was on average 0.6 (min-
max = 0.3-1. 1) in springtails, 1.2 in crickets, and 2.0 in
termites. Mites, spiders, thrips, aphids, beetles, ants and files

76

THE JOURNAL OF ARACHNOLOGY

Figure 1 . — Comparison of the relative capture incidence of 1 1 prey
types.

(larvae and imagoes) were never accepted. The prey capture
incidence thus differed among groups (GEE- b, X",o = 45.7,
P < 0.0001).

Triaeris stenaspis did not build a web for prey capture;
instead, it caught springtails and other prey using the grasp-
and-hold tactic. After an attack, springtails attempted to
escape by means of saltation. Ten percent of springtails
jumped up with the spider attached to the springtail’s back
and hit the top of the experimental arena. All jumping
springtails were large adults with a prey-predator ratio > 1.2.

The attack latency increased significantly with prey size
(GLM-g, Fi^23 = 6.4, P = 0.02, Fig. 2). The number of attacks
required for prey immobilization increased significantly with

Figure 2. — Relationship between the attack latency and the
springtail body size with estimated model.

Figure 3. — Relationship between the number of attacks and prey
size with estimated model.

prey size (GLM-p, X^i — 9.5, P = 0.002, Fig. 3). Triaeris
stenaspis required significantly fewer attacks to catch small
springtails than to catch large springtails. The spiders made on
average 1 .4 attacks (SD = 0.71, n = 17) to catch small springtails
(prey-predator ratio < 0.7), compared to 3.8 attacks (SD = 1 .48,
« = 8) to catch large springtails (prey-predator ratio > 0.7).

DISCUSSION

We found a moderate diversity of potential prey for T.
stenaspis on the surface of the greenhouse soil. All of these
arthropods were expected to be potential prey for T. stenaspis,
at least at the life stage when of suitable body size. Triaeris
stenaspis is a ground dweller that inhabits various structures of
the soil and encounters all of these taxa. Although we did not
record prey capture by T. stenaspis in the field, acceptance
trials clearly indicate that T. stenaspis likely utilizes only a very
small portion of available prey.

Little is known about the prey consumption of goblin spiders.
There is only one report of the natural prey of T. stenaspis: Weber
(1957) stated that it consumed Cyphomyrmex costatus (Mann
1922) ants and springtails. Our laboratory study confirmed the
spiders feeding on springtails but not on ants. Since Weber (1957)
observed only ant remnants in the spider surrounding, not the
actual feeding on ants, we propose that T. stenaspis was not
feeding on ants. Ants are dangerous prey and feeding on them
requires morphoIogi.cal and behavioral specialization (e.g., Pekar
et a!. 2008, 2011). We have not observed any adaptation useful
for capture of ants in this spider species. Triaeris stenaspis is thus
likely myrmecophilous but not myrmecophagous.

In the laboratory, T. stenaspis consumed springtails almost
exclusively and haphazardly preyed on other prey. Spiders
uniformly used the grasp-and-hold predatory tactic to capture
springtails, termites, and crickets. Several other spiders use
this tactic (Nentwig 1987). The grasp-and-hold tactic seems
efficient for the capture of mobile prey, because after a bite
such prey could escape the spider. This is particularly

KORENKO ET AL.—TRIAERIS STENASPIS: A SPRINGTAIL SPECIALIST?

77

important for species capable of fast escape, such as springtails
with furca.

The venom of T. stenaspis seems to be extremely effective in
immobilizing springtail prey. Springtails with bodies larger
than the spiders were not able to jump more than once. The
spider injected the venom behind the head, near the central
neural system, and the paralysis latency was only a few
seconds. Thus T. stenaspis was able to immobilize the large
springtail within a few seconds.

Most cursorial spider species prey on much smaller prey
than themselves (Nentwig 1987). Triaeris stenaspis was able to
catch springtails larger than itself, even capturing one termite
double its size. The ability to capture much larger prey is
typical for many stenophagous predators (e.g., Pekar et al.
2008) that possess various adaptations to capture their specific
prey.

Springtails are consumed by many spider species either of
cursorial or web-building habit, such as corinnids (Pekar &
Jarab 2011), linyphiids (Sunderland et al. 1986; Nyffeler &
Benz 1988; Alderweireldt 1994), lycosids (Hallander 1970;
Gettmann 1978; Punzo 2006), salticids (Guseinov et al. 2004;
Huseynov et al. 2005), theridiids (Ibarra- Nunez et al. 2001)
and thomisids (Guseinov 2006). The spiders differ in the way
they capture springtails. Although web-builders such as
Lepthy’phantes (Linyphiidae) rely on the use of webs, cursorial
species such as Erigone, Oedothorax (both Linyphiidae) or
Mexcala (Salticidae) grasp springtails with their forelegs and
chelicerae (Alderweireldt 1994; Pekar & Haddad 2011). The
linyphiid Bathyphantes similUmus (L. Koch 1879) captures
springtails by means of two different strategies (Rybak 2007):
juvenile individuals rely on the web (a springtail gets entangled
in a web by its hairs), while adult spiders grasp springtails with
their chelicerae.

For spiders, many springtails are palatable prey, except for
some species that are toxic (Toft & Wise 1999). Springtails,
however, possess efficient defenses: species having furca can
escape by jumping either from a web or from a spider’s
forelegs. For a predator that is of similar or smaller size, the
springtails can jump after being grasped. Such “rodeo-riding”
on the springtail may carry a high risk of injury to the spider.
Springtails with a spider attached to their back jumped high
enough to hit the lid of the experimental arena. This strong
knock could cause serious injury to the spider’s soft abdomen.
However, three scuta located on both sides of the soft
abdomen in T. stenaspis seem to prevent such injury. Whether
the scuta can be considered morphological adaptations used
for defense against prey remains to be investigated.

Our study found that springtails are readily accepted as prey
by T. stenaspis, even when the prey is longer than the spider. It
is yet to be discovered whether T. stenaspis is a collembolan
specialist and whether such prey-specificity is a fundamental
property of all populations of this species or only a case of
local specialization. Although data gathered so far do not
provide clear evidence for specific adaptations, we found a
high efficiency of capture of collembolan prey. This efficiency
is attributed to their powerful grasp-and-hold tactic and the
fast-ensuing paralysis from the spider’s bite. Riding the
“rodeo” on the springtail back is a behavioral trait serving
to avoid the loss of the escaping prey. Strong scuta on the
spider abdomen could be a morphological trait that protects

the spider’s soft parts against injury during “rodeo.” The short
paralysis latency suggests that the venom is used for fast
immobilization and minimizing the duration of the dangerous
“rodeo.” Finally, in our previous study, we reared this spider
species on a monotypic diet of springtails (Korenko et al.
2009); the spiders suffered low mortality, were able to develop
completely and produce viable offspring. They also seem to
possess physiological adaptations to utilize a monotypic
springtail flesh. Whether these traits have evolved as an
adaptation to collembolan prey remains to be proven.

ACKNOWLEDGMENTS

We would like to thank L. Brian Patrick, one anonymous
referee, and Elizabeth Jakob for their fruitful comments,
which improved the quality of our manuscript. SK was
supported by the project of European Science Eoundation and
Ministry for Education and Youth of the Czech Republic
CZ. 1.07/2. 3. 00/30. 0040 and "Ucelova podpora na specificky
vysokoskolsky vyzkum" provided by Ministry for Education
and Youth of the Czech Republic.

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Manuscript received 10 December 2012, revised 4 September 2013.

2014. The Journal of Arachnology 42:79-85

Use of locomotor performance capacities reflects the risk level associated with
specific cue types in two cursorial spider species

Joseph A. Dillon' and Jonathan N. Pruitt’-^: ‘Department of Ecology and Evolutionary Biology, University of Tennessee,
Knoxville, Tennessee 37996 USA; -Department of Biological Sciences, University of Pittsburgh, Pittsburgh
Pennsylvania 15260 USA

Abstract. Understanding when and how animals use their performance capacities can yield insights into the selection
pressures driving high performance. Using two species of cursorial spiders, Schizocosa ocreata (Hentz 1844) and Rahidosa
rabida (Walckenaer 1837) (Araneae: Lycosidae), we investigated the escape speeds exhibited by individuals of various body
sizes in response to three aversive stimuli (jets of air, seismic cue, prodding) to determine how individuals use their
performance capacity in response to different stimuli. We found that large individuals of both species exhibited their
highest observed escape speeds in response to jets of air, whereas smaller individuals exhibited their fastest observed escape
performances in response to prodding. We hypothesized that differences in escape behavior may reflect differences in risk
associated with each cue type: fast moving jets of air may announce the arrival of an avian predator, and large individuals
may be at greater risk of avian predation owing to their more conspicuous body size; whereas smaller individuals may be
more susceptible to arthropod predators, which attack from the level of the spider, similar to a prod. We then performed an
unreplicated mark-recapture, avian-exclosure experiment for both species, where we tracked individuals’ persistence for
30 d. Consistent with our predictions, we found that larger individuals enjoyed greater persistence in our avian exclosure
treatment, but their advantage was lost when avian predators were allowed to enter. Our results suggest that these spiders
express their highest performances in only their most dire situations.

Keywords: Behavioral compensation, foraging mode, locomotion, Lycosidae

Variation in performance capacities has been linked with
fitness components in a variety of species. For instance, higher
running speed may be associated with greater survivorship
(Calsbeek & Irschick 2007; Pruitt 2010), stronger bite forces
may help individuals establish dominance (Lailvaiix et al.
2004; Perry et al. 2004) and greater endurance may help
animals obtain prospective mates (Stoltz & Andrade 2010;
Stoltz et al. 2009). Although everyday tasks require that
animals use their performance capacity somewhat regularly,
identifying the specific selection pressures driving maximum
performance can be difficult. This difficulty stems, in part,
from the fact that the same actions are used across many tasks:
capturing prey, avoiding predators, maintaining territories
and displaying courtship (Irschick & Garland 2001 ). Thus, any
number of context-specific selection pressures could drive the
evolution of animals’ maximum performance. One way to gain
insights into the selective pressures driving maximum perfor-
mance is to understand when and how animals use their
performance capacity. The hypothesis underlying this notion
is that individuals are expected to approximate maximum
performance capacity in situations most important for their
survival or reproductive success and express sub-maximal
performance in other, less dire situations (Irschick 200'0a,b;
Irschick & Garland 2001; Husak & Fox 2006; Pruitt & Husak
2010). Here we define individuals’ “maximum” or “highest”
performance as the peak performance individuals exhibited
across all observed contexts (after Husak & Fox 2006),
although we acknowledge that even these values may fall short
of absolute potential.

A small number of studies have shown that individuals tend
to express their highest performance in the ecological contexts
most pertinent to their success. For example, within a

^Corresponding author. E-mail: pruittj(^pitt.edu

population of collared lizards (Crotaphytus coUaris), females
exhibit their highest observed running speeds when escaping
predators. Males, however, reserve their highest speeds for
territorial encounters. Neither sex uses high speeds when
attempting to capture prey (Husak & Fox 2006). Males and
females experience differing selective pressures in this system, as
evidenced by differences in how the sexes use their performance
capacities. Males are territorial and risk suffering a potentially
high cost to their fitness if they do not rapidly respond to
intruding rivals (Husak et al. 2006; Husak et al. 2008), whereas
females are non-territorial and have little selective pressure to
respond intensely to rival females. Thus, strength of selection on
locomotor importance may differ considerably across demo-
graphic groups within a single population.

Similarly, in the funnel-web spider Agelenopsis aperta
Gertsch 1934, individuals show population-level divergence
in how they use their maximum running speeds (Pruitt &
Husak 2010). This species occupies a habitat mosaic of arid
and riparian zones; arid zones are characterized by few
foraging territories, low prey availability, and few predators,
whereas riparian habitats are lush environments with many
suitable foraging territories, high prey availability and many
predators (Riechert & Hedrick 1990; Riechert 1993; Riechert
et al. 2001). Consistent with our understanding of this species’
ecology, offspring from parents collected in arid habitats
exhibited their highest observed running speeds during
territorial encounters, but responded slowly to simulated
predator threats. In contrast, offspring from parents collected
from riparian sites expressed their highest observed speeds in
response to simulated predator threats, but were slower to
attack rivals (Pruitt & Husak 2010). Here again, data suggest
animals scale the use of their performance capacities to the
significance of the task or challenge at hand.

79

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THE JOURNAL OF ARACHNOLOGY

A primary insight gleaned from the studies detailed above is
that different populations or demographic groups within them
experience divergent selection pressures, and these pressures
are echoed in how animals use their performance capacities.
However, the possibility that individuals may differ in their
responsiveness to different cue types within a context (e.g.,
predator avoidance) has been largely overlooked. Here we
explore this topic using two species of temperate wolf spider,
Schizocosa ocreata (Hentz 1844) and Rabidosa rabida (Walck-
enaer 1837 (Araneae, Lycosidae). Specifically, we test whether
individual variation in body size is associated with how
individuals respond to different cue types. For example, large
individuals might exhibit slow running speeds in response to
cues resembling arthropod predators, but high-speed respons-
es towards cues resembling avian/vertebrate predators. We
then test whether performance matches risk of predation in the
field using an unreplicated mark-recapture, predator exclusion
experiment. Documenting associations between performance
use and body size has important general implications for
understanding how animals of different body states (e.g., age,
viability, reproductive status) use their performance capacities.

METHODS

Collection and laboratory maintenance. — Wolf spiders (fam-
ily Lycosidae) are cursorial, non-web building spiders that
traverse leaf litter and low-lying vegetation in search of prey.
We collected spiders for use in our aversive stimuli trials in
March-April {Schizocosa) and July-August (Rabidosa) of
2010. Schizocosa ocreata (n — 46) were collected amid fallen
leaf litter from a deciduous forest habitat (35°47'56"N,
84°14'01"W), and Rabidosa rabida (n = 28) were collected
among mixed herbs and grasses from an early successional
meadow habitat (36°00'49"N, 84°02'32"W) in East Tennessee.
We selected our two test species because they occur commonly
at our test sites and because of their divergent ecologies; they
occupy different habitats, exhibit divergent phenologies, and
differ distinctly in body size (see Results section). Spiders were
collected at night using a spotlight. They were spotted, chased
into pill vials, and transported to a laboratory at the University
of Tennessee, Knoxville. They were housed individually in 490-
ml opaque deli cups, provided a maintenance diet of two
three-week old crickets once weekly, and maintained under
ambient lighting conditions. A moist paper towel was
provided as a water source. Upon reaching maturity (~1-
3 weeks), individuals were selected for use in our aversive
stimuli trials, and all aversive stimuli trials were completed
within two weeks of individuals’ final molts. Only mature
females were used in our aversive stimuli trials and mark-
recapture experiments.

Individuals’ body measurements were collected one day
after their first routine feeding as mature individuals. Each
individual was weighed to the nearest 0. 1 mg, (Mettler-Toledo
XP205) and its cephalothorax length and abdomen length and
width were measured to the nearest millimeter using Leica
digital imaging software and a stereomicroscope (Leica M80).

Aversive stimuli trials occurred three days after a routine
feeding, and 24 h elapsed between trials. We used a
standardized feeding-level for our trials because previous data
indicate recent meal size (via the resulting increase in body
mass) can negatively impact spiders’ escape performance

(Pruitt 2010), and we limit our investigation to recently
matured females. Sex and reproductive status have significant
impacts on spiders’ escape performance (Pruitt and Troupe
2010). To avoid potential confounding of trial order, the
sequence of the trials was alternated among individuals. Thus,
each individual was tested three times, once with each
stimulus.

Aversive stimuli. — Aversive stimuli trials were run at 2000-
2200 at low light conditions (30-50 lux) to mimic the
nocturnal/crepuscular nature of most wolf spiders (Schizocosa:
Cady 1983; general wolf spider ecology: Foelix 1996; but see
Uetz et al. 1999). Spiders were placed on a 30-cm track in a
clear plastic collection vial and allowed 30 sec to acclimate
before the vial was lifted. Spiders were then given another
30 sec of acclimation before an aversive stimulus was applied.
Tracks were lined with graphing paper with 0.5 cm demarca-
tions. Tracks were 6 cm wide and the walls extended 8 cm up
on all sides. We video-recorded spiders’ escape responses using
an infrared Bullet Security Camera (Sony CSP-LR560IR) and
noted individuals’ flight speed (cm/s) over 15 cm of track.
Data from trials where spiders paused or turned were removed
from our analyses, as is standard in the animal performance
literature. In such instances (n = 6), spiders were given 15 min
to recover before the trial was repeated.

Our three aversive stimuli were a prod, a puff of air, and a
seismic cue. For our prod test, we touched the rear end of the
spider with a thin (3 mm wide) paintbrush. This prod was
designed to mimic the tactic cue of an approaching predatory
spider or insect. For our ‘puff test, we used a camera lens
cleaning bulb to apply rapid jets of air on the dorsal, posterior
portion of the spiders from 5 cm distance; two fast,
consecutive puffs were applied. Puffs of air have been used
to simulate the approach of an avian predator in a number of
investigations on spiders (Riechert & Hedrick 1990; Riechert
1993; Riechert et ai. 2001; Pruitt et al. 2008; Jones et al.
2011a,b). This cue is relevant because most spiders lack acute
vision and instead detect the approach of avian predators
using minute vibratory-sensing setae, the trichobothria (Foelix
1996). We applied a seismic cue by dropping a 1.2 kg biology
textbook from Im off the ground, 30 cm away from the spider.
This seismic cue was modeled to resemble the distant approach
of a large vertebrate (e.g., a deer, coyote or a human collecting
spiders). After we recorded their reaction, spiders were
returned to their individual cups.

Selection on body size. — To assess whether individuals of
different body sizes were more or less susceptible to avian
predation, we estimated selection on body size in field
exclosures from March-July 2008 (March for S. ocreata and
July for R. rabida). We established four 3 m X 3 m exclosures:
two to assess selection on body size for R. rabida at a meadow |,|

site (Riechert Farm: 36°00'49"N, 84°02'32"W) and two to 1

assess selection on S. ocreata at a deciduous forest site (House
Mountain: 35°47'56"N, 84°14'01"W). Thus, our predator
present/absent treatment was unreplicated within each species,
but two parallel studies were conducted with two different
species.

Exclosures were lined with aluminum flashing around the
perimeter; flashing extended 40 cm above the ground and
15 cm below. To prevent immigration and emigration across
the aluminum border, herbaceous plants were trimmed back

DILLON & PRUITT— LOCOMOTOR BEHAVIOR MATCHES RISK

81

m

E

JO

■D

&

0

Q.

(/5

0

Q.

CO

o

m

UJ

Prod

Puff Seismic

m

E

S’

■o

0

0

a

m

0

a

0

o

0

LU

Prod

Puff Seismic

Cue Type

Figure 1. — Box plot depicting the raw (cni/s) escape speeds
exhibited by both test species (Schizocosa oo'eata, Rahiclosa rabida)
in response to our three aversive stimuli. Vertical shaded bars
represent interquartile range and vertical lines represent the 90‘*’ and
lO"’ percentiles. Within BT compositions, bars not sharing letter
flagging are significantly different at a = 0.05 using post-hoc
Tukey tests.

on either side of the flashing using pruning shears, and both
sides of the flashing were topped with a 7-cm-wide strip of
Tanglefoot tree pest barrier. For each pair of exclosures, one
was left with an open top (avian predators present) and the
second was topped with a grid of two perpendicular series of
monofilament fishing lines placed every 4 cm in either direction.
Fishing lines were anchored into a wooden frame, which
we fastened atop the aluminum flashing, thus generating a
monofilament ‘table top’ that effectively excluded birds (Hubbs
& Boonstra 1997; Krebs et al. 1995). Although we did not
directly observe avian predation in this study, birds are known
to be important predators for at least one of our test species
(Schizocosa ocreata: Lohrey 2007; Lohrey et al. 2009) and are
thought to be major predators of spiders in general (Foelix
1996; reviewed in Riechert & Hedrick 1990; Gunnarsson 2007).
Although our exclosure design likely excluded other vertebrate

Table 1. — Summary of our general linear models predicting
relative speed in response to three aversive stimuli. Relative speed
was obtained by dividing the flight speed (cm/s) obtained by an
individual in response to each stimulus by the mean tlight speed
exhibited across all three stimuli; d/’ indicates degrees of freedom.

Schizocosa

f ocreata

Source

df

F

P

Combined Model, R" = 0.36

5

3.866

0.03

Intercept

1

58.791

<0.001

Body Size

1

0.45

0.87

Stimulus type

2

10.227

<0.001

Body Size*Stimulus Type

2

7.2

<0.001

Error df

91

Rabidosa

rabida

Source

df

F

P

Combined Model, R‘ = 0.34

5

7.289

<0.001

Intercept

1

40.826

<0.001

Body Size

1

0.03

0.99

Stimulus type

2

13.006

<0.001

Body Size*Stimulus Type

2

13.455

<0.001

Error df

57

predators (e.g., bats, raccoons) and not just birds, we will refer
to our exclosures as ‘avian exclosures’ throughout this paper.

Before initiating an experiment, we systematically sampled
each exclosure by sifting through leaf litter and removing all S.
ocreata and R. rabida therein. Non-focal arthropods (spiders
or otherwise) were replaced within our exclosures before the
start of our marked-recapture experiment. We removed 41 and
26 S. ocreata from our exclosures at House Mountain, and 12
and 5 R. rabida at the Riechert Farm. A pool of mature female
conspecifics was collected from adjacent habitats, measured
using digital calipers, individually marked, and placed within
our exclosures. Individuals’ assignment to treatment (avian
predators present/absent) was determined randomly using
statistical software. We placed 35 S. ocreata in each plot at
House Mountain and 20 R. rcdtida in each plot at the Riechert
Farm. Spider densities were a compromise between 1) our
desire to replicate natural densities of both test species at our
test sites, and 2), our desire to maximize the statistical power
of our experiment. Each test spider was assigned a unique
letter/number combination, and a corresponding tag was
adhered to its prosoma using non-toxic liquid adhesive
(Testors). Care was taken to use a minimal amount of
adhesive, since excessive adhesive may reduce spider mobility.
Exclosures were left undisturbed for 30 d.

After the 30-d selection period, we resampled each plot and
collected all individuals therein. Plots were sampled for four
days by sifting through leaf litter and turning over each
individual leaf. Individuals that were not recovered during our
four-day search period were assumed to be dead. Importantly,
all of the spiders recovered from within our plots were
marked, thus indicating a low probability of emigration or
immigration across our exclosure barriers.

Statistical analyses. — To test for differences in escape speed
(cm/s) in response to our three aversive stimuli, we used one-
way ANOVAs with post-hoc Tukey tests. To assess the
relationship between body size and performance, we used

82

THE JOURNAL OF ARACHNOLOGY

•

Prod

0

Puff

A

Seismic

12 14 16 18 20 22 24 26

Body Size (mm)

Figure 2. — Relationship between individuals’ body size (abdomen + cephalothorax length) and relative speed exhibited (speed displayed with
a given stimulus/average speed displayed across stimuli) in response to various stimuli for Schizocosa ocreata (top) and Rabidosa rabida (bottom).

repeated measure ANOVAs and analyzed each species
independently. We included stimulus type, body size (cepha-
lothorax length + abdomen length) and their interaction term
in our models, and used ‘relative speed’ as our response
variable. Relative speed was calculated by dividing the flight
speed (cm/s) obtained by an individual in response to each
stimulus by the mean flight speed exhibited across all three
stimuli.

To assess the effects of body size on survival in S. ocreata
and R. rabida, we calculated selection gradients for each
species independently by transforming trait values to mean
zero and unit variance, and survival scores (1,0) were
transformed into relative fitness (individuals’ fitness/average
fitness of their cohort). Selection gradients (i.e., the change in
expected fitness per standard deviation of trait value) were
calculated for each exclosure, independently using multiple

DILLON & PRUITT— -LOCOMOTOR BEHAVIOR MATCHES RISK

83

Table 2. — Summary of our combined models predicting spiders’
persistence in our avian exclosure, mark-recapture experiments. The
effects of body size on persistence differed significantly among
treatments (predators present vs. absent).

Scbizocosa ocreata

Source

df

F

P

Combined Model

3

10.35

0.002

Intercept

1

0.11

0.74

Treatment

1

5.09

0.02

Body Size

1

0.19

0.66

Treatment*Body Size

1

6.55

0.01

Rabidosa rahida

Source

df

F

P

Combined Model

3

11.51

<0.001

Intercept

1

0.51

0.64

Treatment

1

5.85

0.03

Body Size

1

0.43

0.45

Treatment*Body Size

1

6.36

0.01

linear regression (Lande & Arnold 1983; Calsbeek & Irschick
2007). To test whether selection gradients differed among
exclosures (avian predators present vs. absent treatment) we
used a combined multiple logistic regression model with
treatment, body size, and the interaction term treatment X
body size as predictor variables. For all analyses we used
logistic regression for our significance tests (after Janzen &
Stern 1998) and multiple linear regression to estimate selection
gradients (after Calsbeek & Irschick 2007). We did not include
non-linear selection terms in our models owing to limited
degrees of freedom, but visual inspection of the data indicated
no non-linearity.

RESULTS

We detected significant differences in the escape speeds

exhibited in response to our three aversive stimuli for S.
ocreata (F2.93 = 4.21, P = 0.02), but not R. rcibida (A.e? =
1.48, P = 0.16). In S. ocreata, individuals exhibited higher
escape speeds in response to rapid jets of air (mean speed =
39.95 cm/s, SE = 1.31) than to seismic cues (mean speed =
35.23 cm/s, SE = 1.11), and their responses to prodding (mean
speed = 38.36 cm/s, SE = 1.12) were intermediate and
indistinguishable from responses to the other stimuli (Fig. 1).

Our combined models predicting individuals’ relative escape
speeds were significant for both S. ocreata - 3.86, P —
0.03) and R. rabida (F5,62 = 7.29, P <0.001). We detected
significant effects of stimulus type and the interaction term
stimulus type X body size for both species (Table 1). From
examination of the interaction plot between body size and
stimulus type (Fig. 2), we found a strong positive relationship
between body size and individuals’ relative escape speed in
response to puffs of air. In contrast, we observed negative
trends in the relationship between relative escape perfomiance
and body size with both the prod and seismic cues (Fig. 2). That
is, larger individuals responded more slowly to prod and seismic
cues, but responded faster after experiencing a puff of air.

Our combined models predicting persistence in our mark-
recapture avian exclosure experiment were significant for both

species: 5. ocreata (F3 67 == 10.35, P — 0.002) and R. rahida
(-^3,37 = 11.51, P <0.001). We detected a significant effect of
treatment (avian predators present vs. absent) and a signifi-
cant interaction between individuals’ body size and treatment
for both species (summarized in Table 2). From examination
of body size distribution of the “surviving” versus “dead”
spiders in each treatment (Fig. 3), we found that surviving
individuals tended to be larger in our predator exclusion
treatments. However, this trend disappeared (S. ocreata) or
was reversed (R. rahida) in our predator inclusion treatment.

DISCUSSION

Documenting how animals employ their performance
capacities can help to elucidate the selective pressures driving
performance. Specifically, data from a wide range of taxa
suggest that animals express their highest speeds in the
contexts/situations most vital in determining their direct
fitness and survival (Domenici & Blake 1997; Husak & Fox
2006; Husak et al. 2008; Irschick & Garland 2001; Pruitt &
Husak 2010). Our data here demonstrate that even within a
single context (anti-predator behavior) animals may differ in
their responsiveness. Specifically, we demonstrate that, in R.
rabida and S. ocreata, larger individuals tend to exhibit higher
running speeds in response to puffs of air, whereas smaller
individuals of both species tended to exhibit greater burst
speeds in response to mechanical stimuli (i.e., prodding).
Concordantly, we found that in the absence of avian
predators, larger individuals enjoy higher survivorship in the
field. In contrast, when avian predators were not excluded, the
advantage of large body size was lost or reversed. Our data
add an additional point of note to standard investigations on
animal performance and indicate experiments that use only a
single cue type may overlook important condition- or trait-
dependent variation in performance utilization.

Individuals of different body sizes tended to express their
highest observed performances in response to different
aversive stimuli, and we infer that this finding reflects
differences in the selection pressures they experience. In both
species, larger spiders were more likely to express their highest
observed speeds in response to puffs of air. Given that birds
are primarily visual predators, it is plausible that larger spiders
are more likely to be spotted and attacked owing to greater
conspicuousness, and thus large individuals express higher
escape speeds in response to sudden jets of air. Certainly,
behavioral data in at least one of our test species (S. ocreata)
have demonstrated that individuals exhibit anti-predator
behavior in response to bird songs (Lohrey et al. 2009), and
a number of bird species have been observed feeding directly
on S. ocreata (Lohrey 2007); thus, we argue that birds are
likely some of the more important vertebrate predators of
these spiders. In contrast, smaller spiders are perhaps less
conspicuous to avian predators, but are no doubt more
susceptible to attacks by other spiders and predaceous insects,
which attack cursorially (i.e., similar to a prod). Hence, we
argue that the greater responsiveness of smaller individuals to
the prod stimulus may reflect a greater threat of arthropod
predation.

Given that larger individuals tended to exhibit higher speeds
in response to puffs of air, we predicted that larger individuals
would suffer greater mortality as a result of avian predation.

84

THE JOURNAL OF ARACHNOLOGY

c

o

c

o

I °

c -a

s<

> ra
^ 1-
o o

DQ JZ

o

re

£

a

0)

o

Total Survived Died Total Survived Died

c

0

C

re

I °

C -D

re <

N ^

^ re

o o

CQ ^

o

re

Q.

re

o

Total Survived Died Total Survived Died

Figure 3. — Relationship between individuals’ body length and survivorship in an avian predator exclusion experiment for the wolf spiders
Schizocosa ocreata (top) and Rahidosa rahida (bottom). Larger body sizes were associated with higher survivorship in the absence of large
vertebrate predators, and this advantage was lost with large vertebrate predators were present. Vertical shaded bars represent interquartile range
and vertical lines represent the 90"’ and lO"’ percentiles.

Concordantly, we found that larger individuals of both species
exhibited greater persistence in our avian exclusion treatments.
However, this advantage was diminished or reversed in our
avian inclusion treatment (Fig. 3). Thus, our results are
consistent with the interpretation that avian predation is a
major selective force on large spiders, and is one plausible
driver of escape speed in these animals. These findings are at
odds with the results of Wise and Chen (1999), whose data
suggested that avian predators were not infiuential in
Schizocosa population dynamics. In contrast, the data herein
and those of Lohrey and collaborators (Lohrey 2007; Lohrey
et al. 2009) do document significant effects/responses of

Schizicosci to the threat of avian predation. Our parallel
prediction that smaller individuals are more likely to fall
victim to predation by other arthropods remains untested.
However, it seems plausible that heterospecific arthropods
and/or cannibalism by larger conspecifics could impose
selection on small individuals. Consistent with this hypothesis,
smaller individuals in our predator exclusion treatment were
generally less likely to survive our 30-day selection period,
perhaps as a result of size-dependent cannibalism or predation
by other arthropods. Data from congeners of S. ocreata
provide some evidence that invertebrate predators impose
significant effects on individual survival (e.g., Punzo 1997).

DILLON & PRUITT— LOCOMOTOR BEHAVIOR MATCHES RISK

85

However, we caution that all of these results must be
interpreted as tentative, since our predator exclosure treat-
ments v/ere not replicated within each species.

Finally, our results bring to light an important methodo-
logical concern for the animal performance literature.
Specifically, the vast majority of studies on animal perfor-
mance assess individuals’ behavior in only a single ecological
context (typically an escape response) and use only a single
aversive stimulus such as chasing the animal with a broom/
brush (e.g., Prenter et al. 2012; Prenter et al. 2010; Pruitt
2010). A potential criticism of these studies is that they could
limit our understanding of animal performance by assessing
performance under a narrow range of conditions and thus lead
to erroneous conclusions.

ACKNOWLEDGMENTS

We are indebted to Susan E. Riechert for her assistance with
the experimental design and for allowing us to use her farm as
a study site. We would also like to thank Kyle Demes, Carl
Nick Keiser, and two anonymous reviewers for comments
on an earlier version of this manuscript. Voucher specimens
for our studies have been deposited in the University of
Tennessee’s invertebrate zoology collection.

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Manuscript received 18 September 2013, revised 28 December 2013.

2014. The Journal of Arachnology 42:86-104

A review and redescription of the cosmopolitan pseudoscorpion Chelifer cancroides

(Pseudoscorpiones: Cheliferidae)

Mark S, Harvey: Department of Terrestrial Zoology, Western Australian Museum, Locked Bag 49, Welshpool DC,
Western Australia 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 Professor: School of Animal Biology, University of Western Australia, Crawley, Western Australia 6009,
Australia; Adjunct Professor: School of Natural Sciences, Edith Cowan University, Joondalup, Western Australia 6027,
Australia. E-mail: mark.harvey@museum.wa.gov.au

Abstract. The taxonomy of the cheliferid pseiidoscorpion genus Chelifer Geoffroy 1762 is reviewed with a single
cosmopolitan species, Chelifer cancroides (Linnaeus 1 758), with the subspecies C cancroides orientalis Morikawa 1954 from
Japan newly synonymised with C. cancroides. Adults and the final two nymphal stages (tritonymph and deutonymph) are
redescribed based on numerous specimens from Europe, North America, Asia and Australasia. The large size variation
evident in the samples is documented. The latero-ventral process of the tarsal claws characteristically found in adults
(except leg I of the male) is lacking in nymphs, a pattern that is also confirmed in the genera Lissochelifer Chamberlin 1932,
Lophochernes Simon 1878 and Parachelifer Chamberlin 1932.

Keywords: Taxonomy, morphology, variation, new synonymy

The two pseudoscorpions described and named by Linnaeus
(1758) in the 10'*’ edition of Systema Naturae were both included
in the mite genus Acarus Linnaeus 1758. Acanis cancroides
Linnaeus 1758 was recorded from Europe and A. scorpioides
Linnaeus 1758 from America. Both species were transferred to
Pludcmgium Linnaeus 1758 by Linnaeus (1767) and then to
Scorpio Linnaeus 1758 by Fabricius (1775), but these genera are
now used exclusively in the arachnid orders Opiliones and
Scoipiones, respectively. In the meantime the genus Chelifer was
established in an anonymous work by Geoffroy (1762) to
accommodate A. cancroides and the mite A. longicornis Linnaeus
1758, and has been used as a valid genus ever since despite being
proposed in a publication that was eventually ruled as an
unavailable work due to the inconsistent use of binominal system
(International Commission on Zoological Nomenclature 1954).
The genus name was conserved in a ruling by the International
Commission on Zoological Nomenclature (1989) with the type
species confirmed as A. cancroides. Since Geoffroy’s publication,
346 additional species-group names have been described within
the genus Chelifer and all except C. cancroides orientalis
Morikawa 1954 have been either removed to other genera or
synonymized with C. cancroides (e.g., Chamberlin 1931b, 1932;
Beier 1932a, 1932b). The genus is currently monotypic (Harvey
2013), although 14 other species originally described in Chelifer
are regarded as nomina duhia (Harvey 2013).

Chelifer cancroides (Linnaeus 1758) is the most widely
distributed pseudoscorpion species in the world and has been
recorded from 58 countries in all major biogeographic regions
(summarized by Harvey 2013). Published records indicate that
it occurs in a variety of habitats, but is most frequently found
in human dwellings, associated buildings and bird’s nests (e.g.,
Artault de Vevey 1901; Ewing 1911; Levi 1948; Beier 1963;
Turienzo et al. 2010).

Chelifer cancroides is also one of the best known pseudo-
scorpions and has been used for a variety of studies including

growth, feeding (Vachon 1932, 1933, 1934a), respiration
(Slama 1995) and oogenesis and genital morphology (Vachon
1934b, 1936; Badian & Ogorzalek 1982; Badian 1987;
Jgdrzejowska et al. 2013).

Despite its widespread distribution and the abundance of
preserved specimens in some museum collections, there are
relatively few published illustrations of C cancroides and there
is no comprehensive modern description. To facilitate the
recognition of this species, C. cancroides is redescribed based
on a variety of specimens collected from four continents.

METHODS

This study is based upon the examination of specimens that
are lodged in the American Museum of Natural History, New
York (AMNH); Australian National Insect Collection,
Canberra (ANIC), California Academy of Sciences, San
Francisco (CAS); Florida State Collection of Arthropods,
Gainesville (FSCA); Naturhistoriska Riksmuseet, Stockholm
(NHRS); Queensland Museum, Brisbane (QM); Queen
Victoria Museum and Art Gallery, Launceston (QVMAG);
Tasmanian Museum and Art Gallery, Hobart (TMAG) and
Western Australian Museum, Perth (WAM). In addition to
the specimens of C. cancroides examined for this study
(Appendix 1 ), specimens of other cheliferid genera were also
examined to determine differences in tarsal claw morphology
between adults and nymphs (see Appendix 1).

Many specimens used in this study had been previously
prepared as permanent slide mounts in Canada Balsam by
other researchers including J.C. Chamberlin, C.C. Hoff and
W.B. Muchmore. The methods they used to prepare the slides
include the removal of soft body tissue through the immersion
of the specimen in potassium hydroxide (KOH) and dismem-
berment of the specimen to facilitate examination of
important morphological features as documented by Cham-
berlin (1931a) and Hoff (1949). Additional specimens exam-
ined were also studied using temporary slide mounts prepared

86

HARVEY— REVIEW OF CHELIFER CANCROIDES

87

by immersion of the specimen in lactic acid at room
temperature for several hours to days and mounting them
on microscope slides with 10 or 12 mm coverslips supported
by small sections of 0.25, 0.35 or 0.5 mm diameter nylon
fishing line. After study the specimens were returned to 75%
ethanol, with the dissected portions placed in 12 X 3 mm glass
genitalia microvials (BioQuip Products, Inc.). Specimens were
examined with a Leica MZ-16A dissecting microscope and an
Olympus BH-2 or a Leica DM2500 compound microscope,
the latter fitted with interference contrast, and illustrated with
the aid of a drawing tube attached to the compound
microscopes. Measurements were taken at the highest possible
magnification using an ocular graticule. Terminology and
mensuration mostly follow Chamberlin (1931a), with the
exception of the nomenclature of the pedipalps, legs and with
some minor modifications to the terminology of the trichobo-
thria (Harvey 1992), chelicera (Judson 2007) and faces of the
appendages (Harvey et al 2012). Terminology for the male
genitalia are taken from Vachon (1938b) and Legg (1975).

The length and width of the pedipalpal femur and chela
were measured for every specimen, even though occasionally
one or more measurements were not possible due to missing,
damaged or poorly aligned appendages. Some specimens,
including ail of the Asian specimens (which included the
smallest specimens; see “Variation and the identity of Chelifer
cancroides orientalis”), and the six largest and the six smallest
specimens of each sex (based on chela length) of the larger
group were measured in detail to prepare the species
description and capture the greatest range of variation.
Additional features such as the number of setae on the
tergites and the posterior margin of the carapace were also
recorded for these specimens. Means and standard deviations
for the pedipalpal femur and chela measurements listed above
were calculated using the AVERAGE and STDEVA functions
in Excel (Microsoft Office Professional 2010). The same
program was used to prepare Figs. 39-44.

SYSTEMATICS

Family Cheliferidae Risso 1827
Chelifer Geoffroy 1762
Chelifer Geoffroy 1762:617-618.

Obisium Illiger 1798:501 (synonymised by Westwood 1836:10)
(see Judson 2012 for the nomenclatural history of this
genus).

Type species. — Chelifer: Acarus cancroides Linnaeus 1758,
by subsequent designation of Latreille, 1810:484.

Obisium: Acarus cancroides Linnaeus 1758, by subsequent
designation of Westwood, 1836:10.

Diagnosis and description, — See below under C. cancroides.
Remarks. — The genus-group name Chelifer was first pro-
posed by Geoffroy (1762) in an anonymous publication that
was not strictly binominal. The publication was placed on
the International Commission of Zoological Nomenclature’s
Official Index of Rejected and Invalid Works in Zoology in
1954 (Opinion 228). After receiving a submission to validate
the name Chelifer (Harvey 1987), the name was conserved by
the Commission in Opinion 1542 (International Commission
on Zoological Nomenclature 1989). Geoffrey’s (1762) publi-

cation was later placed on the Official List of Works Approved
as Available for Zoological Nomenclature in Opinion 1754
(International Commission on Zoological Nomenclature 1994).
Geoffroy included two species in Chelifer, Acarus cancroides
Linnaeus 1758 and A. longicornis Linnaeus 1758, and the
Commission recognised Acarus cancroides as the type species by
subsequent designation of Latreille (1810).

Chelifer is the type genus of Cheliferidae and all coordinate
family-group names (Cheliferoidea, Cheliferinae and Chelifer-
ini). The tribe Cheliferini is characterized by the presence in
the male of a lateral rod in which the anterior margin is deeply
invaginated and usually contains a sclerotic rod-like process
(Fig. 32); coxal sacs of the male, when present, lack a clearly
differentiated medial atrium (Fig. 12) and the median
cribriform plates of the female are distinctly paired (Fig. 34)
(e.g., Beier 1932a; Chamberlin 1932; Hoff 1956). In contrast,
the other cheliferine tribe, Dactylocheliferini, has uninvagi-
nated lateral rods and lacks a sclerotic rod; the coxal sacs,
when present, usually have a differentiated atrium; and the
median cribriform plates are unpaired (e.g., Beier 1932a;
Chamberlin 1932; Hoff 1956). This tribal classification
represents one of the few within the Pseudoscorpiones
characterised solely by genitalic features. There are, however,
some cheliferid genera that have characteristics of both tribes,
including Mexichelifer Muchmore 1973 that has the invagi-
nated lateral rods and paired median cribriform plates
characteristic of the Cheliferini and the distinct atrium of the
coxal sac found in Dactylocheliferini (Muchmore 1973).

A distinctive feature of C. cancroides is the presence of a
latero-ventral process on the tarsal claws of adults (Fig. 26)
with the exception of leg I in males, which is modified to assist
in mating (Figs. 24, 25). Several other cheliferid genera also
possess such processes including the cheliferins Cubachelifer
Hoff 1946, Mesochelifer Vachon 1940, Parachelifer Chamber-
lin 1932 and Tyrannochelifer Chamberlin 1932 (e.g., Beier
1932a; Chamberlin 1932; Hoff 1946, 1956; Mahnert 1981;
Zaragoza 2009), and the dactylocheliferins Lissochelifer
Chamberlin 1932, Lophochernes Simon 1878, Mucrochelifer
Beier 1932 and Stenochelifer Beier 1967 (Chamberlin 1932;
Beier 1932a, 1967a). This process was first reported to be
lacking in the nympha! stages of C. cancroides by Hoff (1949),
an observation that is confirmed in the present study (Figs. 27,
28). It is also absent in nymphs of two different species
attributable to the genus Lissochelifer from northern Aus-
tralia, and a species of Lophochernes from Vanuatu (Figs. 35,
36). Hoff (1964) reported simple claws in nymphs of three
species of Parachelifer, in which the adults possessed claws
with ventral processes. This observation is also here confirmed
in a species of Parachelifer from Florida (Figs. 37, 38) and has
been observed by J. Zaragoza (in litt., 21 June 2012) in
nymphs of Mesochelifer fradei Vachon 1940. It is not known
whether the nymphs of the other cheliferid genera listed above
also lack such processes, but the pattern observed so far
suggests that they are completely restricted to adults.

Most cheliferids have five setae on the cheliceral hand. All
of the specimens of C. cancroides examined in this study have
four cheliceral setae. This state is not, however, unique within
the family. Species of the monotypic cheliferin genera
Kashimachelifer Morikawa 1957 and Mexichelifer, and the
monotypic dactylocheliferin genera Piignochelifer Hoff 1964,

THE JOURNAL OF ARACHNOLOGY

Figure F — Chelifer ccmcroides (Linnaeus), living male from near Prebbleton, New Zealand (image courtesy of B. Donovan).

Siiioc/ielifer Beier 1967 and Tetrachelifer Beier 1967 also bear
only four setae (Morikawa 1957; Hoff 1964; Beier 1967a;
Muchmore 1973). Some species of Rluicochelifer Beier 1932
have only four cheliceral setae (Mahnert 1980; Callaini 1983;
Dashdamirov & Schawaller 1995), but most have five setae.
Some species of Hysterochelifer Chamberlin 1932 and
Paisochelifer Hoff 1946 are reported to have either four or
five cheliceral setae, and some specimens also rarely have a
sixth seta located between setae shs and bs (Hoff 1950, 1956).
The missing seta in Chelifer, Piigiiochelifer, Rluicochelifer and
Tetrachelifer is shs, which can be readily determined by
comparison with other cheliferids with a full complement of
five setae. The seta Is appears to be absent in Kashhiiachelifer
based on illustrations of the chelicera by Morikawa (1957,
1 960), but the missing seta of Mexichelifer and Sinochelifer has
not yet been determined (Beier 1967a; Muchmore 1973).

Chelifer cancroides (Linnaeus 1758)

(Figs. 1-34, 39^4)

Acarus cancroides Linnaeus 1758:616.

Chelifer europaeus de Geer 1 778:355-357, plate 19, Figs. 14, 1 5.
Chelifer hermanni Leach 1817:49, plate 142, Fig. 3.

Chelifer sesamoides Audouin 1826:174-175, plate 8, Fig. 4.
Chelifer ixoides Hahn 1834:53, Fig. 140 (as Chelifer ixioides [sic]).
Chelifer graiudatus C.L. Koch 1843:37, Fig. 111.

Chelifer grandimanus C.L. Koch 1843:38-39, Fig. 778.
Chelifer rhododactyliis Menge 1855:32, plate 4, Fig. 6.

Chelifer serratus Stecker 1874:235-236.

Chelifer cancroides denlatiis Ewing 1911:73.

Chelifer cancroides orientalis Morikawa 1954:73-75, Figs. 2a-e.

New synonymy.

For a full bibliographic treatment, see Harvey (2013).

Material examined. See Appendix 1.

Diagnosis. — Adults of the genus Chelifer and the sole
included species C. cancroides can be distinguished from all
other cheliferids by the following combination of moipholog-
ical features, none of which, however, are unique to the genus:
cheliceral hand with 4 setae, with seta shs absent (Fig. 14); tarsal
claws of all legs, with the exception of leg I of males, with latero-
ventral process (Fig. 26); subterminal tarsal setae denticulate
(Figs. 24, 26); carapace with large setose tubercles (Figs. 9, 10);
carapace and tergites I-VII or VIII of male with distinct lateral
keels (Figs. 7, 9); coxa IV of males strongly arcuate and with a
large lateral process (Figs. 11, 12); coxa IV of males with coxal
sac, which lacks a differentiated atrium (Fig. 12); male genitalia
with rams horn organs and an anteriorly invaginated lateral rod
forming a median depression, in which lies a sclerotic rod
(Figs. 32, 33); and female genitalia with paired spermathecae
and median cribriform plates (Fig. 34).

Description. — Adults from near Pittsburg, Kansas. USA
(AMNH Hoff slides 186.2, 8-41 86.4): Color, sclerotized
portions generally dark red-brown, males generally darker
than females.

HARVEY— REVIEW OF CHELIFER CANCROIDES

89

Figures 2-6. — Chelifer cancroides (Linnaeus), male from Tasmania, Australia (QVMAG 13_53185): 2. Dorsal; 3. Ventral; 4. Detail of
carapace and abdomen, dorsal; 5. Carapace, dorsal; 6. Detail of coxae and abdomen, ventral.

Chelicera: With 4 setae on hand and 1 subdistal seta on
movable finger (Fig. 14); seta sbs absent; seta bs dentate,
remaining setae acuminate; seta bs shorter than others; with 2
dorsal lyrifissures and 1 ventral lyrifissure; galea of d and ?
with 5 terminal rami (Figs. 16, 17); rallum of 3 blades, the
most distal blade with several serrations on leading edge, other
blades smooth (Fig. 15); serrula exterior with 17 (d, ?) blades;
lamina exterior present (Fig. 14).

Pedipalp (Fig. 18): Surfaces of trochanter, femur and patella
coarsely granulate, chela including fingers mostly smooth,
prolateral face very lightly granulate; patella with 2 small sub-
basal lyrifissures; trochanter 2.25 (d), 1.97 (?), femur 5.67 (d),
5.18 (?), patella 4.20 (d), 3.87 (?), chela (with pedicel) 4.61 (d),
4.30 (?), chela (without pedicel) 4.39 (d), 4.09 (?), hand
(without pedicel) 2.07 (d), 1.91 (?) X longer than broad,
movable finger 1.16 (d), 1.20 (?) X longer than hand. Fixed

chelal finger with 8 trichobothria, movable chelal finger with 4
trichobothria (Fig. 21): eb and esb situated basaily, ib and ist
sub-basally, est and isb sub-medially, et and it siibdistally, est
situated slightly distal to isb, and et slightly distal to it; t
situated siibdistally, st situated midway between sb and /, and
sb situated much closer to b than to st; patch of microsetae
present on external margin of fixed chelal finger near et.
Venom apparatus present in both chelal fingers, venom ducts
long, terminating in nodus ramosus midway between et and
est in fixed finger and between / and st in movable finger
(Fig. 21). Chelal teeth (Fig. 21) slightly retrorse, becoming
rounded basaily; fixed finger with ca. 49 (d), 50 (?) teeth;
movable finger with ca. 52 (d), 48 (?) teeth; accessory teeth
absent.

Carapace (Figs. 9, 10): 0.98 (d), 1.00 (?) X longer than
broad; with 1 pair of rounded corneate eyes, which lack a

90

THE JOURNAL OF ARACHNOLOGY

Figures 7, 8. — Clielifer cancroides (Linnaeus), carapace and abdo-
men," left side: 7. Male (AMNH S-4186.3); 8. Female (AMNH S-
2707). Scale lines = 0.5 mm.

tapetum; with 91 (d), 98 (9) setae, arranged with 42 (d), 45 (9)
(including 4 near anterior margin) in anterior zone, 37 (d), 40
(9) in median zone, and 12 (d), 11 (9) in posterior zone;
postero-lateral corner of d with triangular protuberance
surmounted by 1 seta; with numerous lyrifisstires; with 2 deep
furrows, posterior furrow situated closer to posterior carapace
margin than to anterior furrow; anterior furrow situated ca.
0.43 (d), 0.55 (9) mm from anterior margin, and posterior
furrow situated ca. 0.15 (d), 0.10 (9) mm from posterior
margin.

Coxal region: Maxillae and coxae slightly granulate;
manducatory process rounded, with 2 apical acuminate setae,
median seta much smaller than lateral seta, with 1 small

sub-oral seta, and 19 (d), 20 (9) additional setae; median
maxillary lyrifissure rounded and situated submedially; poste-
rior maxillary lyrifissure rounded. Coxa IV of male (Figs. 11,
12) strongly arcuate and with large lateral processes; large coxal
sac without atrium; coxal sac glandular setae long. Chaetotaxy
of coxae I-IV: d, 11: 13; 43: ca. 70; 9, 13: 11: 15: ca. 60.

Legs: Trochanter and patella of leg III and IV with dorsal
setiferous granules; junction between femora and patellae I
and II strongly oblique to long axis; junction between femora
and patellae III and IV very angulate; femora III and IV much
smaller than patellae III and IV; femur + patella of leg IV 3.17
(d), 3.34 (9) X longer than broad; tarsus IV with sub-distal
tactile seta, TS ratio = 0.74 (d), 0.78 (9); subterminal tarsal
setae dentate (Figs. 24, 26); claws of legs (except legs I of d)
with latero-ventral process (Fig. 26); leg I of d with prolateral
claw curved and unmodified, and retrolateral claw slender
with dorso-medial sharp process (Figs. 24, 25); tarsus I of d
not thickened and without dorsal process or spur (Fig. 24);
arolium shorter than claws, not divided (Figs. 24, 26).

Abdomen: Tergites IV-XI of male and I-XI of female with
median suture line fully dividing each tergite (Figs. 7, 8);
tergites I-III without suture line (Fig. 7); sternites V-XI with
medial suture line fully dividing each sternite. Tergal
chaetotaxy: d, 11: 14: 16: 16: 19: 21: 20: 20: 19: 19: 14: 2; 9,
15: 17: 17: 19: 20: 22: 20: 22: 20: 19: 14: 2; tergites IV-X
biseriate, remainder uniseriate; all setae thickened and
strongly dentate; tergites I-VIII of d with lateral triangular
keel surmounted with 1 or 2 seta (Fig. 7). Sternal chaetotaxy:
d, ca. 100: (0) 20 [2+3] (0): (1) 6 (1): 15: 17: 16: 15: 14: 14: 12: 2;
9, 20:(0) 11 (0):(1) 10(1): 18: 17: 17: 18: 17: 18:8:2; uniseriate,
except for sternites II and III, and the lateral discal seta on
sternites IV-XI; most setae acicular, but setae on last two
tergites becoming slightly clavate and denticulate; d sternite II
with arcuate posterior margin (Fig. 29) and with numerous
setae, some bifurcate; d sternite III enlarged with arcuate
anterior margin and with scattered setae (Fig. 29), some
bifurcate (Fig. 30); glandular setae on d sternite III strongly
bifurcate; 9 sternite II with pair of median setae and numerous
pairs of posterior setae (Fig. 31). Spiracles with helix. Anal
plates (tergite XII and sternite XII) situated between tergite XI
and sternite XI. Pleural membrane finely wrinkled-plicate;
without any setae.

Genitalia: Male (Figs. 32, 33): lateral apodemes extending
laterally; rams horn organs present; lateral rods medially
joined and anteriorly invaginated forming a median depres-
sion with a sclerotic rod. Female (Fig. 34): with one pair of
lateral cribriform plates and 2 pairs of median cribriform
plates; with paired thin-walled spermathecae.

Variation: pedipalp: trochanter 1.82-2.07 (d), 1.67-2.18 (9),
femur 4.78-5.85 (d), 4.78-5.67 (9), patella 3.42^.20 (d), 3.61-
4.29 (9), chela (with pedicel) 3.86-5.07 (d), 3.56-5.10 (9), chela
(without pedicel) 3.88^.41 (d), 3.35-4.85 (9), hand (without
pedicel) 1.85-2.25 (d), 1.66-2.31 (9) X longer than broad,
movable finger 0.89-1.20 (d), 0.96-1.30 (9) X longer than hand
(without pedicel). Femur length mean = 1.201, standard
deviation (SD) = 0.087 (d), mean = 1.222, SD = 0.081 (9);
femur width mean = 0.224, SD = 0.018 (d), mean = 0.236, SD
= 0.017 (9); chela (with pedicel) length mean = 1.752, SD =
0.139 (d), mean = 1.795, SD = 0.1 16 (9); chela width mean =
0.398, SD = 0.043 (d), mean = 0.413, SD = 0.040 (9).

HARVEY— REVIEW OF CHELIFER CANCROIDES

91

Figures 9-17. — Chelifer cancroides (Linnaeus): 9. Carapace, dorsal, male (AMNH S^186.3); 10. Carapace, dorsal, female (CAS JC-
1598.01002); 1 1. Coxae IV, ventral, male (AMNH S-2706); 12. Left coxa, IV, showing coxal sac, ventral, male (AMNH SM186.3); 13. Coxae IV,
ventral, female (CAS JC-1598. 01002); 14. Left chelicera, dorsal, male (AMNH S^080.2); 15. Rallum, male (AMNH S-4186.4); 15. Galea, male
(AMNH S-3509); 16. Galea, female (AMNH S-4186.2). Scale lines = 0.1 mm (Figs. 15-17), 0.2 mm (Figs. 12, 14), 0.5 mm (Figs. 9-11, 13).

Carapace 0.91-1.04 (d), 0.95-1.09 (?) X longer than broad;
posterior margin with 11-18 (tJ), 10-14 (?) setae; keels usually
very prominent, but smaller specimens with keels barely
noticeable. Legs: femur + patella IV 2.72-3.84 (d), 3.07-3.75
(?) X longer than broad. Abdomen: tergites II and III of male
sometimes divided; tergal chaetotaxy: d, 12-16: 14-17: 14-20:
15-21: 16-26: 18-26: 19-26: 18-25: 18-23; 17-24; 12-20; 2; ?,
12-17: 14-18: 14-19: 14-20: 16-22: 17-23: 16-24: 17-22: 17-
22: 16-21: 12-16: 2; d sternite III with glandular setae ranging
from [3 + 1] to [4 + 5],

Dimensions: Male from near Pittsburg, Kansas (AMNH,
Hoff slide S^186.4) followed by all other males (where
applicable): Body length 2.78 (2.38-3.50). Pedipalps: trochan-
ter 0.505/0.225 (0.405-0.615/0.21-0.315), femur 1.105/0.195

(0.935-1.355/0.185-0.265), patella 0.925/0.22 (0.79-1.15/0.21-
0.285), chela (with pedicel) 1.590/0.345 (1.29-2.01/0.30-0.48),
chela (without pedicel) 1.515 (1.24—1.875), hand (without
pedicel) length 0.715 (0.635-0.895), movable finger length
0.830 (0.60-1.04). Chelicera 0.270/0.130, movable finger length
0.165. Carapace 0.895/0.910 (0.81-1.09/0.85-1.14); eye diam-
eter 0.060. Leg I: femur 0.305/0.175, patella 0.450/0.140, tibia
0.44/0.10, tarsus 0.39/0.085. Leg IV: femur + patella 0.84/0.265
(0.68-1.04/0.185-0.34), tibia 0.665/0.130, tarsus 0.480/0.085,
TS = 0.355.

Female from near Pittsburg, Kansas (AMNH, Hoff slide S-
4186.2) followed by all other females (where applicable): Body
length 3.63 (2.18-3.71). Pedipalps: trochanter 0.570/0.290
(0.445-0.64/0.215-0.335), femur 1.270/0.245 (1.005-1.42/

92

THE JOURNAL OF ARACHNOLOGY

Figures 18-28. — Chelifer ccmcroides (Linnaeus): 18. Left pedipalp, dorsal, male (AMNH S^186.3); 19. Left pedipalp. detail of prolateral face
of femur, male (AMNH S-4186.3); 20. Left pedipalp, detail of retrolateral face of trochanter, male (AMNH S^186.3); 21. Right chela, lateral,
male (AMNH S^186.3); 22. Left chela (reversed), lateral, tritonymph (CAS JC-257. 01002); 23. Right chela, lateral, deutonymph (AMNH S-
1995.3); 24. Right tarsus I, lateral, male (AMNH S^186.3); 25. Claws of left tarsus I. ventral, male (AMNH S-4186.3); 26. Right tarsus I,
lateral, female (AMNH S-4186.2); 27. Left tarsus I, lateral, tritonymph (CAS JC-257. 01002); 28. Right tarsus I, lateral, deutonymph (AMNH
S-1995.3). Scale lines = 0.1 mm (Figs. 19, 20, 24-28), 0.5 mm (Figs. 21-23), 1.0 mm (Fig. 18).

0.19-0.285), patella 1.045/0.270 (0.835-1.21/0.21-0.315), chela
(with pedicel) 1.870/0.435 (1.405-2.08/0.305-0.54), chela
(without pedicel) 1.780 (1.36-1.94), hand (without pedicel)
length 0.830 (0.705-0.90), movable finger length 1.000 (0.68-
1.125). Chelicera 0.315/0.165, movable finger length 0.210.

Carapace 1.070/1.075 (0.85-1.17/0.79-1.20); eye diameter
0.065. Leg I; femur 0.360/0.190, patella 0.540/0.165, tibia
0.505/0.100, tarsus 0.495/0.075. Leg IV: femur + patella 1.020/
0.305 (0.80-1.20/0.225-0.355), tibia 0.800/0.150, tarsus 0.580/
0.105, TS = 0.450.

HARVEY— REVIEW OF CHELIFER CANCROIDES

93

Figures 29-34. — Chelifer cancroides (Linnaeus): 29. Sternites III and IV, male (AMNH S-4186.4); 30. Detail of setae; 31. Sternites III and IV,
female (AMNH S^186.2); 32. Genitalia, ventral, male (AMNH S-2706); 33. Genital and coxal region showing distended rams horn organs,
ventral, male (CAS JC-1 523.01001); 34. Genital region, female (AMNH S-4186.2). Abbreviations: da = dorsal apodeme; ddv = dorsal
diverticulum; la = lateral apodeme; Ir = lateral rod; mr = median rod; rho = rams horn organs. Scale lines = O.i mm (Fig. 32), 0.2 mm (Figs. 29,
31, 34), 0.5 mm (Fig. 33).

Tritonymph from New York, USA. (CAS JC-257. 01002):
Color: sclerotized portions generally pale red-brown.

Chelicera: With 4 setae on hand and 1 subdistal seta on
movable finger; seta sbs absent; seta bs dentate, remaining
setae acuminate; seta bs shorter than others; galea broken,
rami not visible; rallum with 3 blades, distal blade with
spinules on anterior face, remaining blades smooth; serrula
exterior with 14 blades.

Pedipalp: Trochanter 2.03, femur 4.98, patella 3.67, chela
(with pedicel) 4.47, chela (without pedicel) 4.23, hand (without
pedicel) 2.04 X longer than broad, movable finger 1.09 x
longer than hand (without pedicel). Fixed chelal finger with 7
trichobothria, movable chelal finger with 3 trichobothria
(Fig. 22): eb, esb, ib and ist situated sub-basally, esb situated
closer to et than to esb, et situated closer to the end of the
finger than to it, t situated subdistally, and st situated midway

94

THE JOURNAL OF ARACHNOLOGY

Figures 35-38. — Tarsal claws showing lack of ventrolateral process in nymphs: 35, 36. Lophochernes sp. from Vanuatu (V/AM T1 18590): 35.
Left tarsus I, lateral, female; 36. Left tarsus I, lateral, tritonymph. 37, 38: Parachelifer sp. from Florida (CAS JC-209. 0200 1-2): 37. Tarsus IV,
lateral, male; 38. Left tarsus 1, lateral, tritonymph. Scale lines = 0.05 mm (Fig. 35), 0.1 mm (Figs. 36-38).

between b and t; patch of microsetae present on external
margin of fixed chelal finger near et. Venom apparatus present
in both chelal fingers, venom ducts long, terminating in nodus
ramosus midway between et and est in fixed finger and slightly
distal to t in movable finger. Fixed finger with 36 teeth;
movable finger with 40 teeth.

Carapace: 1.04 X longer than broad; with 1 pair of rounded
corneate eyes; with 50 setae, arranged with 24 (including 4
near anterior margin) in anterior zone, 18 in median zone, and
8 in posterior zone; with 2 deep furrows, posterior furrow
situated closer to posterior carapace margin than to anterior
furrow.

Coxal region: Chaetotaxy of coxae I-IV: 4: 4: 6: 8.

Legs: Femur + patella of leg IV 2.74 X longer than broad;
tarsus IV with sub-distal tactile seta, TS ratio = 0.72;
subterminal tarsal setae dentate; claws of legs without latero-
ventral process.

Abdomen: Tergal chaetotaxy: 10: 11: 11: 12: 11: 14: 13: 14:
12: 10: 4: 2; tergites without lateral keels. Sternal chaetotaxy:
2: (0) 6 (0): (1) 7 (1): 9: 10: 10: 9: 8: 6: 2: 2.

Dimensions: Body length 1.89. Pedipalps: trochanter 0.408/
0.201, femur 0.861/0.173, patella 0.704/0.192, chela (with
pedicel) 1.261/0.282, chela (without pedicel) 1.192, hand
(without pedicel) length 0.575, movable finger length 0.625.
Carapace 0.806/0.773. Leg I: femur 0.237/0.154, patella 0.352/
0.141, tibia 0.326/0.099, tarsus 0.326/0.086. Leg IV: femur +
patella 0.718/0.262, tibia 0.519/0.140, tarsus 0.384/0.090, TS =
0.275.

Dentonymph from Jensen. Utah, USA. ( AMNH Hojf slide S-
1995.3): Color, sclerotized portions generally pale red-brown.

Chelicera: With 4 setae on hand and 1 subdistal seta on
movable finger; seta sbs absent; seta bs dentate, remaining
setae acuminate; seta bs shorter than others; galea with 4 distal
rami; rallum with 3 blades, distal blade with spinules on
anterior face, remaining blades smooth; serrula exterior with
13 blades.

Pedipalp: Trochanter 1.75, femur 4.83, patella 2.46, chela
(with pedicel) 4.61, chela (without pedicel) 4.43, hand (without
pedicel) 2.17 X longer than broad, movable finger 1.08 X
longer than hand (without pedicel). Fixed chelal finger with 6
trichobothria, movable chelal finger with 2 trichobothria
(Fig. 23): eb situated basally, ib and ist subbasally, esb and it
submedially, et subdistaliy, it situated closer to est than to et; b
situated basally and t situated subdistaliy; patch of microsetae
present on external margin of fixed chelal finger near et.
Venom apparatus present in both chelal fingers, venom ducts
long, terminating in nodus ramosus midway between et and it
in fixed finger and distal to t in movable finger. Fixed finger
with 37 teeth; movable finger with 38 teeth.

Carapace: 0.96 X longer than broad; with 1 pair of rounded
corneate eyes; with 39 setae, arranged with 21 (including 4
near anterior margin) in anterior zone, 14 in median zone, and
4 in posterior zone; with 2 deep furrows, posterior furrow
situated closer to posterior carapace margin than to anterior
furrow.

Coxal region: Chaetotaxy of coxae I-IV: 4: 5: 4: 4.

Legs: Femur + patella of leg IV 2.87 X longer than broad;
tarsus IV with sub-distal tactile seta, TS ratio = 0.68;
subterminal tarsal setae dentate; claws of legs without latero-
ventral process.

HARVEY— REVIEW OF CHELIFER CANCROIDES

95

E

E '-35

3

E

CD 1-20

ro '''s

Q.

Q.

Q.

0.95

Chelifer cancnoides, males

•'ll

i ■ ■ ■“

l;!: .

■ s

39

1.45

1-40

E 1.35
— I 1.30
p 1.25

76 1 15

CO 1 10

Q.

^ 1.05
®

0_ 1-00
0.95

Chelifer cancroides, females

■ ' ■ I " ^

. li"'

” " - I I !

40

0.17 0.18 0.19 0.20 0.21 0.22 0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.3 0-1^ 0.19 0.20 0.21 0.22 0.23 0.24 0.25 0.26 0.27 0.28 0.29 0.30

Pedipalpa! femur W (mm)

Pedipalpal femur W (mm)

2.20
'g' 2.10

E 2-00

_J 1.90

"ffl 1-80
o

1.70

ffl

Q. 1.60
.C

1.50

o

Chelifer cancroides, males

B"

■ ■ nil

i .■.L'l': ' ■

■■■ ■

41

0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 0.52 0.54

Chela (with pedicel) W (mm)

E

200
"Z? 1-90

0 1-20
CL

^ 1.60

1.40

03

^ 1.30

o

Chelifer cancroides, males

■ ■iB

„ ."ill

43

0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45

Pedipalpal femur L (mm)

E 2 00

-g 1.80

'-a

Q.1.60
£ 1.50

5

6

Chelifer cancroides, females

■ ■ b"""
.-■"-■IB s® ■ :

5 iiIHi ■

I ■ ■

■■ ■

42

0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 0.52 0.54

Chela (with pedicel) W (mm)

g 2.10

E

_ 2.00
1.90

8

“g 1.70

Q-

^ 1.60

CD 1-40
^ 1-30

O

1.20

1.10

Chelifer cancroides, females

” .a*?'-

44

0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25 1.30 1.35 1.40 1.45

Pedipalpal femur L (mm)

Figures 39^4. — Size variation in adult specimens of Chelifer cancroides (Linnaeus): 39, 40. Pedipalpal femur length versus width; 41, 42.
Pedipalpal chela (with pedicel) length versus width; 43, 44. Pedipalpal chela length (with pedicel) versus femur length. Asian specimens = open
symbols; x = unknown locality; others = closed symbols. Measurements taken from Morikawa (1954) for specimens of C. cancroides
orientalis (triangles).

Abdomen: Tergal chaetotaxy: 6: 6: 6; 6: 6: 7: 6: 7: 6: 6: 6: 2;
tergites without lateral keels. Sternal chaetotaxy: 0: (0) 4 (0):
(1)4(1): 6: 6: 6: 7: 6: 6: 5: 2.

Dimensions: Body length 2.07. Pedipalps: trochanter 0.285/
0.163, femur 0.608/0.126, patella 0.505/0.205, chela (with
pedicel) 0.945/0.205, chela (without pedicel) 0.945, hand
(without pedicel) length 0.445, movable finger length 0.482.
Carapace 0.635/0.661. Leg I: femur 0.176/0.108, patella 0.265/
0.102, tibia 0.235/0.071, tarsus 0.260/0.065. Leg IV: femur +
patella 0.513/0.179, tibia 0.371/0.100, tarsus 0.287/0.070, TS =
0.195.

Remarks. — Type material of Acarus cancroides.- The orig-
inal description of Acarus cancroides by Linnaeus (1758) was a
few words of Latin: “A[carus]. antennis cheliformibus,
abdomine ovato depresso” (i.e., an Acarus with chelate
pedipalps, abdomen ovate, flat), which fails to provide any
diagnostic features by which the species can be recognized.
Apart from any specimens that Linnaeus (1758) may have had
access to, the type series also consists of those specimens
examined by the authors cited by Linnaeus (1758) listed under
the name A. cancroides (International Commission on
Zoological Nomenclature 1999, Article 72.4.1). These five

96

THE JOURNAL OF ARACHNOLOGY

literature sources (Frisch 1730; Seba 1734; Linnaeus 1746;
Rosel 1755; Clerck 1758) referring to a species he believed was
A. cimcroides are here discussed in chronological order.

Frisch (1730) contains two pages of text discussing ‘Die
Scorpion-Spinne’, along with a very rudimentary drawing of a
pseudoscorpion. The long pedipalps suggest that the species
may indeed represent C. cancroides, but the provenance of the
specimen is uncertain and the identification is somewhat
tenuous. This citation was also listed by earlier Linnaeus
volumes including Linnaeus (1746). The whereabouts of
Frisch’s collection - if it still exists - is unknown.

Seba (1734) presents a small illustration of an unidentifiable
pseudoscorpion along with a Latin description and a Dutch
figure caption that specifically mentions pseudoscorpions
living in old walls and old wood. The habitat data suggests
that he was referring to C. cancroides. Albertus Seba (1665-
1736) resided in Amsterdam after moving there in 1696, and it
is possible that the pseudoscorpion specimens were obtained
locally. Much of Seba’s early collections were purchased by
Peter the Great in 1716 and transferred to Saint Petersburg,
forming the basis for the Kunstkamera Museum (Engel 1937).
Seba later developed a second collection, which may have
included the pseudoscorpion illustrated in his 1734 volume.
After his death the collection was auctioned to a variety of
parties, with only a small proportion of it nowadays traceable
(Boeseman 1970). The fate of any pseudoscorpions that might
have been present in Seba’s collection is unknown.

Linnaeus’ Fauna Svecica (1746) was an early companion
volume for Systeina Naturae, the first edition of which was
printed in 1735 (Linnaeus 1735). Pseudoscorpions were listed
in his group “Acarus” as “Scorpio-araneus” (Linnaeus 1735).
Under Acarus cancroides, Linnaeus (1758) refers to Species
1187 of Fauna Svecica which is “ACARUS pedibus primi
paris cheliformibus’’ (= “a mite with first pair of legs
cheliform’’), and the Latin text accompanying the entry
translates as “lives in houses that have been closed for a long
time, not exposed to air, in chests and cellars” and “easily
recognized from the rest by the crablike claws of the first legs
and by the backwards gait, living on book lice” (FI.D.
Cameron, in litt. March 2012). The reference to book lice can
be traced through his original Latin expression “pediculo ligni
antiqui” which is the name applied to Species 1168 in
Linnaeus (1746). This species was later described as Tenues
pulsatorium Linnaeus 1758, which is nowadays known as
Trogium pulsatorium (Linnaeus 1758), a small psocopteran,
which is a well-known minor pest in houses and other human
facilities where they feed on fungal hyphae (e.g., Hall 1988;
Smithers 1996; Turner & Ali 1996; New 2005).

Rosel (1755) provided nearly three pages of text describing
the habits of a pseudoscorpion, which was depicted in
superbly detailed color paintings (Tab. LXIV) of a male,
female and a brood-sac. This pseudoscorpion has all the
hallmarks of C. cancroides, including the general habitus and
the proportions of the pedipalpa! segments, and there is little
doubt of its identity. The illustrations are included in a single
plate entitled “Scorpio Minimus”, but this Latin name does
not appear in the text. After Rosel’s death in 1759, the book
series was updated and translated into Dutch by C.F.C.
Kleeman. These volumes lack any mention of a publication
date, but the entire four-volume series was published between

1764 and 1768. The precise publication date of the third
volume, which contains the section on pseudoscorpions, is not
known. This volume (Rosel von Rosenhof «fe Kleemann [1764-
1768?]) reprinted the same figure (Tab. LXIV) that was
originally printed by Rdsel (1755). This figure was once again
captioned “Scorpio Minimus” and like the earlier volume this
name is not mentioned in the text. Although Rosel’s original
publication (Rdsel 1755) predates the starting point of
zoological nomenclature in 1758 (International Commission
on Zoological Nomenclature 1999), the Dutch version (Rosel
von Rosenhof &. Kleemann [1764-1768?]) may be deemed an
available work and the name “Scorpio Minimus” may be
deemed to be an available species-group name. However, there
is ample evidence elsewhere in the volume indicating that the
figure headings were simply Latin translations of Dutch
vernacular names, with “Scorpio Minimus” arising from the
Dutch expression “Den Kleinsten Scorpioen” (Rosel von
Rosenhof & Kleemann [1764-1768?], p. 317). Elsewhere in the
same volume there are various different freshwater crusta-
ceans depicted by Rosel (1755) and Rosel von Rosenhof &
Kleemann ([1764—1768?]) under the name “Astacus Fluviati-
lis”. It is clear that these names are simply Latin translations
of vernacular names and should not be treated as available
species-group names. Indeed, I can find no instance of Rosel
von Rosenhof & Kleemann being used as the author of any
animal species. The specimens that may have formed the basis
for Rosel’s plates cannot be traced, and it is assumed that
Rdsel’s collections are lost, as are those of Kleemann (Horn
et al. 1990).

Linnaeus (1758) listed Clerck (1758) as the fifth and final
bibliographic citation for Acarus cancroides. Clerck (1758)
published small paintings of a pseudoscorpion and a
harvestman that he included in the Swedish and Latin text
under an entry on “the so-called two-eyed spiders” (transla-
tion). Neither the pseudoscorpion nor the harvestmen were
scientifically named (Holm 1978), unlike the spiders treated
elsewhere in the volume which are deemed by the International
Commission on Zoological Nomenclature to be the only
animal names that predate Linnaeus (1758). The pseudoscor-
pion is not easily recognizable, but it may be the neobisiid
Neobisiwn carcinoides (Hermann 1804), which occurs in
Sweden (e.g., Tullgren 1899; Lohmander 1939; Harvey
2013). There are no pseudoscorpions in the Clerck collection
lodged in the Swedish Museum of Natural History, Stockholm
(Dr T. Kronestedt, in litt. 15 May 2012), and any specimens
examined by Clerck are regarded as lost.

With the loss or unavailability of specimens used by Frisch
(1730), Seba (1734), Rdsel (1755) and Clerck (1758), some of
which are unlikely to represent C. cancroides, the only possible
type specimens are those in Linnaeus’ own collection. The only
surviving known specimens apparently examined by Linnaeus
are lodged in the collection of the Linnean Society of London,
bearing the numbers 7004 and 7005. Images of these specimens
are provided on the Linnean Society’s website (http;//www.
linnean-online.org/24329/ and http://linnean-oiiline.org/24330/,
accessed 27 February 2013). Two images are provided of
specimen 7004, one of the body and the other of the right
pedipalp and labels. The specimen lacks the left pedipalp, and is
pinned with a standard entomological pin through the middle
of the body, damaging or distorting much of the specimen. Dr

HARVEY— REVIEW OF CHELIFER CANCROIDES

97

M. Judson (in litt., 7 August 2013) kindly informed me that he
has examined this specimen, which is a nymph. The two labels
include an old hand-written label “cancroides” and a more
recent printed or typed label “6 Chelifer cancroides (L.)”.
Specimen 7005 is also pinned through the middle of the body,
and is accompanied only by a single printed or typed label “7
Chelifer cancroides (L.)”. It lacks both pedipalps apart from the
left trochanter and femur which are attached to the body. Dr
Judson confirms this specimen is a female. Little morphological
data can be obtained from the images of the two specimens to
ascertain whether these specimens conform to modern diagno-
ses of the species.

One of these specimens was apparently examined by O.P.-
Cambridge (1892), who compared it to specimens collected
from human edifices in Britain and deemed them to be
conspecific with C. cancroides. O.P. -Cambridge (1892, p. 221)
clearly referred to a single specimen in the Linnean Society
collection but it is impossible to ascertain to which specimen
he was referring. O.P. -Cambridge (1892) regarded Chelifer
hermanni Leach 1817 to be distinct from C. cancroides, citing
its slightly smaller size, more slender pedipalps and different
habitat, occurring under tree bark rather than associated with
humans. Chelifer hermanni has since been treated as a
synonym of C. cancroides (e.g., Kew 1911; Beier 1932a).
These specimens are considered to represent the only surviving
syntypes of Acarus cancroides, and further examination is
required to obtain more accurate measurements and observa-
tions on their morphology.

The provenance of Linnaeus’ specimens of Acarus can-
croides is not certain, as he only stated “Habitat in Europae
umbrosis suffocatis”, which can be translated as “lives in dark
constricted places of Europe” (H.D Cameron, in litt. March
2012). Linnaeus (1746) specifically mentioned these pseudo-
scorpions living in houses and feeding on psocopterans, and it
is possible that his material was found inside buildings.
Linnaeus resided for most of his life in Sweden but spent some
time abroad, principally in Harderwijk, nowadays located
within the Netherlands (Blunt 1971). Chelifer cancroides is
commonly found in or near human dwellings (e.g., Beier 1963;
Mahnert 1981; Zaragoza 2009), and it is not uncommon in
southern Sweden where it has been frequently recorded from
houses (e.g., Tullgren 1899, 1906; Lohmander 1939). However,
the provenance of the specimens in the Linnean collection is
uncertain, although Linnaeus clearly stated they came from a
European location.

The identities of the other described species of Chelifer
traditionally treated as synonyms of C cancroides (listed
above) are slightly doubtful, as in many cases the type material
is lost or has not been examined by recent authorities on the
group. Indeed, some may actually represent specimens of
Mesochelifer ressli Mahnert 1981 rather than C. cancroides
(see Mahnert 1981; Zaragoza 2009). Exceptions include the
type material of C. hermanni, which was examined by O.P.-
Cambridge (1892) and who, as mentioned above, suggested it
represented a distinct species. Kew (1911) and later authors
treated C. hermanni as a synonym of C cancroides.

Chelifer europaeus is traditionally listed as a synonym of C.
cancroides and appears to have been introduced as a synonym
of the latter species (de Geer 1778). However, Welter-Schultes
& Wieland (2012) have recently proposed that volumes 3-7

of Memoires pour servir d I’histoire des insectes and the
companion publication by Retzius (1783) be treated as
available works, but that the majority of the polynominal
names proposed in them be suppressed. Chelifer europaeus was
not listed amongst the names to be suppressed.

Variation and the identity of Chelifer cancroides orientalis:
Substantial size variation was found amongst the adults
examined for this study, with the chela (with pedicel) ranging
from 1.24—2.02 mm in males and 1.38-2.08 mm in females
(Figs. 39^4); the largest specimens are approximately 50%
larger than the smallest specimens. The smallest adults include
all of the specimens from East Asia (China and specimens
intercepted in Florida but originally from South Korea), three
males intercepted on ships in Australia, five specimens from
North America (one each from Canada, Indiana, New York,
Oregon and Tennessee) and one with no collection data
(Figs. 39^4). There are, however, no other apparent mor-
phological features that unequivocally separate these speci-
mens from the others. Males of C. cancroides typically have
triangular keels on the postero-lateral corners of the carapace
and the anterior tergites (Fig. 7). Two of the smallest male
specimens examined have highly reduced carapaceal and tergal
keels. Both specimens have the process on the claw of leg I in a
more dorsal position but this may be an artifact of the slide
preparation. Curiously, these small specimens (CAS JC-
2234.01001 and ANIC) were both taken in quarantine
samples. Other small male specimens taken at quarantine
(other ANIC specimens) have normal shaped keels and tarsal
claws, as do the other small males observed in this study.

The smaller specimens from Asia coincide with the
description and dimensions provided by Morikawa (1954)
for the specimens he used to describe C. cancroides orientalis
(Figs. 39^4). This subspecies was based on specimens
collected from Sapporo City, Hokkaido Prefecture, and
Mukaijima Island near Onomichi, Hiroshima Prefecture
(Morikawa 1954), and was later recorded from Asahikawa,
Hokkaido Prefecture (Morikawa 1960). The specimens from
Sapporo were taken from a honey-bee hive and those from
Mukaijima Island were collected “at a cliff by the seashore”
and “in the books” (Morikawa 1954, 1960). Unfortunately
these specimens are not lodged in a public institution and have
not been available for study (Dr H. Sato, pers. comm.).
Morikawa (1954) suggested that the specimens differed from
the nominate subspecies in several features including smaller
body length, carapace broader than long, and the pedipalps
very long in comparison with the body length. Distinguishing
taxa using body length meristics is extremely inadvisable, as
this measurement is easily affected by a variety of factors
including whether the specimen was gravid, how well fed the
specimen was, the mode of preservation and whether or not is
has been treated for permanent slide-mounting. Concentrated
preservatives will contract the abdominal membranes and
artificially foreshorten the length of the specimen. Compara-
tive measurements of complete structures such as the carapace
or individual pedipalpal segments provide more reliable data
to discriminate between species. Such measurements are,
however, also subject to alteration if the structure is flattened
or spread during the slide preparation process. The flattening
is especially noticeable if the coverslip is not supported in some
fashion such as by thin glass rods, glass beads or small strands

98

THE JOURNAL OF ARACHNOLOGY

of fishing line. Such distortion of the carapace will produce
slightly altered carapaceal ratios.

The few specimens examined for this study from East Asia,
as well as the type specimens reported by Morikawa (1954),
have slightly smaller and narrower pedipalps than most of the
remaining specimens from Europe, Russia, North America
and Australasia (Figs. 39^4). As there are no other detectable
differences between the Asian and non-Asian populations, C.
cancr aides orienialis is here regarded as a junior synonym of C.
caiicroides. Further specimens of the Asian populations are
required to better document the intraspecific variation
observed in this study.

Postembryonic development. — Two of the three nymphal
stages are represented in the material examined for this study,
including several tritonymphs and deutonymphs. The number
and position of the individual trichobothria of these nymphs
and the adults (Figs. 22, 23) are identical to those presented by
Vachon (1934c), who also documented the protonymph. Other
morphological features are also similar including the number
of setae on the posterior zone of the carapace, 4 in
deutonymphs {n = 1), 8 in tritonymphs (/? = 1), and 10-18
in adults; Vachon (1934c) reported 6-9 in deutonymphs, 8-12
in the tritonymph, and 11-16 in adults.

Distribution. — Chelifer cimcroides is widely distributed and
frequently recorded in Europe, Central Asia, North Africa
and North America. There are, however, far fewer records in
other regions of the world and documentation of specimens
from the southern temperate regions are even scarcer. The few
sub-Saharan African records include the Democratic Republic
of Congo (Zaire) (e.g., Beier 1955; Beier 1959), Ethiopia
(Simon 1904), Kenya (e.g., Beier 1944, 1967b; Mahnert 1988),
Malawi (Beier 1944), South Africa (e.g., Ellingsen 1910; Beier
1929; Hewitt & Godfrey 1929) and Tanzania (Beier 1944).
Millot (1948) recorded a species from Madagascar that was
claimed to be very close to C. caiicroides, which Legendre
(1972) appears to have accepted as C. caiicroides. The actual
identity of this material should be checked to ascertain its
status. Similarly, there are few records from Central and South
America with specimens only reported from Argentina (Simon
1895; Ceballos & Ferradas 2008), Brazil (Ellingsen 1910),
Chile (Simon 1887, 1895; Cekalovic K. 1976;), Cuba (Banks
1909; Franganillo Balboa 1936) and Mexico (Villegas-Guz-
man & Perez 2005).

The only East Asian records are from the Kamchatka
Peninsula, Russia (Redikorzev 1935), Japan (Morikawa 1954,
1960), Mongolia (Krumpal & Kiefer 1982) and Vietnam (Beier
1951, 1967a), but among the specimens examined for this
study were two females collected in Tsinan’ (now Jinan), in
Shandong Province, China, which represents the first record
of C. caiicroides from China. The specimens from Kamchatka
identified by Redikorzev (1935) were examined and can be
confirmed to be correctly identified as C. caiicroides. Also, a
pair collected in Florida in “straw scuffs from South Korea”
represents the first record of C. caiicroides from that country.

The only undisputed published records from the Austral-
asian region are of several specimens collected in New
Zealand, which were taken from “timber in insectary” at
Owairaka (North Island) in 1945 and from a “nest of Stiiriiiis
vulgaris" at Kaikoura (South Island) in 1971 (Beier 1976). A
newly identified female from New Plymouth (North Island)

collected in 1924 from an ants’ nest (CAS JC-544.01001)
represents the earliest recorded specimen from the Austral-
asian region. Recently, large numbers of C. cancr aides were
obtained from nests of the lucerne leafcutting bee Megachile
rotimdata (Fabricius 1787) in Christchurch (South Island) (B.
Donovan, in litt. December 2012), some of which were
examined for this study. The bee is native to Eurasia and
was deliberately introduced into New Zealand in 1971 to assist
in the pollination of lucerne (Howlett & Donovan 2010).

An early record of C caiicroides from Mount Lofty, near
Adelaide, South Australia, by Beier (1930) was discounted by
Harvey (1981) who regarded the identification of the single,
apparently juvenile, specimen (“1 semiad. ?”) as doubtful.
Harvey (1981) noted that C caiicroides was not included in
subsequent synopses of the Australian fauna by Beier (1948b,
1966), and it is likely that the specimen from Mount Lofty was a
misidentified member of the cheliferid genus Protochelifer Beier,
which is common in southern and eastern Australia. The four
male specimens of C. caiicroides reported here from the
Launceston region in northern Tasmania represent the first
undisputed occurrence of C. caiicroides from Australia. The
specimens were collected inside a house in 1930, from straw in
1977 and without habitat data in 1985, indicating that the species
is established in the area and has persisted for several decades.

Chelifer caiicroides has been reported from a variety of
states of the USA and provinces of Canada, and among the
specimens examined for this study are several new state or
provincial records: Connecticut, New Jersey, South Carolina,
Tennessee and Washington in the USA, and New Brunswick
and Saskatchewan in Canada.

The male specimen from near Baker, Oregon listed as C.
caiicroides by Benedict & Malcolm (1979) has been reexam-
ined and found to belong to the genus Parachelifer. However,
it was not possible to identify the specimen to species due to
the poor condition of the slide preparation.

Harvey (1991) listed distribution records from Ghana and
India, which were repeated in subsequent on-line catalogs
(e.g., Harvey 2011). This Ghana record cannot now be verified
and is presumed to be an error by Harvey (1991). The Indian
record was based on Sharma & Sharma (1975), who reported
many specimens of a species suggested to represent a new
species of Chelifer from Jammu and Kashmir State in
northern India, principally from buildings. Until further
specimens are examined to verify the identification of these
populations, it seems prudent to remove C. caiicroides from
the list of recorded Indian species.

Habitat. — Chelifer caiicroides has been frequently reported
from houses and associated human edifices such as barns,
stables, bee hives and chicken coops (e.g., Vachon 1935; Beier
1939, 1948a, 1963; Hoff 1949; Levi 1953; Legg & Jones 1988),
where they feed on a variety of small invertebrates (e.g.,
Kastner 1931; Vachon 1932; Schlottke 1933, 1940; Levi 1953).

Records of Chelifer caiicroides from caves appear to be non-
existent, but Mr J. Zaragoza (in litt., 6 August 2013) has
informed me of a sample of seven males, five females, and a
tritonymph obtained by Berlese funnel extraction of dried bat
guano from Cova dels Moseguellos cave, Vallada, Valencia,
Spain (UTM 696400 4308700). The specimens do not exhibit
any particular adaptations to cave life, although some are in
the extreme range of the measurements given above.

HARVEY— REVIEW OF CHELIFER CANCROIDES

99

Specimens have been very occasionally found on humans
(Hermann 1804; Andre 1908, 1909a, 1909b; Artault de Vevey
1901; Vachon 1938a), and some have been observed to feed on
the bed bug Cimex lectidarius Linnaeus 1758 (Hemiptera:
Cimicidae). Frickhinger (1920) reported observations made by
a Geraian prisoner of war held in 1915-1918 near Yekaterin-
burg, Sverdlovskaya oblast, Russia, who watched specimens of
C. cancwides feeding on bed bugs. The pseudoscorpions emerged
at night from the walls of the dwelling, but were never
encountered in beds. Kaisila (1949) reported that C cancwides
has been observed feeding on bed bugs in Finland. A male C.
cancwides collected in Wageningen, Netherlands in 1927 (CAS
JC-281.01001) was taken from a '"Cimex infested house”, with
an adult bug mounted on the slide alongside the pseudoscorpion.

ACKNOWLEDGMENTS

I am very grateful to Bruce Halliday (ANIC), Lorenzo
Prendini (AMNH), Charles Griswold and Anthea Carmichael
(CAS), G.B. Edwards and Gay Fortier (FSCA), Gunvi Lindberg
(NHRS), Robert Raven and Owen Seeman (QM), Craig Reid
and the late Brian Smith (QVMAG), and Kirrily Moore
(TMAG) for access to the specimens lodged in their institutions.
H. Donald Cameron (University of Michigan) provided excellent
assistance with Latin translations, Juan Zaragoza (Universidad
de Alicante, Spain) provided infoiTnation on Mesochelifer fradei
and the Spanish cave populations, and Chris Buddie (McGill
University, Montreal, Canada), Barry Donovan (Christchurch,
New Zealand), the late Jacqueline Heurtault (Museum National
d’Histoire Naturelle, Paris, France), Izabela Jgdrzejowska
(University of Wroclaw, Poland) and Frantisek Stahlavsky
(Charles University, Prague, Czech Republic) kindly donated
specimens to the Western Australian Museum. Barry Donovan is
also thanked for permission to use his image of a living specimen
as Fig. 1, and Giulio Gardini (do dell’Universita degli Studi di
Genova, Italy), Mark Judson (Museum National d’Histoire
Naturelle, Paris, France), Volker Mahnert (Museum d’histoire
Naturelle, Geneve, Switzerland) and Juan Zaragoza (Alicante)
kindly provided comments on a draft of the manuscript.

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Manuscript received 4 August 2013, revised 23 September 2013.

APPENDIX 1

The specimens studied in this study.

Chelifer cancroides (Linnaeus 1758)

AUSTRALIA: Tasmania, 2 <5, Cressy, 6 December 1985, A. Marker
(QVMAG); 1 3, Launceston, 5 July 1930, in house (bath), collector
not stated (TMAG, J31 10); 1 3, Launceston, 18 March 1977, in straw
(one of many), A. Colvill (QVMAG, 13:6864).

CANADA: New Brunswick, 1 ?, Saint Johns, no date, ex R.V.
Chamberlin (CAS JC-1 166.01001 ); Ontario, 2 d, 2 9, Westport, 29
September 1973, guano, S. Peck (FSCA WM3482.01-2); 1 9, Lake of
the Woods, Sioux Narrows, April 2001, W. Kobel (FSCA
WM85 12.0 1001); Quebec, 1 9, Montreal, 30 September 2005, in
house, C. Cloutier (WAM T67083); Saskatchewan, 1 d, 1 9, Lady
Lake, 13 Oetober 1931, D.J. Buckle (FSCA WM2050).

CHINA: Shandong, 1 9, Tsinan [= Jinan], 7 December 1922, on
table, 2nd floor of house, Jacot (CAS JC-141 8.02001 ); 1 9, Tsinan
[=Jinan], 19 February 1925, in house, Jacot (CAS JC-141 8.01001);
Province unknown, 1 d, from China, at quarantine in New York, 2
November 1949, alive in shipment, dried orange peel, collector not
stated (CAS JC-2234.01001).

CZECH REPUBLIC: Hlavni niesto Praha, I 9, Praha, Leopoldova
Street, 12 May 1998, inside building, F. Stahlavsky (WAM T78673).

“CZECHOSLOVAKIA”: 1 9, at quarantine in Miami, Florida, 26
July 1948, in mail shipment, dry mushrooms, B.P. Stewart & R.C.
Watson (CAS JC-2 177.0 1001).

DENMARK: 1 3, no further locality, no date, collector not stated
(CAS JC-655.01001) (det. by Schiddte as Chelifer granulatus).

FRANCE: 1 d, 2 9, 1 tritonymph, no further data (WAM T78672).

GERMANY: 1 9, from Germany at Cleveland, Ohio, 3 May 1945,
in hay packing material, U.R. Kuhn (CAS JC-2022. 01001 ); 1 d, 1 9,
Germany, no further data, no date, A. Walcsuch (CAS JC-

1481.01001-2); 1 d, in wood wool in packing case from Germany;
intercepted in Australia, 30 September 1936 (QM W674).

ITALY: 1 d, ex Italy, at quarantine in New York, 15 September

1947, alive in garlic bulb, Fonner & Hidalgo (CAS JC-21 55.01001); 2
9, 1 tritonymph, ex Italy, at quarantine in New York, 29 October

1948, in basket of grapes. Inspector Plummer (CAS JC-218L01001-
3).

NETHERLANDS: Gelderland, 1 d, Wageningen. 20 December
1927, in Cimex infested house, F. Spruyt (CAS JC-281 .01001 ); 1 3, 1
9, Wageningen, 10 July 1928, from covers and straw of bee hives, F.
Spruyt (CAS JC-1474.01001-2).

NEW ZEALAND: Canterbury, 5 d, 1 km S. of Prebbleton, 19
November-20 December 2012, in nests of the lucerne leafcutting bee
Megachile rotundata in shed, B. Donovan (WAM T120948, T130746);
Taranaki: 1 9, New Plymouth, July 1924, in ant’s nest, W. Smith (CAS
JC-544.01001).

POLAND: 20 3, 10 females, 1 tritonymph, laboratory colony in
Wroclaw, originally from Lower Silesia, 7 March 2013, 1. Jgdrze-
jowska (WAM T130754-130755).

SOUTH KOREA; 1 d, 1 9, Florida; Palm Beach County: Farmer’s
Market, 12 March 1962, in straw scuffs from South Korea, R.A.
Long (FSCA WM3185.01001-2).

RUSSIA: Kamchatka Krai, 1 d, 2 9, Petropavlosk, 26 January 1921
(NHRS, GULI00000403).

SWEDEN: Blekinge, 1 3, Baggeboda, 19 June 1940, Butovitsch
(NHRS, GUL1000004105); Gotland, 1 3, Gotska Sandon, A. Jansson

(NHRS, GULI000004104); Smdland, 1 9, locality label not legible, 9 |

June 1946 (NHRS, GULI000004106); Province not determined, 2 3, A [
9, without precise data, 3 July 1946 (NHRS, GULI000004107); 1 I
tritonymph, without precise data (NHRS, GULI000004100); 2 d, 2 9, j
without precise data (NHRS, GULI0000040101, T. Thorell collection). !

USA: Alabama, 1 9, Lee County: Auburn, no date, R.V. I
Chamberlin (CAS JC-657.01001); Alaska, 1 3, Denali Borough:
Mount McKinley, [Denali] National Park, 21 February 1958, from
mouse feces in hotel, Corson (AMNH, Hoff S-3587); California, 1 9, |

El Dorado County: Herus, Placerville, 16 October 1912 (CAS JC-

662.01001) ; 1 3, Santa Clara County: Mayfield, no date, F. Sproyt

(CAS JC-279. 01001 ); 1 9, Santa Clara County: Stanford University,
no date, J.C. Chamberlin (CAS JC-1791. 01001); 1 3, Tulare County:
Visalia, no date, ex R.V. Chamberlin (CAS JC-1 760.01001);
Connecticut, 1 9, Fairfield County: Stamford, Bartlett Tree Research
Laboratory, 2 October 1945, S.W. Bromley (CAS JC-215L01001); 1
3, Tolland County: Storrs, University of Connecticut, herbarium,
November 1945 (AMNH, Hoff S-2634); District of Columbia, 1 9,
Washington D.C., no date, ex R.V. Chamberlin (CAS JC-286. 03001);
Florida, 1 9, Miami-Dade County; Miami, International Airport, 27 ‘

May 1960, on stable fioor, J.L. Weaver (FSCA Hoff 8^126); Idaho: 4

3, 3 9, Bear Lake County: Fish Haven, Bear Lake, 5 Sept. 1921,
‘under bark of balsam log’, ‘under bark of balsam post’ or ‘bark of
logs or slabs of fir’, J.C. Chamberlin, B. Cain (CAS JC-676. 01001-2,

5, JC-676. 02001-^); 2 d, 1 9, Canyon County; Notus, 25 August 1932,

W. Ivie (CAS JC-1350.01001-3); 1 d. Twin Falls County: Castleford ;
Plot, 1932, debris, site of old barn, D.E. Fox and R.L. Piemeisel (CAS
JC-1 178.02001-2); 1 9, Twin Falls County: Twin Falls, 28 May 1935, I
from house (CAS JC-919. 01001); Illinois, 1 9, Champaign County:
Champaign, 29 July 1940, attacking people, R. Lehman (CAS JC-
995.01000; 1 9, Chicago, 12 October 1910, A.W. Slocom (CAS, Hoff
6051-S-533); 1 9, Cook County: Glencoe, 22 June 1942, E. Best
(CAS, Hoff 6052-S-534); 1 3, Cook County: Glencoe, 12 September
1941, M. Best (CAS, Hoff 6049-S-531); 1 tritonymph, Effingham i
County: Shumway, 1 November 1937, L.E. Richter (CAS, Hoff 6047-
S-529); 1 3, Greene County; Roodhouse, 1938, from cattle, F.W.
Helm (CAS JC-1057. 01001); 1 9, Lake County: Waukegan, May
1940, H. Sorensen (CAS, Hoff 6055-S-536); 1 d, 1 9, north-central
Illinois, 24 May 1938, in dwelling, C.L. Metcalf (CAS JC-

1051.01001- 2); Indiana, 1 3, Kosciusko County: Winond Lake, 3 |

July 1927, on weeds, Nester (CAS JC^23. 01001); 1 3, LaGrange j
Country: Shipshewana, no date, under tin of chicken coop, Nester !
(CAS JC^ 10.01 00 1 ); 1 d, Monroe County: Bloomington, 27 May
1927, oak fence rail, Nester (CAS JC-419. 01001 ); 1 9, Monroe
County: Bloomington, 1 June 1927, in house, Nester (CAS JC-

421.01001) ; Kansas, 1 3, Crawford County: 4 miles E. and Vi mile S.
of Pittsburg, 2 June 1963, B. Branson (AMNH, Hoff SM187.1); 2 3, 1
9, Crawford County: 5 miles E. and 3 miles S. of Pittsburg, 22 May
1964, in barn, B. Branson (AMNH, Hoff SMI 86.2-4); 1 d, i
McPherson County: Mound Ridge, 24 August 1934, in flour mill, I
N.E. Good (CAS JC-832.02001); 1 d, 1 9, Riley County: Manhattan, li
3 July 1933, bottom poles of haystack, R.C. Smith (CAS JC-

1201.01001- 2); Kentucky, 1 3, Breathitt County: Quicksand, 25 June
1925, ex C.R. Crosby (CAS JC-1523.01001 ); Maine, 1 9, Hancock
County: Brookline, no date, ex R.V. Chamberlin (CAS JC- ,

1506.01001) ; Massachusetts, 2 9, Middlesex County: Groton, 8 June S

1937, in barn swallow nest, E.A. Mason (CAS JC-1040. 01001-2); :

Michigan, 1 3, Ingham County: East Lansing, July 1925, in house,
R.H. Pettit (CAS JC-542. 01001 ); Missouri, 1 3, Greene County;
Springfield, no date, ex R.V. Chamberlin (CAS JC-1202. 01001 );
Montana, 1 3, Garfield County; Jordan, 15 May 1927, collector not
stated (CAS JC-632. 01001); 1 3, Missoula County: Missoula, 1 May j
1958, H.F. Pollmann (AMNH, Hoff S-4256); 1 d, Ravalli County: '
Hamilton, 1 March 1934, W.L. Jellison (CAS JC- 1096.01 00 1 ); 1 9,
Ravalli County: Hamilton, 12 September 1932, laboratory, C.B.
Philip (CAS JC-1 1 12.01001); 1 9, Ravalli County, 20 May 1934, W.L. |

HARVEY— REVIEW OF CHELIFER CANCROIDES

103

Jellison (CAS JC-1 121.01001); 1 <?, no locality or date, Brunson
(AMNH, Hoff S-2530); 1 d, no locality or date, Brunson (AMNH,
Hoff S-2533); Nevada, 1 d, Elko County: Elko, 21 May 1935, in
flour, M.W. Menke (CAS JC-829.0I001); New Hampshire, 2 ?,
Grafton County: New Franconia, no date, ex R.V. Chamberlin (CAS
JC-1487. 01001-2); 1 <3, Grafton County: Wentworth, 20 April 1957,
H.M. van Deusen (AMNH, Hoff S-3509); New Jersey, 1 3, Bergen
County: Ramsey, Gertsch (AMNH, Hoff S-1482); 1 ?, Bergen
County: Ramsey, September 1948, Gertsch (AMNH, Hoff S-1518); 1
$, Bergen County: Ramsey, June 1941, Gertsch (AMNH, Hoff S-
1480); 1 3, Ocean County: Lakehurst, 2 October 1909 (AMNH, Hoff
S-2648.1); New Mexico, I 3, Bernalillo County: Albuquerque, 20
April 1961, from milk filter testing equipment (AMNH, Hoff S-
41.70); New York, 1 $, Albany County: Albany, 6 August 1914, sandy
woods, ex S.C. Bishop (CAS JC-1592.01001); 1 ?, Albany County:
Lincoln Pond, Huyck Preserve, Rensselaerville, 6 July 1949, J.C.
Bishop (AMNH, Hoff S-2655); 1 $, Albany County: Voorheesville,
27 June 1934, barn swallow nest, D. Stoner (CAS JC-824. 01001); 1 9,
Chenango County: New Berlin, 22 August 1916, collector not stated
(CAS JC-241.0100I); 1 $, Dutchess County: Poughkeepsie, 19 August
1939, J.H. Fulton (CAS JC-99 1.0 1001); 3 3, 4 9, Genesee County:
Bergen Swamp, 6 September 1965, collector not stated (FSCA
WM827. 01001-6, 8); 1 9, Monroe County: Penfield, July 1960, in
house, collector not stated (FSCA WM359.01001); 1 9, Nassau
County: Sea Cliff, no date, ex R.V. Chamberlin (CAS JC-298. 07002);
1 3, Nassau County: Sea Cliff, no date, ex R.V. Chamberlin (CAS
JC-298. 04001); 1 9, Nassau County: Sea Cliff, no date, ex R.V.
Chamberlin (CAS JC-298.07001); 1 9, 1 tritonymph, Nassau County:
Sea Cliff, no date (CAS JC-257.01001-2); 1 9, Nassau County:
Westbury, Long Island, June 1949, D.G. Nichols (AMNH, Hoff S-
2656); 1 9, New Y ork County: New Y ork (AMNH, Hoff S- 1 54 1 ); 1 9,
Oneida County: New Hartford, 12 March 1901, S.C. Bishop (CAS
JC-1688. 01001); 1 9, Steuben / Yates Counties: Lake Keuka, May
1904, June 1904, ex C.R. Crosby (CAS JC-1656.01001); 1 3, 1 9,
Suffolk County: Babylon, 14 September 1930, F. Spruyt (CAS JC-
1598.01001-2); 1 3, Sullivan County: Beaver Kill, 25 August 1947,
R.B. Fischer (AMNH, Hoff S-2657); 1 3, Sullivan County: Beaver
Kill, 14 July 1946, R.B. Fischer (AMNH, Hoff S-2654); 1 3, 1 9,
Tompkins County: Groton, February 1960, in hay barn, H. Dietrich
(AMNH, Hoff S-4080.1-2); 2 3, 1 9, Tompkins County: Ithaca, 1
August 1887 (CAS JC-661. 01001-3); 1 9, New York, Tompkins
County: Ithaca, April 1966, collector not stated (FSCA
WM899.01001); 1 3, Tompkins County: Slaterville (now Slaterville
Springs), 6 July 1929, P.R. Needham (CAS JC-1572. 01001 ); North
Carolina, 1 3, Currituck County: Shawboro, 14 April 1937, J.K.
Duncan (CAS JC-1027. 01001); Ohio, 1 3, Lawrence County: South
Point,20 April 1934, in poultry house, H.C. Mason (CAS JC-

855.01001) ; 1 3, Columbus, no date, in cellar under seed, C.M. Weed
(CAS JC-1541. 01001); Oregon, 1 3, Baker County: 10 miles W. of
Baker, 7 August 1963, J.S. Buckett (CAS, EB.E-581.01001); 1 9,
Baker County: Huntington, 5 May 1934, W.L. Jellison (CAS JC-

1109.01001) ; 1 9, Benton County: Alsea, November 1920, J.E. Davis
(CAS JC-1186.01001); 1 3, Benton County: Corvallis, 13 May 1936,
on human being, in bathroom, Wheeler (CAS JC-83 1.0 1001); 1 9,
Benton County: Corvallis, 14 May 1936, on human being, in
bathroom, N. Larson (CAS JC-831. 02001); 1 9, Benton County:
Corvallis, 20 April 1940, from house, S.J. Couper (CAS JC-

1087.01001) ; 1 9, Benton County: Corvallis, May 1936, Prof. Scullen
(CAS JC-1 185.01001); 1 9, Benton County: Corvallis, 20 April 1940,
from house, S.J. Couper (CAS JC-1087.01002); 1 9, Benton County:
Corvallis, 18 April 1938, E. Crumb (CAS JC-1082.0I001); 1 3,
Benton County: Corvallis, spring 1935, J.M. Pierson (CAS JC-

1875.01001) ; 1 9, Benton County: Corvallis, 26 May 1935, C.E. Cody
(CAS JC-1 872.01001); 1 3, Benton County: Corvallis, 14 June 1939,
in college building, V. Shattuck (CAS JC-9 15.02001); 1 9, Benton

County: Corvallis, 20 March 1937, in shaving soap container in
bathroom, D. Edwards (CAS JC-898.02001); 1 3, Clackamas County:
Wilsonville, no date, in house, G. Danforth (CAS JC-1 062.0 1001); 1
9, Clackamas County: Wilsonville, May 1938, from house, G.
Danforth (CAS JC-1062. 01002); 1 3, Deschutes County: Redmond,
28 April 1939 (AMNH, Hoff S-2025.1); 1 9, Deschutes County:
Redmond, 28 April 1939 (AMNH, Hoff S-2025.2); 1 3, Harney
County: P Ranch, 1 mile E. of Frenchglen, 12 May 1972, dung & hay,
E.M. Benedict (CAS, EB.E-32. 01001); 1 9, Jackson County:
Medford, 7 July 1935, L.G. Centner (CAS JC-1454.01001); 1 3,
Lane County: 2.5 miles E. of Cheshire, 4 December 1971, shed, hay,
nests and swallow debris, E.M. Benedict (CAS, EB-195. 01001); 1 3,
Marion County: Salem, 15 September 1945, from dead peach limb, J.
Schuh (CAS JC-2246. 01001); 1 9, Multnomah County: Gresham, 10
June 1944, found in bed in house, J. Schuh (CAS JC-205 1.0 1001); 1 9,
Wasco County: The Dalles, 23 June 1939, in house, presented by D.C.
Mote (CAS JC-850. 02001); 1 3, no further data, summer 1922, in
sugar bowl, B.C. Cain (CAS JC-680. 01001 ); South Carolina, 1 9,
Pickens County: Clemson, 21 October 1962, J.A. Payne (FSCA
WM528. 01001); Tennessee, I 9, Anderson County: Oak Ridge, 11
July 1960, A. Lawler (FSCA WM360.01001); Utah, 1 9, Box Elder
County: Park Valley, 9 September 1932, R.V. Chamberlin (CAS JC-

1605.01001) ; 1 3, Cache County: Logan, 8 August 1940, in chinchilla
nest, G.F. Knowlton (CAS JC-996.01001); 1 9, Cache County:
Logan, 20 March 1913, H.R. Hagen (CAS JC-627.01001); 1 9, Salt
Lake County: East Millcreek, no date, under board, old hen coop,
J.C. and O.W. Chamberlin (CAS JC-1209. 01001); 1 3, Salt Lake
County: Salt Lake City, no date, R.V. Chamberlin (CAS JC-

1212.01001) ; 9 3, 5 9, Salt Lake County: Salt Lake City, September
1921, in ruins of old hen coop, B.C. Cain (CAS JC-228. 01001, 2, 1 1-
19, 24-26); 1 3, Salt Lake County: Salt Lake City, 14 April 1923,
laboratory, ex R.V. Chamberlin (CAS JC-1 234.0 1001); 1 3, Salt Lake
County: Salt Lake City, September 1921, in old hen coop, B.C. Cain
(CAS, no number); 1 3 (genitalia & chelicerae only). Salt Lake
County: Salt Lake City, no date, collector not stated [CAS JC-
258.01020 (2 slides)]; 1 3, Salt Lake County: Salt Lake City, no date,
ex R.V. Chamberlin (CAS JC-1723. 01001); 1 9, 1 deiitonymph,
Uintah County: Jensen, 7 November 1952, ex nest of Peromyscus
maniculatus, D.E. Beck (AMNH, Hoff S-1995.1-3); 1 9, Utah
County: Goshen, no date, R.V. Chamberlin (CAS JC-282. 01001); 1
9, Washington County: Saint George, 1926, ex R.V. Chamberlin
(CAS JC-1226.02001); 1 3, Emery County: San Rafael, 20 April 1928,
A.M. Woodbury (ex R.V. Chamberlin) (CAS JC-1230.01001); 1 3, 1
9, Emery County: San Rafael River, 20 April 1928, A.M. Woodbury
(ex R.V. Chamberlin) (CAS JC-1215. 02001-2); Virginia, 1 3, Falls
Church City: Falls Church, no date, R.V. Chamberlin (CAS JC-
3.04001); Washington, 1 9, Pierce County: Puyallup, 27 March 1934,
W.W. Baker (CAS JC-1 8 12.0 1001); 1 3, Pierce County: Puyallup, 20
June 1932, C.W. Geteendaner (CAS JC-1923.01001); 1 9, Pierce
County: Puyallup, no date, L.L. Stitt (CAS JC-2101. 01001); 1 9, no
further data, no date, ex R.V. Chamberlin (CAS JC-1560.01001);
Wisconsin, 1 3, Crawford County: Prairie du Chien, June 1949, Levi
and Smethurst (AMNH, Hoff S-2706); 1 3, 1 9, Dane County:
Madison, Levi (AMNH, Hoff S-2717.1-2); 1 9, Dane County:
Verona, February 1947 (AMNH, Hoff S-2715); 1 3, Marathon
County: Wausau, 27 January 1950, in kitchen, Levi (AMNH, Hoff S-
2674); 1 9, Marathon County: Wausau, May 1952, in house, Levi
(AMNH, Hoff S-2707); 1 9, Oneida County: Hazelhurst, 19 August
1949, Levi (AMNH, Hoff S-2679); 1 9, Sauk County: Badger, April
1944, in house, Levi (AMNH, Hoff S-2675).

UNITED KINGDOM: England, 1 3, 1 9, Seabrook, Essex, no
date, C. Warburton (CAS JC-660. 01001-2).

NO DATA: 1 9, ex R.V. Chamberlin (CAS JC-1 536.02002);
2 3 (CAS JC-29.02001-2); 1 9, ex R.V. Chamberlin (CAS

104

THE JOURNAL OF ARACHNOLOGY

JC-1 528.01001); 1 <J, from the ship ‘Mararita’ at Newcastle, New
South Wales, Australia, 31 August 1964, R.G. Winks (ANIC); 1 S,
from the ship ‘Alexandros’ at Newcastle, New South Wales,
Australia, 5 September 1964, R.G. Winks (ANIC); 1 d, from the
ship ‘Alexandros’ at Geelong, Victoria, Australia, 18 September
1965, H. Caple (Department of Primary Industries Inspector)
(ANIC).

Lissochelifer sp. ex Western Australia

AUSTRALIA: Western Australia, 3 <3, 2 ?, Mt Trafalgar,
15°16'50"S, 125°04'05"E, 12 June 1988, under bark, B.Y. Main
(WAM T78668); 1 tritonymph, Mt Trafalgar, 15°17'S, 125°04'E,
June 1988, litter, J. Majer (WAM T78669).

Lissochelifer sp. ex Queensland

AUSTRALIA: Queensland, 11 d, 7 9, 2 tritonymphs, Palmerville
Station, 16°00'S, 144°05'E, 30 June 1997, under bark of rotting logs,
F.D. Stone (WAM T1 18592).

Lophochernes sp. ex Vanuatu

VANUATU: 1 9, 2 tritonymphs, Vanuatu, 28 August 2000
(quarantine intercept in Australia) (WAM T1 18590); 1 d, Vanuatu,
17 July 2000 (quarantine intercept in Australia) (WAM T1 18591).

Parachelifer sp.

USA: Florida, 1 3, 1 tritonymph, Ormond [probably Ormond
Beach], no date, R.V. Chamberlin (CAS JC-209. 0200 1-2).

2014. The Journal of Arachnology 42:105-1 10

A new trogloMtic ideoroncM pseudoscorpion (Pseudoscorpiones: Ideoronddae) from southern Africa

Mark S. Harvey: Department of Terrestrial Zoology, Western Australian Museum, Locked Bag 49, Welshpool DC,
Western Australia 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 Professor: School of Animal Biology, University of Western Australia, Crawley, Western Australia 6009,
Australia; Adjunct Professor: School of Natural Sciences, Edith Cowan University, Joondalup, Western Australia 6027,
Australia. E-mail: mark.harvey@museum.wa.gov.au

Gerhard Du Preez: Unit for Environmental Sciences and Management, Potchefstroom Campus, North-West University,

. Private Bag X6001, Potchefstroom 2520, South Africa

Abstract. The first blind African species of Ideoroncidae is described from a cave in northwestern Botswana,

. r Botswanoncus ellisi, representing a new genus and a new species. Apart from the complete lack of eyes, it is also unusual in
having the lowest recorded number of trichobothria of any adult ideoroncid with 17 on the fixed finger and nine on the
movable finger.

Keywords: Taxonomy, morphology, Botswana, new genus

The pseudoscorpion family Ideoroncidae comprises 13
genera and 70 species found in Africa, Asia and the Americas
(Harvey 2013; Harvey & Muchmore 2013). The majority occur
in tropical ecosystems, but some occur in drier regions such as
the southwestern USA and Mexico, Chile and Argentina, and
the deserts of the Middle East. Ideoroncids have the combina-
tion of two unique morphological features, supernumerary
trichobothria and the sub-basal position of the median
maxillary lyrifissure (Harvey 1992; Harvey & Muchmore
2013). Another unusual feature, the division of the median
genital sac of the male genitalia into two distinct parts, is also
found in some species currently attributed to the family
Syarinidae (e.g., Vachon 1938, 1954, 1969; Chamberlin 1952;
Mahnert 1980; Harvey 1992).

The African ideoroncid fauna (Fig. 3) consists of four
genera, Afroroncus Mahnert 1981, Dhanus Chamberlin 1930,
Nannoroncus Beier 1955 and Negroroncus Beier 1931 (Harvey
2013). The genera Nannoroncus and Afroroncus are restricted to
forested habitats in Kenya (Mahnert 1981). Three species of the
genus Dhanus are known from the island of Socotra located off
the Somali coast (Mahnert 2007). This genus is otherwise
known from the Middle East (Afghanistan and Iran), India and
southeast Asia (Harvey 2013). Negroroncus is the most
widespread African genus, being found throughout Kenya,
eastern Democratic Republic of Congo and northern Tanzania,
as well as individual outlying species from Zimbabwe and the
Republic of Congo (Mahnert 1981; Harvey 2013).

Among a small collection of pseudoscorpions taken from a
cave in the Gcwihaba region of northwestern Botswana, the
junior author found a small ideoroncid that completely lacks
eyes (Fig. 4). Eyeless ideoroncids are elsewhere only known
from the New World: Albiorix anophthalmus Muchmore 1999
from a cave in Arizona, USA (Muchmore & Pape 1999),
Ideoroncus anophthalmus Mahnert 1984 and I. cavicola
Mahnert 2001 from Brazil (Mahnert 1984, 2001) and five
species of Typhloroncus 1979 from caves in Mexico and
epigean ecotypes in the US Virgin Islands (Muchmore 1979,

1982, 1986; Harvey & Muchmore 2013). The only cave-
dwelling ideoroncids from Africa are Negroroncus aelleni
Vachon 1958 from the Republic of Congo and N jeanneli
Vachon 1958 from Tanzania (Vachon 1958), but neither
species is completely eyeless, even though the eyes are small
(Vachon 1958).

An initiative by the Botswana government to discover and
explore unknown caves with the aim to promote caving in the
tourism industry led to the discovery of several caves with no
natural openings in the northwestern Gcwihaba region of
Botswana. These caves were discovered through gravimetrical
surveys of dolomitic outcrops. This technique identifies
isolated subterranean cavities that are subsequently penetrated
by means of drilling 700 mm vertical shafts. In 2010 efforts led
to the discovery of a cave system named Diviners Cave. The
drilled shaft, with a surface entrance altitude of 1056 m a.s.L,
penetrates through 50 meters of rock into the cave.
Exploration revealed an extensive cave system (Fig. 1) at
several levels, with chambers of up to up 180 meters in
diameter. Within the sealed cave we found areas where the
roots of wild fig trees {Ficus cordata) penetrate the caves
associated with sand and water drips. In these areas we found
a diversity of invertebrates including diplurans, centipedes and
termites. Although the cave does not reach the water table, the
relative humidity exceeds 95% with a fairly constant temper-
ature of 27°C (±2°C). Prior to drilling to open the cave, the
system was under a high CO2 pressurized atmosphere, making
the circumstances in which the cavernicoles survived quite
different from other natural caves in southern Africa.

Our study of the specimens revealed a number of unusual
features, including the lowest number of trichobothria thus far
recorded for an adult ideoroncid (Table 1), and a short
arolium that lacks a ventral hook-shaped process. Apart from
being a distinctive new species, we also suggest that it
represents a previously imdescribed genus. Therefore, we here
provide a description and name the species to highlight its
distinctive morphology and habitat.

105

106

THE JOURNAL OF ARACHNOLOGY

Figures 1, 2. — 1. The type locality of Botswcmoncus ellisi. Diviners Cave, Botswana (Photo: Gerhard Jacobs); 2. Roots of the fig tree {Ficus
Cordelia): the specimens were collected from the soil below the roots.

METHODS

The specimens examined in this study were mainly sampled
with pitfall traps. Each trap was neatly buried in the soft sand
associated with wild fig tree roots (Fig. 2) and half-filled with
75% ethanol. Also, some specimens were caught by hand or
extracted from soil samples using Berlese funnels. These
specimens are lodged in the Western Australian Museum,
Perth (WAM) and the KwaZulu-Natal Museum, Pietermar-
itzburg (NMSA), and were studied using temporary slide
mounts prepared by immersion of the specimen in lactic acid
at room temperature for several days. They were then
mounted on microscope slides with a 10 mm coverslip
supported by small sections of 0.25 or 0.35 mm diameter
nylon fishing line. After study the specimens were returned to

75% ethanol with the dissected portions placed in 12 X 3 mm
glass genitalia microvials (BioQuip Products, Inc.). Speci-
mens were examined with a Leica MZ-16A dissecting
microscope and a Leica DM2500 compound microscope,
the latter fitted with interference contrast, and illustrated with
the aid of a drawing tube attached to the compound
microscope.

Measurements were taken at the highest possible magnifi-
cation using an ocular graticule. Terminology and mensura-
tion mostly follow Chamberlin (1931), with the exception of
the nomenclature of the pedipalps and legs, and with some
minor modifications to the terminology of the trichobothria
(Harvey 1992), chelicera (Judson 2007) and faces of the
appendages (Harvey et al. 2012).

10°W 0° 10°E 20°E SO’E 40'’E SO'E 60°E ZO'E

HARVEY & DU PREEZ— TROGLOBITIC PSEUDOSCORPION FROM AFRICA

107

Figure 4. — Botswanoncus ellisi, new species, female holotype, prior
to dissection, dorsal aspect.

The description was compiled using the DELTA (DEscription
Language for Taxonomy) Editor computer program, version
1. 0-Beta (available at http://code.google.eom/p/open-delta/)
(Dallwitz et al. 1999).

SYSTEMATICS

Family Ideoroncidae Chamberlin 1930
Genus Botswanoncus gen. nov.

Type species. — Botswanoncus ellisi new species

Diagnosis. — Botswanoncus is the only ideoroncid genus with
a short arolium that lacks a ventral hook on the arolium. It
also differs from other ideoroncids by the presence of only 17
trichobothria on the fixed chelal finger and nine trichobothria
on the movable chelal finger (Table 1) (Figs. 11, 12), and from
all other African ideoroncids by the complete lack of eyes
(Fig. 5).

Description. — Adult female (male unknown}'. Chelicera
(Fig. 9): hand with 5 setae; movable finger with 1 sub-distal

seta; all setae acuminate; galea present, simple, long and
slender; lamina exterior absent; serrula exterior connected to
chelicera finger for only part of length; not modified to form
velum; rallum (Fig. 10) with 4 blades, all with anterior
spinules, basal and sub-basal blades shorter than others.

Pedipalp (Fig. 8): long and slender. Fixed finger with 17
trichobothria (Figs 11, 12): eh, esh, et, ish and it regions each
with 1 trichobothrium; est region with 6 trichobothria; ib
region with 3 trichobothria; ist region with 3 trichobothria; et
slightly distal to it. Movable finger with 9 trichobothria
(Figs. 11, 12)'. b, sb and st regions each with 1 trichobothrium;
t region with 6 trichobothria. Chelal teeth (Fig. 11) juxtaden-
tate, with fixed finger chelal teeth low and flattened, and
movable finger chelal teeth low; venom apparatus present in
both chelal fingers; venom ducts of medium length, terminat-
ing midway between it and est in fixed finger and basal to t in
movable finger; nodus ramosus not infiated.

Cephalothorax: carapace (Fig. 5) sub-rectangular; anterior
margin slightly convex; with 4 setae on anterior margin and 2
on posterior margin; furrows absent; eyes completely absent.
Manducatory process somewhat pointed, with 2 apical setae;
median maxillary lyrifissure situated sub-basally.

Legs: femora I and II much longer than patellae I and 11,
respectively; femora III and IV about same size as patellae III
and IV, respectively; metatarsi shorter than tarsi; subterminal
tarsal seta acuminate; arolium about same length as claws,
with slight medial division (Figs. 6, 7); ventral hook-shaped
process absent.

Abdomen: tergite I with 2 setae; spiracular plates each with
1 seta; medial sternites without suture line; pleural membrane
uniformly longitudinally striate; stigmatic helix present; anus
situated between tergite XI and sternite XI.

Genitalia: female: with gonosac covered in small acetabula.

Remarks. — The arolium of B. ellisi is about the same length
as the claws (Figs. 6, 7), and therefore resembles the New
World genera Typhloroncits and Xorilhia Harvey and Mahnert
2006, the African Negroroncus aelleni Vachon 1958, and the
Asian Dhanus siamensis (With 1906), which have arolia that
are slightly shorter than the claws. The remaining ideoroncid
genera have arolia that are clearly longer than the claws. It
differs from these genera with short arolia by lacking the
ventral hook of the arolium. It further differs from all other
ideoroncids by the reduced number of trichobothria, with only
17 on the fixed finger and nine on the movable finger (Table 1,
Figs. 11, 12).

Etymology. — The genus is named for its occurrence in
Botswana, combined with the last five letters of Roncus, a
common pseudoscorpion stem which has been thought to be
derived from the Latin runco, “living in weeds” (Parker 1982).
It is to be treated as masculine.

Botswanoncus ellisi sp. nov.

Figs. 4-12

Material examined. — Botswana: North-western District', ho-
lotype female. Diviners Cave, Gewihaba region, 20°08'32.2"S,
2n2'36.6"E, 19 October 201 1, G. Du Preez (WAM T125604).
Paratypes: 1 female, same data as holotype except 13 March
2012 (WAM T130804); 1 female, same data as paratype
(NMSA-Pse 026870).

Diagnosis. — As for genus.

108

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Table 1, - The recorded number of trichobothria found in adults of genera of Ideoroncidae. Variant numbers are shown in brackets.

eh

esh

est

et ib

isb ist

it

h sb

st

t

Fixed finger,
total

Movable
finger, total

Reference

Afroroncus 1

1

6

1 4

1 5

1

2 1

1

6

20

10

Mahnert (1981 )

Alhiorix 1

1

6

1 4-5

1 5-6(4)

1

2 1

1

6

20-22

10

Harvey & Muchmore (2013)

Botswcuioncus 1

1

6

1 3

1 3

1

1 1

1

6

17

9

This study

Dliamis 1-3

1

5-9

1 3-6

1 5 12

1

2M 1

1

6-8

23-31

11-14

Mahnert (1984, 2007)

Ideoroncus I

1

6

1 4(5)

1 4-6

1

2 1

1

6

20-21 (22)

10

Mahnert (1984, 2001); Harvey
& Muchmore (2013)

Mahnertiiis 1

1

6

1 5

1 6

1

2 1

1

6

22

10

Harvey & Muchmore (2013)

Miichmoreiis 1

1

6

1 4

1 5

1

2 1

1

6

20

10

Harvey & Muchmore (2013)

Nawioroucus 1

1

6

1 4

1 5

1

2 1

1

6

20

10

Mahnert (1981)

Negroroncus 1-2

1

6-10

1 4

1 5-6

1

2-3 1

1

6-7

20-26

10-12

Mahnert (1981); Vachon (1958)

NIuitraiigici 2

1

9

1 6

1 9-10

1

4 1

1

8

30-31

14

Mahnert (1984)

Pseudalhiorix 1

1

6

1 4

1 5

1

2 1

1

6

20

10

Harvey et al. (2007)

Shnivaua 1

1

6

1 5

1 7

1

3 1

1

7

23

12

Mahnert (1984)

Typliloronciis 1

1

6

1 4-5

1 6-7

1

2 1

1

6

22

10(11)

Muchmore (1986); Harvey &
Muchmore (2013)

Xorilbia 1

1

6

1 5

1 6

1

2 1

1

6

22

10

Mahnert (1984, 1985)

Description. — Adult: Color: pedipalps, carapace, chelicerae
and coxae light red-brown, tergite I and legs pale brown and
remainder light yellow-brown (Fig. 4).

Setae and cuticle: setae long, mostly straight and acicular;
most cuticular surfaces smooth and glossy, with exception of
pedipalps and chelicera, which are finely granulate.

Chelicera (Fig. 9): ca. 50% length of carapace; surface
reticulate; cheliceral hand with 5 setae; movable finger with 1
sub-distal seta; all setae acuminate; galea present, simple, very
long, slender and slightly curved; fixed finger ca. 7 teeth, each
approximately same size, small; movable finger with ca. 8
teeth, each approximately same size, small; exterior and
interior condylar lyrifissures present; serrula interior with 18
blades; lamina exterior absent; rallum (Fig. 10) with 4 blades,
all with anterior spinules; basal and sub-basal blades shorter
than others.

Pedipalp (Fig. 8): long and slender; trochanter and femur
granulate, prolateral margin of patella and chelal hand
granulate, retrolateral surfaces of fingers granulate, all other
surfaces smooth; setae acicular, straight or nearly so;
trochanter with anterior margin rounded, 2.85 x; femur
cylindrical, without trichobothria, 4.57-5.01 x; patella cylin-
drical, with strongly pronounced pedicel, with 3 lyrifissures, 2
at distal end of pedicel and 1 in middle of pedicel, 2.99-3.25 x;
chelal hand ovoid, external chelal condyle small and rounded,
internal chelal condyle small and rounded, chela (with pedicel)
3.91-4.14 X, chela (without pedicel) 3.71-3.92 x, hand (without
pedicel) 1.26-1.32 x, movable finger 1.93-2.54 x longer than
hand (without pedicel). Fixed finger with 17 trichobothria;
movable finger with 9 trichobothria (Figs. 11, 12); eh, esh and
ish in straight row at base of finger; ih region situated dorsally
in the middle of chelal hand; eh, esh, et, ish and it regions each
with 1 trichobothrium; est region with 6 trichobothria; ih
region with 3 trichobothria; ist region with 3 trichobothria; et
slightly distal to if, h, sh and st regions each with 1
trichobothrium; t region with 6 trichobothria; not ventrally
displaced, or st situated much closer to h than to t;
trichobothrium t acuminate. Both fingers straight in lateral
view; chelal teeth juxtadentate (Figs. 11, 12); fixed finger
chelal teeth low and flattened; venom apparatus present in

both chelal fingers, venom duct of medium length, terminating
midway between it and est in fixed finger and basal to t in
movable finger; nodus ramosus not inflated.

Cephalothorax: carapace (Fig. 5) sub-rectangular, 1.20-
1.39 X longer than broad; anterior margin slightly convex;
epistome absent; lateral margins slightly convex; posterior
margin straight; with 18 setae, arranged 4: 4: 4: 2: 2: 2; setae
subequal in length; furrows absent; eyes completely absent.
Manducatory process somewhat pointed, with 2 apical setae,
both setae approximately equal in length; maxilla with 5
additional setae; maxillary shoulder absent; median maxillary
lyrifissure present, situated sub-basally, strongly curved, U-
shaped; posterior maxillary lyrifissure present, strongly
curved. Coxa I about same width as coxa IV; anterior margin
of coxa I with process near foramen; coxae I-IV of? with setae
arranged 4: 5: 4: 4.

Legs: femora I and II much longer than patellae I and II,
respectively; femur I and II without basal swelling; femora I
and II with primary slit sensillum directed transversely; femora
III and IV about same size as patellae III and IV, respectively;
femur + patella IV 3.89 x longer than broad; tibiae III and IV
without obvious tactile seta; metatarsi III and IV with long
tactile seta, situated medially; tarsi III and IV without tactile
seta; metatarsi and tarsi of all legs not fused; metatarsi shorter
than tarsi; subterminal tarsal seta acuminate; claws smooth;
arolium about same length as claws, with slight medial
division (Figs. 6, 7); ventral hook-shaped process absent
(Fig. 7).

Abdomen: tergites straight, without suture line, setal
formula ?, 2: 4: 4: 4: 5: 6: 6: 6: 6: 6: 6 (arranged T ITT IT): 2;
arranged in single rows; sternites, without suture line, setal
formula ?, 6: (1) 2 (1): (1) 4 (1): 8: 8: 8: 8: 8: 8: 6 (arranged
1T2T1): 2; setae of anterior genital operculum (sternite II) of?
very small; posterior tergites and sternites with several tactile
setae; glandular setae absent; pleural membrane uniformly
longitudinally striate.

Genitalia: female: with gonosac covered in small acetabula.

Dimensions (mm): Female holotype (with paratypes in
parentheses, where applicable). Body length 2.28 (2.16-2.25).
Chelicera 0.313/0.132, movable finger length 0.200. Pedipalp:

HARVEY & DU PREEZ— TROGLOBITIC PSEUDOSCORPION FROM AFRICA

109

Figures 5-12. — Botswanoncus ellisi, new species, female hoiotype: 5. Carapace, dorsal; 6. Tip of left tarsus IV showing claws and arolium,
dorsal; 7. Distal end of left tarsus IV, lateral; 8. Right pedipalp, dorsal; 9. Left chelicera, dorsal; 10. Rallum; 1 1. Left chela, lateral; 12. Left chela,
lateral, showing trichobothriai pattern. Scale lines = 0.05 mm (Figs. 6, 7), 0.1 mm (Figs. 9, 10), 0.2 mm (Figs. 5, 11, 12), 0.5 mm (Fig. 8).

trochanter 0.314/0.110, femur 0.685/0.150 (0.659-0.722/0.141-
0.144), patella 0.545/0.182 (0.502-0.550/0.163-0.169), chela
(with pedicel) 1.182/0.302 (1.136-1.250/0.295-0.302), chela
(without pedicel) 1.12 (1.072-1.184), hand (without pedicel)
0.380 (0.390-0.397), movable finger length 0.732 (0.689-
0.768). Carapace 0.564/0.471 (0.584-0.596/0.419-0.435). Leg

I: femur 0.330/0.080, patella 0.160/0.070, tibia 0.250/0.049,
metatarsus 0.152/0.039, tarsus 0.337/0.032. Leg IV: femur +
patella 0.521/0.140, tibia 0.324/0.071, metatarsus 0.228/0.046,
tarsus 0.328/0.040.

Remarks. — Botswanoncus ellisi exhibits some moderate
modifications for cave existence, including the complete lack

110

THE JOURNAL OF ARACHNOLOGY

of eyes (Figs. 4, 5) and pallid coloration (Fig. 4). The
appendages, however, are not especially elongated as in the
other troglobitic ideoroncids Albiorix anophthalmus Much-
more 1999 from Arizona, USA and several Typhloroncus
species from Mexican caves, which have long, slender
appendages consistent with a permanent cave-dwelling life-
style (Muchmore 1982, 1986; Muchmore & Pape 1999; Harvey
& Muchmore 2013).

Only two other pseudoscorpion species have been recorded
from Botswana. Nanolpium siibgrande (Tullgren 1908) and
Beierolpium deserticola (Beier 1964) (Tullgren 1908; Beier
1964), both in the family Olpiidae, making it one of the least
known countries for pseudoscorpion diversity (Harvey 2013).

Etymology. — This species is named for the renowned caver
Roger Ellis, who facilitated GDP’s trip to Botswana. Roger
has devoted over 40 years to the discovering, surveying and
conserving of caves in southern Africa.

ACKNOWLEDGMENTS

We thank the government of Botswana, especially the
President His Excellence Ian Khama, for creating the
opportunity to explore and study the subterranean realms of
the Gcwihaba district. Also, we would like to thank Roger
Ellis and the rest of the Potch Potholers Caving Club for
assisting our cause. Volker Mahnert and Juan Zaragoza
provided very helpful comments on the manuscript.

LITERATURE CITED

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Fauna des siidlichen Afrika. Annals of the Natal Museum
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Chamberlin, J.C. 1931. The arachnid order Chelonethida. Stanford
University Publications, Biological Sciences 7(1): 1-284.
Chamberlin, J.C. 1952. New and little-known false scorpions
(Arachnida, Chelonethida) from Monterey County, California.
Bulletin of the American Museum of Natural History 99:259-312.
Dallwitz, M.J., T.A. Paine & E.J. Zurcher. 1999. User’s Guide to the
DELTA Editor. Accessed 10 January 2012. Online at http://
biodiversity.uno.edu/delta/

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, http://www.museum.wa.gov.
au/catalogues/pseudoscorpions/. Accessed 6 July 2013.

Harvey, M.S., R. Barba D., W.B. Muchmore & A. Perez G. 2007.
Psetidalhiorix, a new genus of Ideoroncidae (Pseudoscorpiones,
Neobisioidea) from central America. Journal of Arachnology
34:610-626.

Harvey, M.S. & W.B. Muchmore. 2013. The systematics of the
pseudoscorpion family Ideoroncidae (Pseudoscorpiones, Neobi-
sioidea) in the New World. Journal of Arachnology 41:229-290.
Harvey, M.S., P.B. Ratnaweera, P.V. Randeniya & 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.

Judson, M.L.I. 2007. A new and endangered species of the
pseudoscorpion genus Lagynochihonius from a cave in Vietnam,
with notes on chelal morphology and the composition of the
Tyrannochthoniini (Arachnida, Chelonethi, Chthoniidae). Zoo-
taxa 1627:53-68.

Mahnert, V. 1980. Pseudoskorpione (Arachnida) aus Hohlen Italiens,
mit Bemerkungen zur Gattung Pseudoblothriis. Grotte dTtalia (4a)
8:21-38.

Mahnert, V. 1981. Die Pseudoskorpione (Arachnida) Kenyas. 1.
Neobisiidae und Ideoroncidae. Revue Suisse de Zoologie 88:535-559.

Mahnert, V. 1984. Beitrag zu einer besseren Kenntnis der Ideor-
oncidae (Arachnida: Pseudoscorpiones), mit Beschreibung von
sechs neuen Arten. Revue Suisse de Zoologie 91:651-686.

Mahnert, V. 1985. Weitere Pseudoskorpione (Arachnida) aus dem
zentralen Amazonasgebiet (Brasilien). Amazoniana 9:215-241.

Mahnert, V. 2001. Cave-dwelling pseudoscorpions (Arachnida, Pseu-
doscorpiones) from Brazil. Revue Suisse de Zoologie 108:95-148.

Mahnert, V. 2007. Pseudoscorpions (Arachnida: Pseudoscorpiones)
of the Socotra Archipelago, Yemen. Fauna of Arabia 23:271-307.

Muchmore, W.B. 1979. Pseudoscorpions from Florida and the
Caribbean area. 9. Typhloroncus, a new genus from the Virgin
Islands (Ideoroncidae). Florida Entomologist 62:317-320.

Muchmore, W.B. 1982. Some new species of pseudoscorpions from
caves in Mexico (Arachnida, Pseudoscorpionida). Bulletin for the
Association of Mexican Cave Studies 8:63-78.

Muchmore, W.B. 1986. Additional pseudoscorpions, mostly from
caves, in Mexico and Texas (Arachnida: Pseudoscorpionida).
Texas Memorial Museum, Speleological Monographs 1:17-30.

Muchmore, W.B. & R.B. Pape. 1999. Description of an eyeless,
cavernicolous Albiorix (Pseudoscorpionida: Ideoroncidae) in Ar-
izona, with observations on its biology and ecology. Southwestern
Naturalist 44:138-147.

Parker, J.R. 1982. What’s in a name? — scorpions and pseudoscor-
pions. Newsletter of the British Arachnoiogical Society 34:1-2.

Tullgren, A. 1908. Pseudoscorpionina (Chelonethi). In: & Schulze, L.
1908. Zoologische und Anthropologische ergebnisse einer For-
schungsreise im westlichen und zentralen Siidafrika ausgefiihrt in
den Jahren 1903-1905. Denskschriften der Medizinisch-Naturwis-
senschaftlichen Gesellschaft zu Jena 13:283-288.

Vachon, M. 1938. Recherches anatomiques et biologiques sur la
reproduction et le developpement des Pseudoscorpions. Annales
des Sciences Naturelles, Zoologie (11) 1:1-207.

Vachon, M. 1954. Remarques morphologiques et anatomiques sur les
Pseudoscorpions (Arachnides) appartenant au genre Pseudobio-
thriis (Beier) (Fam. Syarinidae J.C.C.) (a propos de la description
de P. slrinatii n. sp., des cavernes de Suisse). Bulletin du Museum
National d’Histoire Naturelle, Paris (2) 26:212-219.

Vachon, M. 1958. Sur deux Pseudoscorpions nouveaux des cavernes
de FAfrique equatoriale [Ideoroncidae], Notes Biospeologiques
13:57-66.

Vachon, M. 1969. Remarques sur la famille des Syarinidae J.C.
Chamberlin (Arachnides, Pseudoscorpioiis) a propos de la
description d’lme nouvelle espece: Pseudoblothrus thiebaiidi habi-
tant des cavernes de Suisse. Revue Suisse de Zoologie 76:387-396.

Manuscript received 4 August 2013, revised 23 September 2013.

2014. The Journal of Arachnology 42:111-118

Comparison of scorpion behavioral responses to UV under sunset and nighttime irradiances

Douglas D. Gaffin and Tristan N. Barker: Department of Biology, University of Oklahoma, Norman, Oklahoma 73019 USA.
E-mail: ddgaffin@ou.edu

Abstract. Scorpions are nocturnal arachnids that fluoresce a bright cyan-green when exposed to UV light. Although the
function of this fluorescence remains unknown, some authors have suggested that it may aid the scorpions’ light detection.

Taking advantage of scorpions’ negatively phototactic behavior, we tested the responses of desert grassland scorpions,
Paruroctonus utahensis (Williams 1968), to 395 nm UV light at irradiances corresponding to an hour before sunset (0. 15 pW/
cm^), sunset (0.01 pW/cm"), and moonlight (0.0001 pW/cm"), as well as no light. We found that animals showed the
strongest responses to UV light levels equivalent to sunset. The animals moved more quickly and sporadically under the
higher light levels. In addition, animals were less likely to complete a trial under highest light conditions, suggesting that
UV light may inhibit normal scorpion locomotion. Finally, this study resulted in several methodological refinements,
including automated tracking of the subjects’ movements that should prove useful in future behavioral studies of scorpion
phototactic behavior.

Keywords: Fluorescence, light, orientation, sensory, vision

Scorpions are nocturnal arachnids that fluoresce a bright
green color when exposed to ultraviolet (UV) light due to the
presence of beta-carboline and 4-methyl-7-hydroxycoumarin
in their cuticle (Stachel et al. 1999; Frost et al. 2001). No
functional reason behind their fluorescence has been deter-
mined. Some authors, including Frost et al. (2001) and
Wankhede (2004), have suggested that scorpion fluorescence
may serve no behavioral purpose, while others have proposed
that fluorescence may help scorpions capture prey (Kloock
2005), attract mates, or ward off predators and territorial
rivals (Kloock 2008). Other researchers hypothesize that
fluorescence may play an active role in light detection, helping
scorpions identify shelter or decide when to stay in their
burrows (Camp & Gaffm 1999; Blass & Gaffm 2008; Gaffin
et al. 2012; Kloock et al. 2010).

Scorpion cuticle fluoresces most strongly under 395 nm UV
light, reemitting it as green (~ 500 nm: Fasel et al. 1997;
Kloock 2009). Studies indicate that the medial eyes of
scorpions are most sensitive to green light (peaking around
500 nm: Machan 1968; Fleissner & Fleissner 2001). Parts of
the scorpion metasoma are also sensitive to green light
(Zwicky 1968, 1970a,b; Rao & Rao 1973). It is therefore
tempting to suggest that fluorescence may aid in light
detection by transducing UV light to increase light intensity
in the range of peak sensitivity of their visual system.

A few behavioral studies support this hypothesis. Blass &
Gaffm (2008) showed that scoipions become most active when
exposed to UV or green light, as compared to other wavelengths.
Kloock et al. (2010) found a difference between fluorescent and
fluorescence-reduced scorpions in the variance of time spent in
light-exposed areas, as well as differences in activity levels under
UV light. Gaffm et al. (2012) found that scorpions with medial
and lateral eyes covered were far less likely to react to 505 nm
light, but only slightly less likely to react to 395 nm light. This
last study led to the hypothesis that the cuticle may act as a
whole-body UV photon collector, transducing UV wavelengths
to green wavelengths. This information may allow the scorpion
to detect and turn toward shade when one part of the cuticle
receives diminished light levels.

Taken together, the physiological and behavioral evidence
suggest that UV light plays an important role in scorpion
orientation. Hov/ever, to better understand these effects, we
first need to quantify the levels at which UV light becomes
behaviorally relevant.

Our objective in the current study was to establish a dose
response curve illustrating scorpions’ reactions to irradiances
of UV light corresponding to natural conditions ranging from
early sunset to the middle of the night. Gaffin et al. (2012)
observed significant phototactic behavior when scorpions were
exposed to 0.15 |iW/cm“ UV light, slightly greater than the UV
component of sunlight about an hour before sunset when the
sun is 1 1.4° above the horizon (Johnsen et al. 2006). This light
level is somewhat higher than what scorpions normally
encounter; they become active shortly after sunset (Polis
1980) and are therefore most likely to encounter intensities
of light corresponding to refracted sunlight, starlight, or
moonlight.

We used 0.15 pW/cm” as the high light treatment on our
dose response curve. We selected 0.01 pW/cm^, the UV
component of the sky’s irradiance at sunset (when the sun is at
the horizon: Johnsen et al. 2006), as the second treatment. We
selected 0.0001 pW/cm", the UV component of full moonlight
on a clear night (Johnsen et al. 2006), as the third and lowest
light treatment. Finally, we used no light as the control and the
final point on the curve, where we expected to see no
phototactic behavior. Combined, these four points represented
a relatively even distribution of celestial irradiance values that
would be present from the early evening into the night.

We found that scorpions respond to UV light levels that
correspond to irradiance values found around sunset. Since
this assay uses a negative phototactic locomotor behavior as
the response, the actual threshold of sensitivity is probably
lower than these deterrence levels. Taken together, scorpions
appear capable of detecting UV levels that are consistent with
light levels during early evening.

An additional objective of this study was to improve the
efficiency of the behavioral assay used to detect light
avoidance behavior in scorpions. We have greatly improved

112

THE JOURNAL OF ARACHNOLOGY

Figure I. — Diagram of behavioral set-up. A power source (PS) powers four independently controllable operational amplifier circuits (OA),
which connect to LEDs (SL) that extend through holes in the tops of dark PVC cylinders placed over Petri dish arena. An IR sensitive video
camera below the stage monitors scorpion activity. Infrared light is directed across the bottoms of the arenas from two sources (IR) placed at the
side of the set-up. Video output is relayed to a computer for recording, processing, and analysis.

the visibility of the animals in the behavioral arenas and
applied automated tracking software to assist in the scoring of
behavioral trials and to reduce possible sources of human bias.

METHODS

Animals. — We used 12 male and 12 female adult Parur-
octonus utahensis (Williams 1968) scorpions collected in late
August and early September 2012 from sandy regions of the
northern Chihuahua Desert. Collecting areas ranged from the
Texas-New Mexico border between El Paso and Las Cruces to
areas east of Socorro, New Mexico and near the Sevilleta field
station in La Joya, New Mexico. We deposited a voucher
specimen in the Sam Noble Oklahoma Museum of Natural
History on the University of Oklahoma campus in Norman,
Oklahoma. Animals were kept in the laboratory at the
Sevilleta station and housed individually in plastic food
storage containers (Great Value, 236 ml) that had four
5.6 mm air holes drilled in the corners of their lids. Each
container also held 20 ml of sand from their native habitat

(filtered through a #12 sieve) and a 4 cm X 4 cm square of
paperboard folded into a tent for shelter. The animals were
provided a few ml of water weekly by misting and a wax worm
every other week. The animals were exposed to a 14:10 h
light;dark cycle (on at 0530, off at 1930) using a white
fluorescent bulb (General Electric “Energy Smart” 13 W bulb
- 60 W equivalent) in a work light (Bayco clamp light, 21.6 cm)
plugged into a timer switch. The light was placed 50 cm from
the animals. The room temperature ranged from 20-21 °C
during the day. To increase animal activity on trial nights, a
small heater (Sunbeam compact ceramic heater) was used to
warm the room to 22-24°C.

Behavioral apparatus. — We used a modified version of the
apparatus described in Gaffin et al. (2012). Figure 1 shows a
diagram of the behavioral set-up. We created a circular arena
for the animals by gluing a 5.40-cm diameter Petri dish upside
down in the center of an 8.75-cm diameter Petri dish (15 mm
deep). The larger Petri dish lids were used for covers and
secured to the dish bottoms with small pieces of electrical tape.

GAFFIN & BARKER— SCORPION UV DOSE RESPONSE

113

Instantaneous velocity (mm/s)

Figure 2. — Scorpion behavior in the behavioral test apparatus. A.
Sample plot of animal movements during 10-minute trial under no
light condition; the points are plotted at 0.67 s intervals. The numbers
inside the circle indicate arena coordinates that are referenced in
figure 3A. B. Plot of instantaneous velocity across duration of the
trial shown in A. Instantaneous velocity is calculated as distance
traveled in mm between each frame divided by 0.67 s (time between
frames). The line is a five-point running average of instantaneous
velocity. C. Frequency distribution of the instantaneous velocities for
the trial shown in A.

The arenas were inserted into four holes in a 36 X 30 X 1.5 cm
particleboard stage that was suspended by a PVC frame with
adjustable supports for leveling. The outside upright walls of
the larger Petri dish arenas were covered by black electrical
tape to keep light from entering the sides of the arenas and to
help the dishes fit snugly in the stage holes. The dishes were
lowered into the holes until the arena lids rested flush against
the top of the particleboard stage. We then placed a piece of
PVC pipe (10 cm diameter and 15 cm tall) lined with black
construction paper over each arena and topped this pipe with
a black square of Plexiglas that had a 5-mm hole drilled in its
center to accommodate an LED light. We fitted four such
tubes with LEDs emitting UV light (395 nm, 15° viewing
angle; Super Bright LEDs Inc.). The LEDs were fixed in place
with black electrical tape and connected via patch cables with
mini-hook clips to op-amp circuits to control the intensity
of each LED and allow the light of each arena to
be independently set, so that each trial could include four
separate treatments. We filmed the arenas from below with an
infrared-sensitive camera (Sony Handycam CCD-TRV16 with
‘nightshot’ feature) connected to a computer running a video
capture program (Elgato Video Capture System). To reduce
glare and improve the image, we covered the camera’s IR light
source with two layers of black electrical tape. We directed the
IR light emitted from two surveillance cameras (Swann
NightHawk Day/Night Security Cameras) at 45-degree angles
onto the bottom of the arenas to provide the IR source for the
camera. We taped some thin semi-transparent foam packing
material over the lights to diffuse the IR. To reduce glare and
light contamination of the video image from the trial LEDs,
we taped a circle of black construction paper to the bottom of
the arena to cover the area occupied by the smaller Petri dish.

Light calibrations. — We used an Ocean Optics USB4000-
UV-VIS-ES spectrophotometer (200 pm slit, 600 pm diameter
optical fiber, 3900 pm diameter CC-3 cosine corrector) to
calibrate each LED to the same relative irradiance for each
trial. Since we exposed the animals to LEDs emitting only UV,
we matched the irradiance of these LEDs to the isolated UV
component of natural light levels such as moonlight and
sunlight.

We tested the scorpions’ responses to four light levels. The
highest level, 0.15 pW/cm", is approximately equal to the
ultraviolet component of sunlight when the sun is just over
1 1.4° above the horizon; this level is also about 1500 times the
ultraviolet component of full moonlight (0.0001 pW/cm').
This allowed us to verify that our scorpions demonstrated
behavior similar to that observed under previously tested light
conditions (Gaffin et al. 2012). We compared the scorpions’
behavior under this light to their behavior under the UV
irradiances of sunset (light emitted from the sky when the sun
is at the horizon; 0.01 pW/cm"), full moonlight and no light.
We determined these intensities based on irradiance values
given in Figure 2C of Johnsen et al. (2006) and conversion
factors provided by Johnsen (2012).

Dose response trials. — We conducted these trials in a
windowless room at the Sevilleta field station in September
2012. All animal manipulations were done under dim red light
provided by a headlamp (Energizer Trailfinder 6 LED
Headlight); previous studies showed no apparent behavioral
sensitivity of scorpions to red light (Camp & Gaffin 1999;

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Gaffin et al. 2012). Trials began around 2000 (30 min after the
beginning of the dark cycle) and were completed by 2130.
Trials were run four at a time, with each arena’s light tuned to
one of the four light levels: arena A, no light; arena B,
0.0001 )aW/cm“; arena C, 0.01 pW/cm"; arena D, 0.15 pW/cm“.
Based on the activity levels of P. iitahensis under these
conditions in our pilot studies and as reported in previous
studies (Blass & Gaffin 2008; Gaffin et al. 2012), 24 animals
participated in two sets of trials separated by 15 days. Within
each set of trials, each animal experienced a different intensity
on four successive nights. The light intensity order was
randomized so that the animals were exposed to neither
ascending nor descending intensities; the sequence was
reordered for the second set of trials. Each animal was
therefore exposed to each light condition twice, separated by
about 15 days. One animal died during the 15-day interval and
was removed from all data analyses. Subtracting this animal,
184 trials formed the data set for these experiments (23*4 -h
23*4). Each animal was fed a wax worm seven days before the
start of the first set of trials and another worm one day after
completing the first set of trials.

The protocol for all trials was identical. Each night, the 24
animals were lined up in their numbered containers on the
counter in the dark room. Five minutes before the beginning of
the each trial, the four arenas were cleaned with 70% ethanol
and dried with a Kimwipe, to ensure that the animals could not
detect pheromones or other clues from previous animal use.
One animal was then put into each arena, and the lids were
secured with two small strips of electrical tape. The arenas were
then placed in the holes on the particleboard platform under the
PVC pipe with the correct light intensity, but with the arena
lights switched off Once all four animals were in place, the
video and the lights were turned on. The video was set to turn
off automatically after 10 minutes. Halfway through one trial,
we set up the arenas for the next trial. When the video turned
off, the arena lights were switched off, the video was saved to
the hard drive, and the four trial animals were returned to their
home containers. The next video was readied and the four new
animals were placed on the stage under the correct lights. This
routine was completed for all 24 animals (six groups of four
simultaneous trials per night).

Analysis. — We imported each video (saved in .mp4 by the
Elgato system) to iMovie (Apple Corporation), used the speed
function to increase the playback rate lOX, and saved to .mov
format (Quicktime, medium band). These clips were imported
into ImageJ (Rasband 2012), and the images were cropped to
100 X 100 pixel squares around each of the four arenas; each
cropped image was saved to a separate animated .tif file. Each
file was then imported to Fiji (Schindelin et al. 2012) and
converted to 8-bit format and adjusted with the Image-
Adjust-Threshold function to highlight and extract the
scorpion from the background. Then the outside and the
inner circle areas were cleared to isolate the image to the
scorpion track. We further cleaned the image by digitally
removing small video incongruities outside areas of animal
movement. We used Fiji’s Mtrack2 plugin to track scorpion
movements (settings: minimum object size (pixels) = 50;
maximum object size (pixels) = 99999; maximum velocity =
50; minimum track length (frames) = 2). The software marked
the position of a centroid defined by the animal’s outline. If

there was too much glare contamination for accurate
automated tracking (about 25% of the trials), we tracked the
animals manually using Fiji’s Manual Tracking plugin,
marking each point based on a position just caudal to the
medial eyes. After processing, the ten-minute videos parsed to
900 frames in Fiji. Therefore, each frame represented
0.67 seconds (600 s / 900 frames).

We imported each of the tracked files to Excel for further
analysis. First, we applied the Pythagorean theorem to calculate
the distance (d) moved between each frame in the record:

^/ = V((x2-xi)- + (y2-yi)-)

(where [x2, y2] and [xi, yj] are the scorpion’s coordinates within
the current frame and the previous frame, respectively). We
then removed all movements less than 4 pixels (3.6 mm; 1 pixel
= 0.9 mm) between frames to avoid bias from some jitter
introduced by the automatic tracking program. We then
summed the remaining distances to obtain the total distance
moved for the trial. We set a threshold of at least 100 total pixels
(9 cm) moved for trials to be considered legitimate; this distance
is approximately two-thirds of the way around the arena track
and is roughly equivalent to the movement criterion used in
Gaffin et al. 2012. Scorpions typically show bouts of movement
interrupted by prolonged pauses. This minimum distance was
important to filter random movements from potential stimulus-
induced responses.

Next we calculated the instantaneous velocity of each
movement by dividing each legitimate distance (those > 4.0
pixels between frames) by 0.67 s (the time between frames). We
used these numbers to derive a frequency plot of all of the
instantaneous velocities for each trial. A plot of all 5648
instantaneous velocities obtained during this study shows a
positive skew (mean = 10.91, median = 9.58, mode = 5.54,
standard deviation = 5.18; Pearson’s first and second
skewness coefficients: 1.04 and 0.77, respectively). Because of
the skewed distributions, we calculated the median instanta-
neous velocity for each trial and used those numbers to
determine the mean of each animal’s scores for legitimate trials
for each light level. For a given light level, if an animal had
one trial that was legitimate and another that was not, the
legitimate score was used as the animal’s score. No score was
given if neither trial was legitimate; these trials were not
included in our statistical analyses.

Note that the scorpions’ behavior can be described in terms
of either the total distance traversed or the instantaneous
velocity. In our experience, measuring the total distance
traversed is unsatisfactory, as control animals often walk
slowly and deliberately, covering the same distance overall as
stimulated animals that “sprint” and rest. In this study,
distances traveled by animals exposed to different light levels
were not significantly different (repeated measures ANOVA:
Fr = 1 .000; P — 0.8438); we therefore estimated activity levels
based on instantaneous velocity.

We used circular statistics to test for bias in the animal
arena position in these trials. We first calculated the mean
vector direction for animal positions in each legitimate trial.
We then used these directions to calculate the overall mean
vector direction and length (r). We calculated the z-statistic
and used the Rayleigh test for randomness to determine the
statistical significance of the mean vector.

GAFFIN & BARKER— SCORPION UV DOSE RESPONSE

115

Figure 3. — Activity patterns in behavioral apparatus. A. Average
animal arena position (in degrees) for all 83 legitimate trials shows no
arena or global position bias (0 degrees is toward the top of the video
screen; 290 degrees is geomagnetic north relative to this reference). B.
Females and males show differences in the number of trials that met
the legitimacy criterion.

We also looked at activity differences between males and
females. We used a two-tailed Mann-Whitney U analysis to
test statistical differences in number of legitimate trials per
animal among males and females.

We used a repeated-measures ANOVA and Dunn’s multiple
comparisons post-hoc test to analyze the significance of
differences among the scorpions’ median instantaneous
velocities under different light intensities. In this assay,
scorpion responses appear as spurts of locomotor movement;
as such, we predicted that stimulated scorpions would have
higher median instantaneous velocities. We considered treat-
ments significantly different if the P value was less than 0.05.
Our null hypothesis was that there would be no significant
differences between the scores at different irradiances. We
used InStat 3 statistical software (Graph Pad Software, Inc.,
San Diego, CA, U.S.A.) for the Mann-Whitney U and
ANOVA analyses.

Table 1. — Number of legitimate trials by experimental factor.

Factor

Condition

Legitimate trials

Total trials

Time of night

Early

48

96

Late

35

88

Trial set

First

39

92

Second

44

92

Night order

1

22

46

2

21

46

3

21

46

4

19

46

Gender

Males

54

96

Females

29

88

RESULTS

The new behavioral apparatus is different from the one used
by Blass and Gaffm (2008) and Gaffin et al. (2012) in that the
Petri dish arenas are suspended in holes in a particleboard stage
rather than sitting atop a Plexiglas stage. The arrangement we
used in this study, coupled with diffuse IR light directed from
the side, provided clear images of the scorpions when filmed
through the Petri dish from below. The scorpion images were
distinct enough to use video detection software to automatically
track animal movements, thereby removing the potential bias of
a human observer. A sample plot of an animal moving under
the “no light’’ condition is shown in Fig. 2A. The time course of
this animal’s instantaneous velocities is shown in Fig. 2B and
these data are compiled in Fig. 2C as a frequency plot of the
instantaneous velocities for the 10-min trial.

We made various checks of the new behavioral assay.
Legitimacy (> 9 cm movement) was achieved in 45.1% of the
trials (83 of 184). Although we recognize that considering all
83 legitimate trials includes non-independent observations, we
feel that we can learn something about general patterns of
behavior by examining these data. We found no bias in arena
or global position among the legitimate trials (Fig. 3A: (p =
307°, r = 0.0369, z = 0.1132. P = 0.8935). Table 1 gives the
number of legitimate trials by time of night (roughly, first half
from 2000 to 2045, second half from 2045 to 2100), trial set,
trial night and gender. Pooling across all light conditions,
males had more legitimate trials than females (Fig. 3B: P =
0.0265, Mann-Whitney, two-tailed; U-statistic = 30.0).

Figure 4 shows an example of an animal with legitimate
trials under all four light levels. This composite shows a
general trend in behavior, with animals under no light (4A) or
the lowest light (0.0001 pW/cm"; 4B) conditions making
shorter, steadier movements than the sporadic movements of
animals under the highest light (0.15 pW/cm“) condition (4D).
The 0.15 pW/cm" trial contained the highest proportion of
instantaneous velocities greater than 28 mm/s (right side of
4D). Likewise, the 0.01 pW/cm‘ trials occasionally contained
examples of faster instantaneous velocities, as can be seen in
the initial movements depicted in the middle plot of 4C.

The averages of instantaneous velocities for scorpions under
each light level for all legitimate trials are shown in Fig. 5A.
The graph shows similar patterns for the no light and
0.0001 pW/cm^ trials and a flattening of the distribution
pattern for the 0.15 pW/cm^ trials. The pattern for the

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0.01 (iW/cm^

Instantaneous velocity (mm/s)

Figure 4. — Sample behavioral response of an animal to all four light levels. A.-D. Response to no light, 0.0001 pW/cm~, 0.01 pW/cm' and
0.15 pW/cnr, respectively. Left: plots of animal movements in arena; middle: plots of instantaneous velocities during the 10-min trials (lines =
five-point running averages); right: frequency distributions of instantaneous velocities.

0.01 pW/cm" trials is similar to, but slightly below, the no light
and 0.0001 pW/cm“ trials curves.

Figure 5B shows the distribution of the 83 legitimate trials
based on light treatment. We scored each animal as 0 if neither
its first nor its second trial was legitimate, 0.5 if one of its two
trials was legitimate, and I if both of its trials were legitimate.
A Friedman test (nonparametric repeated measures ANOVA)

across these data showed significant variation among the
treatments {P = 0.0274); Dunn’s multiple comparisons test
showed no significant difference between pairs of treatments.
Figure 5C compares the median instantaneous velocity scores
among the light levels (scores were averaged for animals with
two legitimate trials within a given treatment). Seven of the 23
animals had legitimate trials across all four treatments.

GAFFIN & BARKER-SCORPION UV DOSE RESPONSE

117

A

B

no light 0.0001 0.01 0.15

UV level (jiW/cm^)

Figure 5. — Composite behavioral responses to various UV levels.
A. Averaged instantaneous velocity distributions for the four
experimental light levels (mean ± SE). B. Number of legitimate trials
parsed by light level. C. Median instantaneous velocities by light level
for the seven animals included in our repeated measures ANOVA
analysis (mean ± SE).

However, the no-light values for one animal were more than
two standard deviations greater than the mean; we therefore
removed all trials for this animal from our analyses, reducing
our sample size to six. A repeated measures ANOVA showed
significant overall differences among the light levels (Fr =
9.000; P — 0.0218); there was also a pair-wise difference
between the 0.15 |iW/cm“ and no-light treatments {P < 0.05).

DISCUSSION

The results of these studies are clear: using a negative
phototactic behavioral assay, scorpion locomotor behavior is
highest at UV irradiance levels that correspond to sunset.
Adding additional indicators, such as the distribution of
instantaneous velocities and the number of legitimate trials by
light treatment, suggests that the UV response threshold
detected by this assay is between the 0.15 pW/cm^ and
0.01 pW/cm^ light responses. Pooling across all light treatments,
we also found a difference in the activity of males and females,
which is to be expected since these trials were conducted at the
end of the mating season when males are more active on the
surface and tracking females (Bradley 1988).

This study demonstrates that scorpions respond differently
to different UV levels experienced during the normal activity
time of P. utahensis, the time at sunset when they normally
move to the thresholds of their burrows (Gaffm 2011). Since
the assay used in these trials measures scorpion locomotor
behavior, the absolute detection sensitivity to UV could be
much lower. In addition, these studies suggest that relatively
high UV inhibits normal scorpion locomotion, indicated by
the low number of legitimate trials under 0.15 pW/cm" and
0.01 pW/cm^ levels. This inhibition affects the sensitivity of the
assay because many animals did not move at all during the 10-
min trials at high UV irradiation. Those data were dropped
from the analyses (59 of 92 trials = 64%).

Several recent studies suggest that scorpion cuticle plays a
role in UV detection (Kloock et al. 2010; Camp & Gaffm 1999;
Blass & Gaffm 2008; Gaffm et al. 2012). Kloock et al. (2010)
found that scorpions that had their lluorescence compromised
by photo-bleaching made more transitions between UV light
exposed and unexposed regions of Petri dish arenas than
untreated scorpions; also, fluorescent scorpions reduced their
activity under UV light at intensity levels similar to what we
present here. The authors discuss the possibility that scorpion
fluorescence is related to the detection of moonlight and the
decision to avoid foraging on nights with high moon
illumination; scorpions are less active on the surface during
moonlit nights than during moonless nights (Skutelsky 1996).
However, our results do not support this notion since animals
under UV levels that match the UV composition of full moon
nights showed no difference in behavior from animals under
no-light conditions. This does not mean that the animals are
not detecting and using UV at full moon levels; it simply
suggests that it does not act as a deterrent.

Gaffm et al. (2012) found similar locomotor responses of
scorpions under UV and green wavelengths at the 0.15 pW/
cm^ intensity and differences in behavior under the two
wavelengths when the eyes were covered. The behavior of
eyes-blocked animals changed more when exposed to green
light than to those animals exposed to UV light, suggesting a
possible role for the fluorescent cuticle in UV detection. Gaffm
et al. (2012) suggested the cuticle could serve as a whole-body
light detector for purposes of finding shelter. That is, shading
of any part of the cuticle stimulated by UV would represent
overhead shelter (such as a twig or blade of grass), and a
reflexive turning toward the shaded side would move the
animal’s body under the shelter.

We made several changes to earlier behavioral assay
protocols to improve the efficiency of the trials. Most

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THE JOURNAL OF ARACHNOLOGY

significantly, we dramatically improved the scorpion image by
removing interference from the Plexiglas stage and glare from
direct IR projection of the recording camera. By using diffuse
IR from the side, the image cleared to a point that we could
easily detect scorpions using the public domain ImageJ image-
processing program. Once resolved from the background, the
animals were accurately tracked via Fiji’s Mtrack2 plugin.
Automated tracking greatly reduces scoring time and removes
the potential for human bias. We think similar tracking will be
useful for additional scorpion studies, including those
behaviorally testing for and identifying chemicals that make
up scorpion pheromone secretions (Taylor et al. 2012).

Additional steps need to be taken to determine whether
scorpion fluorescence has an adaptive function in UV
detection. Although our assay has been useful for detecting
a response and a potential deterrence threshold, it is also
laborious, time consuming and requires a large number of
trials to register an effect. It would be helpful to develop a
behavioral assay that measures individual responses to light of
various intensities and wavelengths, perhaps focused on
various body regions. Also, it could be useful to reduce the
fiuorescence through bleaching (Kloock 2009) or other means
to see if the behaviors we observe can be compromised.
Finally, some members of the family Chaerilidae Simon have
been recently reported to lack the fiuorescence phenomenon
(Lourenqo 2012). These animals could be useful in compar-
ative light detection assays with normally fiuorescent animals.

ACKNOWLEDGMENTS

We thank Lloyd Bumm for technical assistance in delivering
and measuring UV light levels, Ashley Davis and John Little
for help with pilot studies of UV induced behavior, and
Marielle Hoefnagels, Caleb Cosper, Danielle Vinnedge, Jay
Vinnedge, Elise Knowlton and Matthew Taylor for valuable
discussions and for critically reviewing the manuscript. We
give special thanks to Greg Blass for the early forays into the
investigation of scorpion fluorescence that helped spawn this
line of research. Einally, we thank the staff of the University of
New Mexico Sevilleta Field Station for laboratory space and
research support; in particular, we thank Ben Specter for help
in refining the behavioral apparatus. This work was supported
by funds from the University of Oklahoma Life account.
These experiments complied with current laws governing
animal care and use in the United States.

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Manuscript received 14 December 2012, revised 22 December 2013.

2014. The Journal of Arachnology 42:119-122

SHORT COMMUNICATION

Fine structure of the stinger (aculeus) in Enscorphts

Rainer Foelix, Bruno Erb and Matt BraunwaMer; Neue Kantonsschule Aaraii, Biology Department, Electron Microscopy
Unit, Zelgli, CH=5000 Aarau, Switzerland. E-mail: r.foelix@gmx.ch

Abstract. A scorpion’s last metasoma! segment (telson) consists of a bulbous base that contains two venom glands and a
curved tip (aculeus) where two venom ducts open to the outside. These two openings lie laterally just before the very tip of
the aculeus; to see both of them at the same time, the stinger has to be looked at “tail-on” from the dorsal side. The two
venom ducts have a distinct cuticular lining, which can be recognized in a transparent exuvia as long tubes (1 mm)
extending from the distal pores back to the venom glands. Whereas the proximal bulb has many long sensory hairs on its
surface, the distal aculeus is very smooth but contains small pits with tiny club-shaped hairs. These are probably contact
chemoreceptors. The advantage of such sunken sensory hairs is certainly that the stinger can penetrate into prey (or foe)
but can still perceive mechanical or chemical stimuli. Additionally, the aculeus bears several slit sensilla and numerous fine
pores of unknown function. The aculeus is thus not only a well-adapted injection device but also contains sensory
structures, which provide information on mechanical and chemical input.

Keywords: Scorpions, stinger, aculeus, fine structure

It is common knowledge that a scorpion delivers its venomous sting
with the tip of its tail (Hjelle 1990; Braunwalder 2005; Mahsberg et al.
2012). However, many arachnologists are unaware that there are in
fact two small venom openings near the very tip. This is
understandable because these pores are quite small (less than 10 pm
in diameter) and hard to detect within the dark tip of the stinger. It is
remarkable that some of the very early microscopists had already
noted both openings. For instance, Antoni van Leeuwenhoek (1700)
described in a letter to the Royal Society: “... when we observe the
sting through the magnifying glass, we find that the sting has an opening
on either side close to the sharply pointed part ...”. A more detailed
picture was given by Maupertuis (1733) by conducting a small
experiment, namely squeezing the bulb of the stinger: “Si I’on presse
fortement la fiole, ...on voil la liqueur qu’elle contient, s’ echapper d
droite & d gauche, par ces deux trous.” (If one presses the bulb

strongly . . . one can see the liquid that it contains, exiting on the left
and right side, through two holes.”) Later, Joyeux-Laffuie (1883)
made histological sections of the stinger (telson) and provided
illustrations of the tip (aculeus) in lateral and dorsal views. Modern
microscopical techniques have rarely been used to examine a
scorpion’s stinger, perhaps with the exception of a SEM (scanning
electron microscope) picture showing both venom openings with
congealed venom oozing out (Farley 1999). We have looked at the
stinger of several species of Euscorpius using light microscopy and
SEM to illustrate the dual opening clearly, and to study the overall
organization of the exterior and interior of the aculeus.

We used mostly exuviae from Euscorpius flavicaudis De Geer 1778,
but also from the species E. italicus Herbst 1800, E. germanus Koch
1837, E. alpha Caporiacco 1950 and E. tergestinus Koch 1837. All
specimens were from scorpions bred in captivity by one of the authors

Figures 1-3. — 1, Lateral view of the last tail segment (telson) in E. flavicaudis showing a bulbous base proximally and the curved stinger
(aculeus) distally. The venom exits near the tip of the aculeus (arrow). Several sensory hairs cover the bulb, but are lacking on the smooth
aculeus. 2, Aculeus tip laterally (E. flavicaudis), showing a subterminal venom opening (arrow). A corresponding opening would be visible on the
other side. 3, A dorsal view of the aculeus tip {E. flavicaudis) shows two elongated venom openings side by side (arrows). Note several small
dimples, containing very short sensory hairs.

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Figures 4-6. — 4, A bleached stinger (E. flaviccmdis) reveals a rather
solid tip and two cuticular tubes inside, which originate from the two
venom glands. 5, Higher magnification of the initial part of the venom
ducts. A very delicate collar (arrowhead) makes the connection to the
distal end of the venom glands lying inside the bulb of the telson.
6, UV illumination of the telson (E. italiciis) causes a bright green
lluorescence, except for the distal stinger, which remains black. Note
the sharp borderline (arrow) between the fluorescent and the non-
lluorescent part of the stinger. (Photo by Bastian Rast).

(MB). For light microscopy, stingers were bleached in lactic acid for
several hours, washed, and then immersed in 70% alcohol for bright
field, dark field and phase contrast examination. Photographs were
taken with a digital camera (Canon 600D) attached to a Leitz Diaplan
microscope. For scanning electron microscopy (SEM) stingers were
dissected under 70% alcohol, then dehydrated in acetone, and after
air-drying, carefully mounted and oriented on aluminum stubs. After
sputter coating with gold they were examined in a Zeiss DSM 950;
digital photographs were taken at 20-5000x.

The last tail segment, the telson, measures 5-6 mm in length in
Eiiscorphis species. Its base is bulbous and contains the two venom
glands; its distal end, the aculeus, is curved and narrows into a needle-
like tip. Microscopic examination shows that the bulb is covered with
sensory hairs, mostly on the ventral side. In contrast, the distal
aculeus appears smooth and does not bear long sensory hairs (Fig. 1).
However, at high magnification small dimples containing tiny club-
shaped sensilla become visible (Figs. 2, 3); they are surrounded by
numerous tiny pores (ca. 0.1 pm diameter; Figs. 3, 7, 9), which can
only be discerned under the electron microscope. These nanopores
occur only on the distal half of the aculeus and may be related to an
equally restricted network of nanometer canals, which are involved in
the deposition of heavy metals (Schofield et al. 2003; see below).

The most conspicuous features of the aculeus are the venom openings,
which are located laterally, about 0.1 mm away from the very tip of the
stinger. Normally, only one opening is visible, because the stinger is
usually seen from the side (Fig. 2). Only if the telson is viewed from
behind (“tail-on”) can both venom openings be observed in one picture.
Although this can be convincingly demonstrated with the SEM (Figs. 3,
7), it is almost impossible with the light microscope. Firstly, the diameter
of these pores is only 6-7 pm, and secondly, the cuticle of the aculeus tip
is rather dark and almost solid, thereby obscuring fine structural details.
We were partly successful, however, if we used preparations that had
been bleached for several hours in lactic acid. In those cases one can
vaguely see the actual venom openings near the stinger’s tip and, much
more clearly, the two venom ducts inside the aculeus, leading back to
venom glands (Figs. 4, 5). This distinct visibility is due to a cuticular
lining of the venom ducts, which is preserved during ecdysis. These ducts
are about 1 mm long and 30 pm in diameter; the wall thickness is about
1 pm. It appears that each duct is connected to the distal end of the
venom gland by a fine cuticular collar (Fig. 5).

Because we did not know whether the two venom ducts would
eventually fuse distally into one channel, we cut off the very end of the
aculeus tip with fine scissors and looked at the cross-sectioned stinger
under the SEM (Fig. 8). All our preparations exhibited two separate
venom ducts, where the medial walls touched but never fused. Both
tubes were embedded in a slightly porous cuticle, which was
peripherally surrounded by the solid wall of the stinger. Thus, despite
its seemingly delicate nature, the aculeus tip must be rather rigid and
resistant to mechanical stress.

As mentioned above, the distal-most mm of the aculeus bears no
projecting sensory hairs, but only tiny sunken sensilla and very small
pores - both of which are not present on the proximal telson (bulb).
However, we did observe a few larger pores that are most likely
openings of dermal glands (Fig. 11). And we also found a few slit
sensilla with their slits oriented perpendicular to the long axis of the
aculeus (Fig. 13). A few short hair sensilla (40 pm long. 4 pm in
diameter; Fig. 12) were detected in the proximal part of the aculeus.
Based on their morphology (blunt tip, distinct socket), they could be
contact chemoreceptors (Foelix & Schabronath 1983; Foelix 1985;
Gaffin & Brownell 2001).

A remarkable property of scorpion cuticle is its bright green
fluorescence in response to longwave ultraviolet (UV) illumination
(Pavan 1954). Although this is true for the entire body cuticle, there is
one exception: the distal (black) end of the aculeus does not fluoresce.
Under daylight illumination the transition into the dark aculeus tip
appears as a gradual change, but under UV light there is a sharp
borderline between the proximal fluorescent telson and the complete-
ly black, non-fluorescent aculeus tip (Fig. 6). We assume that the
cuticle of that distal region lacks the fluorescent substances (P-
carboline and coumarin) that are normally present in the body cuticle
(Stachel et al. 1999; Frost et al. 2001). Unfortunately, hardly anything
is known about the possible biological significance of fluorescence in
scorpion cuticle (Gaffin et al. 2012).

A conspicuous feature of the aculeus is the smooth surface of the
last millimeter of its tip. This is certainly advantageous for an easy
and relatively deep penetration into the prey’s tissues. Any hair
sensilla projecting from the surface are restricted to the proximal part
of the telson. However, several tiny club-shaped sensilla do occur in
the tip region, but they are hidden in tiny dimples. It is quite likely
that they represent contact chemoreceptors. Very similar sunken
sensilla were found on the jaws (maxillae) of ant lions, which are also
used for venom injection into prey (R. Foelix unpublished results).

Additionally, there are several single slit sensilla embedded in the
cuticle of the aculeus, which provide information on mechanical
strain. The aculeus is thus not merely an injection device, but is also
able to integrate mechanical and chemical stimuli. This is also
indicated by the presence of a telson nerve running between the two
venom ducts (Farley 1999).

FOELIX ET AL.— FINE STRUCTURE OF SCORPION STINGER

121

Figures 7, 8. — 7, A posterior view of the aculear tip (£. flavicaiidis) showing the two venom openings and small dimples containing sunken
sensory hairs. 8, If the very tip of the aculeus (E. italicus) is snipped off with scissors, the two venom ducts are seen in cross-section; they are
surrounded by a porous cuticle inside and a solid cuticle wall in the periphery.

The function of the dermal glands on the telson is not really known,
but it is possible that they play a role in courtship. During early
courtship the male scorpion often stings the female into her joint
membranes, leaving the aculeus there for up to 10 min (Francke
1979). It is not clear whether this implies an actual venom injection or
a transfer of other chemical substances, which may originate from the
aculear dermal glands. Another possibility is that these dermal glands
produce a sex pheromone in females; it has been reported that female
cuticle extracts induce courtship patterns (tail wagging and pedipalp
reaching) in male scorpions (Gaffin & Brownell 1992).

The venom ducts are lined by a thin cuticle and are firmly enclosed
by an inner porous and an outer solid cuticle. Thus, the aculeus tip is
rather solid and hence resistant to mechanical damage — a feature that
was already pointed out in early descriptions (^‘fort dur”; Maupertuis
1733). On the other hand it was also claimed that this hardness makes
the stinger brittle and that the very tip can easily break off (Joyeux-
Laffuie 1883). However, our own observations on hundreds of
scorpions showed only a few instances of broken tips and we therefore
conclude that the aculeus tip is not that vulnerable. An increased
hardness of certain cuticular parts in arthropods is often achieved by

Figures 9-13. — 9, Close-up of one venom opening (£. flavicaudis), three sunken sensory hairs and many small cuticular pores. 10, High
magnification of Fig. 9, showing a sunken sensory hair with a folded joint membrane at its base. 1 1, Relatively large pores with a split duct in the
center most likely represent openings of dermal glands. 12, Relatively short sensory hairs projecting from a distinct socket occur on the proximal
aculeus but not in the distal tip region. 13, Several slit sense organs are found on the stinger's surface, lying perpendicular to the long axis of
the telson.

122

THE JOURNAL OF ARACHNOLOGY

an accumulation of heavy metals {zinc, manganese, iron); for
example, in insect mandibles. This has also been demonstrated in
scorpion mouthparts, tarsal claws and the stinger (Schofield 2001,
2005). Since it is known that zinc incorporation into ant mandibles
increases their hardness about three-fold (Schofield et al. 2003), it is
very likely that the high zinc or manganese concentration in the
scorpion’s aculeus will also render it more resistant to wear and tear.
Interestingly, this distal region of heavy metal accumulation is exactly
the same region that is non-fluorescent under UV illumination, yet so
far, we do not know whether there is any causal relationship between
those two phenomena.

It is noteworthy that the venom openings are located subterminally;
that is, not at the very tip as in a pipette, but on both sides of the
aculeus (almost 100 pm from the tip). This arrangement is well known
from other injection devices, such as the cheliceral fangs of spiders,
the venom teeth of vipers or in hypodermic needles (Foelix 2011).
From a technical viewpoint this is a superior solution because a
lateral pore opening is mechanically more stable and cannot be
clogged by tissue when pushed into the prey.

ACKNOWLEDGMENTS

We are indebted to Samuel Furrer (Zoo Zurich) for pointing out
the lack of fluorescence in the tip of the scorpion’s stinger, and to
Bastian Rast for taking excellent photographs of stingers under UV
light. We are grateful to the Neue Kantonsschule Aarau for letting us
use the facilities of their electron microscopy laboratory. And we
thank Douglas Gaffin for help with the literature search, and Jerome
Rovner and Benno Wullschleger for critically reading our manuscript.

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Braunwalder, M.E. 2005. Scorpiones (Arachnida). Fauna Helvetica
13. Centre Suisse de cartographic de la Faune, Neuchatel,
Switzerland.

Farley, R.D. 1999. Scorpiones. Pp. 1 17-222. hi Microscopic Anatomy
of Invertebrates. (F.W. Harrison & R.F. Foelix, eds.). Volume 8A:
Chelicerate Arthropoda. Wiley-Liss, New York.

Foelix, R.F. 1985. Mechano- and chemoreceptive sensilla. Pp. 1 18-137.
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Foelix, R.F. 2011. Biology of Spiders. 3''*^ ed., Oxford University
Press, New York.

Foelix, R.F. & J. Schabronath. 1983. The fine structure of scorpion
sensory organs. I. Tarsal sensilla. Bulletin of the British
Arachnological Society 6:53-67.

Francke, O.F. 1979. Observations on the reproductive biology and
life history of Megacormus gertschi Diaz (Scorpiones: Chactidae:
Megacorminae). Journal of Arachnology 7:223-230.

Frost, L.M., D.R. Butler, B. O’Dell & V. Fet. 2001. A coumarin
as a fluorescent compound in scorpion cuticle. Pp. 363-368. In
Scorpions 2001 In Memoriam Gary A. Polis. (V. Fet & P.A.
Selden, eds.). British Arachnological Society, Burnham Beeches,
Bucks, Great Britain.

Gaffin, D.D. & P.H. Brownell. 1992. Evidence of chemical signaling
in the sand scorpion, Paruroctonus mesaensis (Scorpionida:
Vaejovida). Ethology 91:59-69.

Gaffin, D.D. & P.H. Brownell. 2001. Chemosensory behavior and
physiology. Pp. 184-203. hr. Scorpion Biology and Research. (P.H.
Brownell & G.A. Polis, eds.). Oxford University Press, Oxford.
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2012. Scorpion fluorescence and reaction to light. Animal
Behaviour 84:429^36.

Hjelle, J.T. 1990. Anatomy and Morphology. Pp. 54-56. In The
Biology of Scorpions. (G.A. Polis, ed.). Stanford University Press,
Stanford, California.

Joyeux-Laffuie, J. 1883. Appareil venimeux et venin du Scorpion. A.
Hennuyer, Paris.

Leeuwenhoek, A. 1700. Alle de brieven. Deel 13: 1700-1701. (L.C.

Palm, ed.). N.V. Swets & Zeitlinger, Lisse 1993.

Mahsberg, D., R. Lippe & S. Kallas. 2012. Skorpione. Natur & Tier
Verlag, Munster.

Maupertuis, J. 1733. Experiences sur les scorpions. Pp. 223-229. In
Histoire de V Academie Royale des Sciences. Annee 1731.
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Pavan, M. 1954. Primi dati per la caraterizzazione della sostanza
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Schofield, R.M.S. 2001. Metals in cuticular structures. Pp. 234-256.
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Schofield, R.M.S. 2005. Metal-halogen biomaterials. American
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Schofield, R.M.S., M.H. Nesson, K.A. Richardson & P. Wyeth. 2003.
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and a scorpion. Journal of Insect Physiology 49:31^4.

Stachel, S.L., S.A. Stockwell & D.L.V. Vranken. 1999. The
fluorescence of scorpions and cataractogenesis. Chemistry and
Biology 6:531-539.

Manuscript received 25 August 2013, revised 19 November 2013.

2014. The Journal of Arachnology 42:123-125

SHORT COMMUNICATION

Intense leg tapping behavior by the harvestman Mischonyx cuspidmtus (Gonyleptidae):
an uidescribed defensive behavior in Opiliones?

Barbara Crespo Dias, Elene da Silva Souza, Marcos Ryotaro Hara and Rodrigo Hirata Willemart': Laboratorio de
Ecologia Sensorial e Comportamento de Artropodes (LESCA), Escola de Artes Ciencias e Humanidades, Universidade
de Sao Paulo, Rua Arlindo Bettio, 1000 - Ermelino Matarazzo - 03828-000, Sao Paulo, SP, Brazil

Abstract. We describe for the first time the behavior “Intense Leg Tapping (ILT)” being used in a prey-predator context
between the Neotropical harvestman Mischonyx cuspidatus (Roewer 1913) and the syntopic spider Ctenus ornatus

(Keyserling 1877). Previously, the harvestman’s repeated fast
during conspecific male-male interactions. We suggest it has

Keywords: Ctenidae, defense, deimatic, harvestmen, spider

Prey species often produce signals toward predators according to
the sensory modality these predators use. Several invertebrates and
vertebrates use colors or body marks against visual diurnal predators
(Eisner et al. 2005; Caro 2009; Stevens & Ruxton 2012); moths
produce sounds against nocturnal predators that use ecliolocation to
hunt, such as bats (Conner & Corcoran 2012), and squirrels increase
the temperature of their tail specifically against predators sensitive to
minimum temperature variation, such as vipers (Rundus et al. 2007).
Such signals function either to warn the predator of a potentially
dangerous defense or distastefulness (the case of aposematism) or to
startle the predator (the case of deimatic behaviors), causing it to
hesitate or give up attacking (Edmunds 1974).

Ctenid spiders are nocturnal hunters that feed on a variety of
arthropods by quickly jumping onto them, biting and injecting venom
(Hofer et al. 1994; Wullschleger & Nentwig 2002). Though they may
detect visual stimuli, eyes are not necessary to find prey: they rely
almost exclusively on substrate and air borne vibrations and air
displacement to catch prey detected by the very sensitive metatarsal
organs and trichobothria on their legs (Barth 2002). Therefore, if a
prey was to use any deimatic behavior or send warning signals to such
spiders, one could expect it to use air displacements or vibrations.

Harvestmen are known to defend themselves in several ways. The
list includes anachoresis, the use of chemical deterrents from
repugnatorial glands, fleeing, feigning death, leg autotomy and
retaliation (Gnaspini & Hara 2007). A heavy armature has also been
shown to be effective (Souza & Willemart 2011; Dias & Willemart
2013) and based on their colorful body, some species have been
suggested to be aposematic (Gnaspini & Hara 2007; Pomini et al.
2010). In addition to these, the harvestman Eumesosoma roeweri
(Goodnight & Goodnight 1943) (Sclerosomatidae) has been shown to
avoid chemicals from predators (Chelini et al. 2009). Herein we
describe a putative new defensive behavior in the order Opiliones,
namely the intense dorso-ventral movements of legs II (Intense Leg
Tapping - ILT, sensu Willemart et al. 2009a) of Mischonyx cuspidatus
(Roewer 1913) against a predator sensitive to air displacements and
vibrations, the large wandering spider Ctenus ornatus (Keyserling
1877) (Ctenidae) (Fig. 1).

Individuals of the harvestman M. cuspidatus and the spider C.
ornatus were collected at the Reserva da Cidade Universitaria
Armando Salles de Oliveira, Sao Paulo State (23°33'S, 46°43'W).
They were maintained individually in plastic containers (12 X 8 X
4 cm height for the harvestmen and 20 cm (diameter) X 8 cm height

' Corresponding author. E-mail: willemart@usp.br

dorsoventral movements of legs II had only been described
a defensive function.

for spiders) with soil on the bottom and cotton rolls for humidity. The
harvestmen were fed on wet dog food and the spiders on crickets.
Temperature was ambient (20-25°C) and the light cycle was natural
(approximately 12:12 light:dark cycle).

We made the behavioral observations from May to September 2009
(spiders: six subadult males and 36 adult females; harvestmen: 42
individuals, 23 adult males and 19 adult females) and again in
January 2011 (spiders: 10 females, three adult males and two
immature individuals; harvestmen: three adult females and 1 1 adult
males) under dim light, between 1800 and 2300 h. All the spiders were
starved for 25-30 days before the observations. Each individual spider
and harvestman was used only once, and the sequence was
detennined in random order. The circular arena used for the tests
(20 cm diameter X 8 cm height) had humid soil on the bottom. A
spider was introduced into this arena 8 h before the trial to minimize
stress, and the harvestman was introduced in a vial as far as possible
from the spider, allowed to acclimate for 2 min and then released. We
used a Sony Handycam DCR-TRV361 ‘nightshot’ (hand held to
allow recording from better angles, only one observer) to record the
behaviors related to the approach between the two animals, the
physical interaction and the 10 s subsequent to the interaction to
determine if the spider would start eating the prey. Results are
presented as “mean ± S.D.”

We observed four female and eight male M. cuspidatus displaying
ILT against adult females of C ornatus, out of 56 harvestmen
observed. Intense Leg Tapping consisted of very rapid dorso-ventral
movements of legs II, with either one or both legs. Harvestmen
performed one or several bouts (sensu Lehner 1998) of ILT. The total
number of bouts, pooling across all individuals and single and double
leg bouts, was 74, 43 pointing toward the spider and 31 not pointing
toward the spider. Among harvestmen that displayed ILT, males
displayed 1.67 ± 0.5 single leg bouts and 2.6 ± 2.2 double leg bouts
per interaction with spiders. Females displayed 1.75 ± 0.95 single leg
bouts and 1.25 ± 1.26 double leg bouts per interaction with spiders.
There was no difference between males and females (Mann- Whitney
test: single leg bouts: U = 28.0, P = 0.938, n = 4 and 9; double leg
bouts: U = 19.0, P = 0.283, n = 4 and 8). The mean duration of bouts
was 4.96 ± 0.57 s (min: 3.4; max: 6.3) (data combined for all 74
bouts). Six harvestmen did not displace while exhibiting ILT, four
moved toward the spider and two moved in the opposite direction.
Eight harvestmen made contact with the spider before displaying ILT
(in four cases contact was established because the spider attacked the
harvestmen) and the remaining four did not. The distance between the
anterior portion of the prosoma of spiders and the tip of the closest

123

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Figure 1. — a) The harvestman Mischonyx cuspidatus touches the
spider Ctcmis ornatus with its leg II; b) the spider jumps onto the
harvestman; c) the spider retreats and the harvestman starts
displaying intense leg-tapping (ILT); d) the spider walks away.

leg of the harvestmen when ILT started was 4.4 ± 2.3 cm (max: 9.3,
min: 1.8 cm, n = 12). In six cases, the spider did not move after the
harvestman started displaying ILT. Five spiders moved in the
opposite direction and one spider attacked.

Intense Leg Tapping is performed during male-male fights in the
species Neosadocus maximus Giltay 1928 (Gonyleptidae) (Willemart
et al. 2009a). The function of the behavior in that context remains
unknown, but it was clear that the males only started ILT when
fighting with conspecifics. Now that we have observed the same
behavior by harvestmen in a predator-prey interaction, a very
different context, we suggest that ILT is also a defensive behavior.
We propose two main pieces of evidence. First, we have
accumulated approximately 340 hours of behavioral observations
that were taped in distinct contexts (resting, walking, interacting
with conspecifics of both sexes, interacting with heterospecifics,
foraging, etc.), of several harvestmen species (at least 13), in
addition to field observations since 1999 that were not taped (R.H.
Willemart and members of our laboratory, personal observations).
Intense leg tapping had never been observed except in the context of
male-male fighting and after making contact with a predator (see
Willemart et al. 2009a; this paper). Second, the sensory modality
exploited by the harvestman when displaying ILT is exactly what
these spiders use when hunting, namely air displacement and
vibrations (Barth 2002 and references therein). It could therefore be
analogous to animals that use color, sounds or variation in body
heat as a deimatic behavior or when signaling to predators that use
each of these specific sensory modalities to hunt (Stevens 2007;
Rundus et al. 2007; Ruxton 2009).

In almost half of the bouts in which ILT was observed, the
behavior was not directed toward the spider. We believe this reflects
the limited sensory abilities of the harvestmen and its inability to
accurately detect nearby arthropods (Willemart et al. 2009b). We
unfortunately do not have solid data on how the predator is affected
by ILT. This is also the case for several mechanisms that have been
considered to be defensive in harvestmen, such as nipping behavior,
pinching with chelicerae and pedipalps, tanathosis and aposematism
(Gnaspini & Hara 2007 and references therein). The fact that ILT has
also been observed in conspecific fights is not evidence against the
defensive hypothesis. Deer, antelope, and other mammals with horns
or antlers use these weapons in male-male fights and for defense
(Andersson 1994). Since both sexes of M. cuspidatus have been
observed performing ILT, ILT in the sexual context (only males have
been observed doing it) could be an exaptation (sensu Gould & Vrba
1982) of ILT in the defensive context. This would be more
parsimonious than to believe that it first appeared in a sexual context
among males, after which they also started using in a defensive
context, after which it then appeared again in females for defensive
purposes.

An alternative hypothesis could be that these movements have a
sensory function and that harvestmen are actually trying to gain
information about the predator. Based on previous studies on the
sensory biology of harvestmen (e.g., Willemart & Chelini 2007;
Willemart et al. 2009b), this would be very unusual since laniatorid
harvestmen use slow waving movements of legs I and II in the air or
gently tap the substrate when stimuli are provided. These are very
different from ILT.

Another alternative hypothesis is that ILT against predators is a
displaced, out of context behavior with its origin in male-male fights,
but that females also exhibit this behavior. Further studies with
observations of ILT in multiple contexts would weaken the defensive
hypothesis and maybe strengthen the “displaced behavior” hypothesis.

We have documented that ILT occurs in the context of prey-
predator interaction. The defensive hypothesis could be further tested
by examining exactly what stimuli trigger it or how it affects their
predators, but alternative hypotheses should not be discarded at this
point.

DIAS ET AL.— PUTATIVE NEW DEFENSIVE BEHAVIOR IN HARVESTMAN

125

ACKNOWLEDGMENTS

Erika Portela, Guilherme Gainett and Guilherme Pagoti helped to
collect the spiders. Two anonymous reviewers and Linden Higgins
greatly improved the manuscript. This study was funded by Fundagao
de Amparo a Pesquisa de Sao Paulo (FAPESP).

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Caro, T. 2009. Contrasting coloration in terrestrial mammals.
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Manuscript received 27 February 2013, revised 20 June 2013.

2014. The Journal of Arachnology 42:126-129

SHORT COMMUNICATION

Seasonal patterns of microhabitat selection by a sub-tropical whip spider, Phrynus longipes^ in the

Luquillo Experimental Forest, Puerto Rico

Caroline A. Curtis' and Christopher P. Bloch^: 'Graduate Program in Organismic and Evolutionary Biology, University
of Massachusetts Amherst, Amherst, Massachusetts 01003 USA. E-mail: cacurtis@cns.umass.edu; -Department of
Biological Sciences, Bridgewater State University, Bridgewater, Massachusetts 02325 USA

Abstract. Phrynus longipes (Pocock 1894) is a top predator among arboreal invertebrates in the Luquillo Mountains of
Puerto Rico, but many aspects of its ecology remain poorly understood. We sampled four of the most abundant tree species
in the Luquillo Mountains during the dry and wet seasons of 2008 to evaluate microhabitat preferences of this species. In
the dry season, P. longipes occurred significantly less frequently on a palm, Prestoea acuminata var. montana (Arecaceae),
than the other tree species. Carapace length and the diameter of the tree on which an individual was found were positively
correlated, suggesting competition for substrates. Microhabitat selection shifted in the wet season. Individuals occurred as
frequently on P. acuminata as on any other species. The seasonal shift in substrate use could result from altered distribution
or abundance of prey, an ontogenetic shift in substrate preference or greater competition arising from an increased
abundance of P. longipes.

Keywords: Amblypygi, resource availability, competition, fitness, arboreal predators

Habitat selection contributes to individual fitness by affecting the
ability to acquire resources, survive and reproduce (Rosenzweig 1981;
Huey 1991). Furthermore, intra- and interspecific competition
influence the ability of individuals to occupy optimal habitats
(Whitham 1980; Stamps 1991). Habitat selection is likely to be
affected by temporal changes in food availability, reproductive
activity and the emergence of young, ail of which occur seasonally
(Wolda 1978). Therefore, a comprehensive understanding of habitat
selection requires comparisons among seasons.

Whip spiders (Amblypygi) are nocturnal ambush predators that use
vertical substrates, such as trees or rocks, for hunting and nearby
crevices as mating or retreat sites. Adults of many species defend a
small home range near the structure they employ for hunting
(Weygoldt 2000). Patterns of habitat use are poorly known for most
amblypygid species, but some factors associated with amblypygids
that inhabit tropical forests have been identified. In the central
Amazon Basin, Heterophrynus kmgicornis (Butler 1873) occurs most
often on large trees with buttressed roots (Dias & Machado 2007). In
contrast, Porto & Peixoto (2013) found that individuals were more
likely to maintain their territory on a tree if a burrow was present at
its base, regardless of tree size. The presence of termite nests at a tree
base is another indicator of higher H. longicornis abundance
(Carvalho et al. 2012). In Costa Rica Phrynus parvulus Pocock 1902
selects habitats based on tree surface area, the presence of buttresses
and moss cover (Hebets 2002). Large trees with buttresses might be
preferable if they provide high prey density in the deep leaf litter
between roots (Heyer & Berven 1973), or areas for mating or retreat
sites.

Phrynus longipes (Pocock 1894) inhabits caves and forests on
Hispaniola, Puerto Rico and the Virgin Islands (Quintero 1981), but
relatively little is known about its ecology or behavior. A previous
study of P. longipes in the Luquillo Mountains of northeastern Puerto
Rico found individuals most often on the sierra palm, Prestoea
acuminata var. montana (Arecaceae) (Bloch & Weiss 2002). This is
unusual compared to the habits of other whip spiders, as P. acuminata
has a relatively small diameter compared to other trees in this forest
and lacks buttresses. Two explanations could account for this
unexpected pattern of habitat use. First, neither the sampling
methodology nor the statistical analyses of Bloch & Weiss (2002)

explicitly accounted for tree species abundance, so P. longipes may
have appeared to prefer P. acuminata simply because it was the most
abundant substrate within the sampling plots. Second, the extensive
anthropogenic and natural disturbance history of northeastern Puerto
Rico might contribute to unique habitat characteristics and popula-
tion dynamics. Until the 1930s, much of the land below 600m was
used for farming, logging and coffee cultivation (Thompson et al.
2002). In addition, hurricanes of moderate to high intensity hit the
forest every 50-60 years on average, and storms of lesser intensity
occur more frequently (Scatena & Larson 1991). This disturbance
regime would have necessitated adaptation by organisms to a
heterogeneous, frequently changing environment, and could have
affected patterns of habitat use.

This study evaluated whether P. longipes in the Luquillo Mountains
actually selects substrates differently from other species of forest-
dwelling whip spider, or if the unusual pattern of habitat use observed
by Bloch & Weiss (2002) emerged simply because of a sampling bias.
We compared use of P. acuminata to use of three other abundant trees
in the Luquillo Mountains to determine whether P. acuminata was
occupied more than expected by chance or was simply used in
proportion to abundance. We also compared substrate use between
dry and wet seasons to assess the effects of seasonality.

The Luquillo Experimental Forest (LEF) is an 1 1,300-ha reserve in
the Luquillo Mountains of northeastern Puerto Rico, and is a site in
the National Science Foundation’s Long-Term Ecological Research
Network (Hobbie et al. 2003). This study was conducted near the El
Verde Field Station (18°19'16.83"N, 65°49'10. 13"W), in the north-
western region of the LEF, where elevation ranges from approxi-
mately 300 to 400 m. Total annual precipitation at the El Verde Field
Station averages 335 cm year^' (Heartsill-Scalley et al. 2007).
Precipitation is mildly seasonal, with a relatively dry period from
January to April (McDowell & Estrada Pinto 1988). For 1975-1989,
average monthly precipitation for the dry (January-April) and wet
(May-December) seasons was approximately 22 cm and 35 cm,
respectively.

To determine the most dominant tree species in the area we
sampled based on basal area (Thompson et al. 2002). From these
common species, we selected four of the most abundant within our
sampling area, each of which has a distinct root system. Prestoea

126

CURTIS & BLOCH— MICROHABITAT SELECTION BY PHRYNUS LONGIPES

127

Table 1. — Data for each of the two sampling periods are presented. The number (n) of each tree species (Cs, C. schreheriaiui; Mb, M.
hidentata; Pa, Prestoea acuminata', Sb, Sloanea herteriana) sampled, DBH for each of the four species and two seasons are included, as well as
values combined for both sampling periods. The number of adult and juvenile P. longipes recorded on each species is also listed by tree species
and season.

n

Average DBH (cm)

Maximum DBH (cm)

Phrynus longipes

Adults Juveniles

Dry Season

Cs

25

18.9

36

1

2

Mb

55

19.5

59.6

3

1

Pa

121

10.1

19.1

2

0

Sb

84

7.2

44.6

8

5

Total

285

14

8

Wet Season

Cs

56

20.3

41.3

5

7

Mb

80

18.2

77

3

4

Pa

214

13.6

46

25

36

Sb

115

9.2

38.4

4

18

Total

465

37

65

Combined

Cs

81

19.9

6

9

Mb

135

18.7

6

5

Pa

335

12.3

27

36

Sb

199

8.4

12

23

Total

750

51

73

acuminata has a tightly packed system of prop roots, Cecropia
schreberiana (Urticaceae) has widely spaced branching roots that
angle off from the main stem, Sloanea herteriana (Elaeocarpaceae)
develops buttresses as it grows, and Manilkara hidentata (Sapotaceae)
has a straight trunk devoid of buttresses (Thompson et al. 2002).

Surveys were conducted in March 2008 (dry season) and from June
to August 2008 (wet season). Sampling was conducted by two
researchers and occurred between 20:00 and 02:00. As many trees as
possible of each target species were found, and each was visually
inspected up to a height of 2m, including all visible crevices,
buttresses, and exposed roots. Total body length (TBL, the length
of the abdomen and carapace) of each whip spider was measured
using dial calipers (Table 1). Diameter at breast height (DBH) was
measured using a diameter tape for all trees, regardless of presence or
absence of whip spiders (Table 1).

Currently, there is no documentation of the size at which P.
longipes becomes sexually mature. A congener, Phrynus margin-
emaculatus Koch 1840, is sexually mature when it has reached
approximately half of its average adult size (Weygoldt 2000). Typical
adult size of P. longipes is roughly 35 mm (Weygoldt 2000), so
individuals were considered adults if TBL >18 mm. All remaining
individuals were considered juveniles. Although 18 mm is an arbitrary
threshold, changing this value by a few mm did not substantively alter
the results.

Chi-square contingency table analyses were used to test the null
hypothesis that P. longipes occupied tree species randomly (i.e., in
proportion to abundance). If there was a significant result,
standardized residuals were examined to determine which species
contributed most to the overall significant result. If a standardized
residual exceeded two standard deviations (i.e., —2.0 > z > 2.0), the
associated count was considered to be significantly different from the
expected.

Pearson’s Product-moment Correlation Coefficient was used to test
for an association between TBL of P. longipes and the DBH of trees
on which individuals were found. For each season, analyses were
conducted for all individuals, as well as for adults and juveniles
separately. Further analyses assessed the relationship between TBL
and DBH separately for each tree species.

Microhabitat selection by P. longipes was non-random in the dry
season = 14.24, df = h, P = 0.003; Fig. la). Standardized
residuals indicated that whip spiders were observed more frequently

than expected on S. herteriana (z = 2.61) and less frequently than
expected on P. acuminata (z = —2.38). TBL of P. longipes was
significantly and positively correlated with the DBH of trees on which
it was found (r = 0.68, P = 0.001, n = 22) when all individuals were
included in the analysis and for adults only (r = 0.59, P = 0.026, /i =
14). Furthermore, the significant positive correlation between TBL of
individuals and the DBH of S. herteriana on which they were found
(r = 0.72, P = 0.005, n =13) suggests that, as occurs in other
amblypygid species, larger individuals were selecting and defending
territories on larger trees. However, for the remaining tree species, or
when only juveniles on all trees were considered, TBL was not
significantly correlated with DBH, suggesting that some individuals
were unable to acquire or defend territory on the preferred trees. This
may also correspond to the relatively small size range observed for the
other tree species, especially P. acuminata, which rarely exceeded
20 cm DBH.

In the wet season, substrate use was non-random among the four
tree species (x" = 11.01, df = 3, P = 0.012; Fig. lb). Whip spiders
were observed less frequently than expected on M. hidentata, the
species with the simplest above-ground component of the root system
and the least surface area (z = -2.21). This was also true if the
analysis was restricted to juvenile whip spiders (z = —2.14). Other
species did not differ significantly in frequency of occupancy. TBL
was not significantly correlated with DBH when all individuals and
all trees were tested together (r = -0.05, P = 0.642, n = 102) or for
adults (r = 0.03, P = 0.857, n = 37) and juveniles (r = —0.17, P =
0.179, n = 65) analyzed separately. However, individuals that
occupied S. herteriana displayed a positive correlation between TBL
and DBH (r — 0.59, P = 0.004, n = 22), while TBL was negatively
correlated to DBH for individuals on C. schreberiana {r = —0.63, P =
0.029, n = 12). For individuals found on M. hidentata, a negative
correlation approached significance (r = —0.69, P = 0.085, n = 7),
but TBL of individuals found on P. acuminata was not significantly
correlated to the DBH of the tree on which they were found (/■ = 0.03,
P = 0.828, n = 61).

In the LEF, S. herteriana has similar characteristics (i.e., buttressed
roots and large DBH) to those of trees that are selected by H.
longicornis in northern Brazil (Dias and Machado 2007) and P.
parvulus in Costa Rica (Hebets 2002) and may therefore provide the
same putative benefits. Nevertheless, Bloch & Weiss (2002) report
that a palm, P. acuminata, was the tree most often occupied by

128

THE JOURNAL OF ARACHNOLOGY

Figure 1. — Patterns of substrate use by Phryims loiigipes in the Luquillo Experimental Forest. The proportion of individuals of each tree
species (Cs, C. schreberiaiur, Mb, M. hiclentcitcr. Pa, Prestoea cicuinmatcr, Sb, Sloaiiea herterkmci) occupied by P. longipes. a. Dry season; b. Wet
season. Asterisks above bars indicate significant departures from expected occupancy based on standardized residuals of chi-square
contingency tables.

amblypygids in the LEF. We reconcile the observations of Bloch &
Weiss (2002) with other studies by demonstrating changes in
microhabitat use between sampling periods.

Two lines of evidence are consistent with the hypothesis that large,
buttressed Y berteriana are preferred substrates for P. longipes in the
LEF. First, 5. berteriana was among the most frequently occupied
tree species, regardless of season, especially if only relatively large
trees are considered. In the dry season, P. longipes occupied 86% of S.
berteriana that measured s 20 cm and 53% that measured s 10 cm,
compared to only 7.2% that measured < 10 cm. This pattern was
repeated in the wet season, as P. longipes occupied 75% of S.
berteriana s 20 cm, 58% S: 10 cm, but only 1 1% that were < 10 cm.
Second, the positive correlation between TBL of P. longipes and DBH
of Y berteriana suggests that larger individuals were best able to
defend a territory on large, buttressed trees.

There was a considerable shift in microhabitat use between seasons,
however, with individuals avoiding P. aciiniinata in the dry season but
using it as frequently as C. sclireberiana and 5. berteriana in the wet
season. Several reasonable hypotheses may explain this pattern.
Abundance or quality of prey may change seasonally, making P.
acuminata trunks better hunting sites in the wet season than in the dry
season. For example, foraging activity of P. longipes (Pfeiffer 1996)
and insect abundance (Garrison & Willig 1996) are greater during
rainy than dry periods, suggesting that higher prey capture rates are
possible during such times, even on trees that represent less suitable
habitat during dry periods.

Alternatively, decreased use of P. acuminata in the dry season may
reflect that it is unsuitable for mating, or that an ontogenetic shift in
substrate selection occurred. The courtship display of many species of
whip spider requires a sheltered, level area (Weygoldt 2000), such as
the root surface or the caverns that form under the roots of the most
frequently selected trees. The roots of P. acuminata generally lack
such spaces. The small crevices found between P. acuminata roots are,
however, useful as retreat sites, especially for small individuals, and
may therefore provide higher quality habitat in the non-breeding
season and for juveniles.

The population density of P. longipes was greater during the wet
season than the dry season, as indicated by much higher proportional
occupancy of all four tree species (Figs. la,b). The increase in density
is probably driven by the emergence of juveniles that hatched late in

the dry season or early in the wet season. As a result, competition for
optimal substrates was considerably lower in the dry season than in
the wet season. In the wet season, two adults were observed occupying
the same tree once (on S. berteriana), and an adult was observed on
the same tree as a juvenile in two cases (once each on P. acuminata
and S. berteriana), and multiple juveniles were observed on a single
tree for all species except M. bidentata. This suggests that food was
abundant enough throughout the wet season that it did not influence
habitat selection.

To maximize fitness, individuals must respond to geographic and
temporal changes in resource availability either by selecting a territory
that provides them with high-quality habitat in all seasons or by
moving to a different, higher-quality site when resources decline.
Here, we provide evidence of a shift in substrate occupation from the
wet season to the dry season in a sub-tropical whip spider, P. longipes.
Given the limitations of this study, further research is needed to
determine whether our observations are consistent with a long-term
pattern or if the years we sampled represent an anomaly. Neverthe-
less, our findings are partially consistent with other research on
habitat selection by amblypygids, particularly regarding tree selection
in the dry season. However, the wet-season shift to occupying what
we assume are lower-quality trees indicates that although habitat
characteristics such as tree size are likely to determine whether or not
a tree is suitable habitat, biotic interactions such as those that occur
among P. longipes in the wet season are likely to further influence the
small-scale distribution of a species.

ACKNOWLEDGMENTS

This research was supported in part by grant number DEB-0620910
from the National Science Foundation to the Institute of Tropical
Ecosystem Studies, University of Puerto Rico, and the International
Institute of Tropical Forestry as part of the Long-Term Ecological
Research Program in the Luquillo Experimental Forest. In particular,
we thank M. Willig for the support of his sub-contract through the
University of Connecticut. R. Pitt provided funding for C. Curtis
through the Office of Academic Affairs at Bridgewater State
University. The staff of El Verde Field Station provided valuable
logistical support in Puerto Rico. 1. Castro-Arellano, L. Cisneros, L.
Jones, S. Kieschnick, B. Klingbeil and E. Souza provided assistance in
collecting data.

CURTIS & BLOCH— MICROHABITAT SELECTION BY PHRYNUS LONGIPES

129

LITERATURE CITED

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whipspider Phrynus longipes (Arachnida: Amblypygi) in the
Luquillo Experimental Forest, Puerto Rico: response to natural
and anthropogenic disturbance. Caribbean Journal of Science
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Carvalho, L.S., J. O Gomes, S. Neckel-Oliveira & N.F. Lo-Man-
Hung. 2012. Microhabitat use and intraspecific associations in the
whip spider Heterophrynus longicornis (Arachnida: Amblypygi) in
forest fragments formed by the Tucurui Dam Lake, Para, Brazil.
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Dias, S.C. & G. Machado. 2007. Microhabitat use by the whip spider
Heterophrynus longicornis (Amblypygi, Phrynidae) in Central
Amazon. Journal of Arachnology 34:540-544.

Garrison, R.W. & M.R. Willig. 1996. Arboreal invertebrates. Pp.
183-245. In The Food Web of a Tropical Rain Forest, (D.P.
Reagan & R.B. Waide, eds.). University of Chicago Press,
Chicago, Illinois.

Heartsill-Scaliey, T., F.N. Scatena, C. Estrada, W.H. McDowell &
A.E. Lugo. 2007. Disturbance and long-term patterns of rainfall
and throughfall nutrient fluxes in a subtropical wet forest in Puerto
Rico. Journal of Hydrology 333:472-485.

Hebets, E.A. 2002. Relating the unique sensory system of amblypy-
gids to the ecology and behavior of Phrynus parvulus from Costa
Rica (Arachnida, Amblypygi). Canadian Journal of Zoology
80:286-295.

Heyer, W.R. & K.A. Berven. 1973. Species diversity of herpetofauna!
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Hobbie, J.E., S.R. Carpenter, N.B. Grim, J.R. Grosz & T.R. Seastedt.
2003. The US long term ecological research program. BioScience
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Huey, R.B. 199!. Physiological consequences of habitat selection.
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McDowell, W.H. & A. Estrada-Pinto. 1988. Rainfall at El Verde
Field Station, 1964—1986. CEER-T-228. Center for Energy and
Environment Research. Rio Piedras, Puerto Rico.

Pfeiffer, W.J. 1996. Arboreal arachnids. Pp. 247-271. In The Food
Web of a Tropical Rain Forest. (D.P. Reagan & R.B. Waide, eds.).
University of Chicago Press, Chicago, Illinois.

Porto, T.J. & P.E.C. Peixoto. 2013. Experimental evidence of habitat
selection and territoriality in the Amazonian whip spider Hetero-
phrynus longicornis (Arachnida, Amblypygi). Journal of Ethology
31:299-304.

Quintero, D. 1981. The amblypygid genus Phrynus in the Americas
(Amblypygi, Phrynidae). Journal of Arachnology 9:117-166.

Rosenzweig, M.L. 1981. A theory of habitat selection. Ecology
62:327-335.

Scatena, F.N. & M.C. Larsen. 1991. Physical aspects of Hurricane
Hugo in Puerto Rico. Biotropica 23:317-323.

Stamps, J.A. 1991. The effect of conspecifics on habitat selection in
territorial species. Behavioral Ecology and Sociobiology 28:29-36.

Thompson, J., N. Brokaw, J.K. Zimmerman, R.B. Waide, E.M.
Everham III & D.J. Lodge et al. 2002. Land use history,
environment, and tree composition in a tropical forest. Ecological
Applications 12:1344-1363.

Weygoldt, P. 2000. Whip spiders (Chelicerata: Amblypygi): Their
biology, morphology and systematics. Apollo Books, Denmark.

Whitham, T.G. 1980. The theory of habitat selection: Examined and
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Manuscript received 28 August 2013, revised January 6 2014,

2014. The Journal of Arachnology 42:130-132

SHORT COMMUNICATION

Natural prey of the crab spider Xysticus mavmovatus (Araneae: Thomisidae) on Eryngium plants

Elchin Fizuli oglu Huseynov: Institute of Zoology, National Academy of Sciences, block 504, passage 1 128, Baku 370073,
Azerbaijan. E-mail: hun-vey-bin@rambler.ru

Abstract. The natural prey of medium-sized juvenile (ca. 3 mm) crab spiders Xysticus marmoratus Thorell 1 875 inhabiting
Eryngium hiebersteinianum plants was studied on the Absheron Peninsula, Azerbaijan. The percentage of specimens found
feeding on prey was low (3.4%). Xysticus marmoratus is a polyphagous predator with representatives of four arthropod
orders found in its diet. The primary food of X. marmoratus was ants (Formicidae), which accounted for 83.3% of the total
number of prey. The length of prey killed by X. marmoratus ranged between 0.87-7.50 mm (mean 2.96 mm) and constituted
from 28.5-300.0% (mean 96.9%) of the length of their captors. The most frequently captured size group of prey was 50-
70% the length of the spiders.

Keywords: Flower-dwelling, diet, myrmecophagy, prey length

With over 2000 described species, Thomisidae is one of the largest
families of spiders (Platnick 2013). However, despite the family
members’ great diversity and potential predatory significance, few
studies have addressed the natural prey of thomisids. A survey of
arachnological literature revealed that quantitative data on the
natural diet are available for only fifteen species of crab spiders
(Broekhuysen 1948; Nyffeler & Benz 1979; Tarabaev 1979; Morse
1981, 1983; Ricek 1982; Lubin 1983; Deanetal. 1987; Agnew & Smith
1989; Castanho & Oliveira 1997; Schmaihofer 2001; Romero &
Vasconcellos-Neto 2003; Guseinov 2006; Huseynov 2007 a, b). Crab
spiders (Thomisidae) do not use silk for prey capture; instead, they lie
in ambush and wait until prey comes within reach of their long
raptorial forelegs (Foelix 1996).

Xysticus marmoratus Thorell 1875 has not previously been the
subject of ecological or behavioral investigation. Xysticus marmoratus
is distributed from Eastern Europe to Central Asia (Marusik &
Logunov 1994). It is a small crab spider (adult body length 4-6 mm),
with adult males usually slightly smaller than females. In Azerbaijan,
X. marmoratus occurs in arid habitats, including semi-deserts and
steppes. Xysticus marmoratus has an annual life cycle. Adult
specimens are found in October and November and inhabit grass
litter. In contrast, juveniles are very abundant on flowering plants
throughout the summer (Huseynov unpubl. data).

This investigation was carried out on the Absheron Peninsula,
Azerbaijan. The three primary study sites were located near the
villages of Shagan, Gres, and Bina (40°27'30"N 50°04'08"E), where
over 95% of the total observation time was spent. Additionally, two
secondary study sites were located near Gala village and Ganly-Gyol
Lake. The study sites were areas of ephemeral semi-desert covered
with dwarf shrubs Eryngium hiebersteinianum Nevski, Alhagi pseu-
doalhagi (MB), Noaea mucronata (Forsk), and several herbs and
grasses. The sites near Shagan, Bina, and Ganly-Gyol were
additionally characterized by planted pines, Pinus eldaricus Medw.,
while the others were treeless.

All individuals of Xysticus observed during the study period were
immature. They were abundant only on Eryngium biebersteinianunr,
therefore, observations were concentrated exclusively on this plant.
The prey of spiders was sampled during three successive years: 1997 (2
July-9 August), 1998 (14 June-25 July), and 1999 (14 June-31 July).
Fifty surveys were conducted during these periods, which took about
113 hours. During the surveys, E. hiebersteinianum plants were
thoroughly searched for Xysticus, and the mouthparts of each
individual spider found were inspected with a hand-lens of 4X
magnification to avoid overlooking small prey. Spiders with prey in

their chelicerae were captured with a transparent cup, placed in
separate vials containing 75% ethyl alcohol and brought back to the
laboratory for measurement and prey identification. Spiders without
prey were left in the field. All surveys were done in the daylight hours
between 11:00 and 21:00. Most of these surveys were conducted
between 1 1:00 and 17:00. The number of prey collected from Xysticus
increased after 18:00. Therefore, a five-day series of double surveys
was undertaken in July 1999 at Bina. On each of these days, one
survey was made during the first half of the day (11:00-16:00) and
another during the second half of the day (18:00-21:00).

All Xysticus individuals observed in the field were of approximately
the same size and had a similar color pattern, suggesting that they
belonged to the same species. To identify the species, I collected forty
living specimens from Eryngium in July 2006 at Bina (where 85% of
all spiders were observed in 1997-1999) and reared them in the
laboratory until they reached maturity. Spiders were kept separately
in glass vials (50 mm long, 8 mm diameter) under a natural light-dark
regime. Two types of prey were offered to these spiders. “Innocuous”
prey included various specimens of Diptera, and “dangerous” prey
were workers of the ant Lasius alienus Forster 1850. The prey insects
were collected in the garden of the Institute of Zoology, Baku, and
only those between 50-100% of the spiders’ body length were used in
the feeding experiments. Individual spiders were fed three times a
week with one of the prey types in a random order. Interactions
between spiders and prey were monitored for two hours, and the prey
was recorded as accepted if it was captured and consumed by spider
during this period. I considered the prey to be consumed by the spider
if it was not released immediately and was held in the chelicerae for
at least 15 minutes after capture. Thirty-five juveniles survived
to adulthood in the laboratory, and all these proved to be X.
marmoratus. Voucher specimens of X. marmoratus and their prey
items were deposited in the Institute of Zoology of Azerbaijan
Academy of Sciences.

In total, 2,495 specimens of X. marmoratus were observed in the
field, 84 of which (3.4%) had prey in their chelicerae. One juvenile was
consuming two prey items simultaneously. Thus the actual number of
feeding events was 85. Individuals of X. marmoratus fed significantly
less frequently during the first half of the day (6 prey records of 583
spider observations) than during the second half (38 of 614) (yp i =
21.1; P < 0.001).

One X. marmoratus individual dropped its prey before it could be
captured, so 84 prey items were collected for dietary analysis. These
were distributed among four orders of arthropods, including three
from the class Insecta (Hymenoptera, Coleoptera, and Diptera) and

130

HUSEYNOV— PREY OF XYSTICUS MARMORATUS

131

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Size categories

Figure 1. — Distribution of prey of Xysticus marmonitus in
different size categories (body lengths of prey expressed as percent-
ages of the body lengths of their captors).

one from the class Arachnida (Araneae). By far the dominant prey
order was Hymenoptera, which accounted for 92.9% of total prey.
Most of the Hymenoptera captured were ants (ca. 90%). They
included representatives of subfamilies Formicinae [43 Pkigiolepis sp.,
8 Cataglyphis aenescem (Nylander 1849), 3 Cataglyphis setipes (Fore!
1894), 1 Stenamma sp.)], Myrmecinae (7 Messor denticiihitm Santschi
1927, 7 Cardiocondyla sp.) and Dolichoderinae (1 Tapinoma sp.).
Except for two winged males of M. denticulatus, all ants were
workers. Other Hymenoptera consisted of three halictid bees
(Halictus sp., Nomioides sp., Sphecodes sp.), three stinging wasps (2
Bethylidae and 1 Thiphiidae) and two parasitic wasps (Braconidae
and Scelionidae). The remaining arthropods included three adult
beetles (2 Dermestidae and 1 Bruchidae), two flies (Chloropidae and
Milichiidae) and a conspecific spider (juvenile X. numuorcUm).

Of 620 ants and 442 dipterans offered to spiders in the laboratory, 542
ants (87.4%) and 397 dipterans (89.8%) were eaten by X. inamioratus,
the rates of acceptance of these prey groups being very similar.

Eighty-one natural prey items were measured. Their lengths varied
from 0.87-7.50 mm (mean ± SD: 2.96 ± 1.77 mm) and constituted
from 28.5-300.0% (96.9 ± 57.5%) of the length of their captors,
which ranged from 2.25-3.80 mm (3.05 ± 0.30 mm). The size
distribution of the prey in relation to the sizes of their captors is
shown in Fig. 1. Most of the prey did not exceed the length of their
captors (70.4%, ii = 57). These included Pkigiolepis, Cardiocondyla,
Tapinoma and Stenamma ants, bethylid and scelionid wasps, as well
as beetles, flies, and a conspecific spider. The most frequently
captured (60.5%, n = 49) were medium-sized arthropods from 50-
100% spider body length, while small prey, not exceeding half the
length of the spiders, were represented by only eight items (9.9%).
About one third of the prey of X. marmoratus consisted of large
arthropods exceeding the length of their captors. These prey consisted
of Cataglyphis and Messor ants, thiphiid and braconid wasps, and
halictid bees. Many of the large prey (23.5%, n = 19) exceeded 150%
of the body length of their captors.

The percentage of X. marmoratus individuals found while feeding
was low (< 10%), as is typical of cursorial spiders (Nyffeler & Breene
1990) and crab spiders in particular (Nyffeler & Benz 1979; Dean et al.,
1987; Romero & Vasconcellos-Neto 2003; Guseinov 2006; Huseynov
2007a). The difference in prey capture rate of X. marmoratus at
different times of the day is likely related to the fiuctuation of ant
activity on Eryngium throughout the day. In the summer, on the
Absheron Peninsula, most ants are inactive during the first half of day.

apparently because of high surface temperature, and start to forage
only after 18:00, when the temperature decreases. In the evening large
numbers of ants, especially Pkigiolepis sp., appeared on Eryngium,
which might result in increased prey capture by spiders.

This investigation has shown that X. marmoratus is a polyphagic
predator feeding on a wide range of arthropods. The heavy prevalence
of worker ants in its diet is unusual, since these insects possess
effective defensive equipment, such as strong mandibles, a hard
cuticle, poisonous stings or formic-acid spray (Blum 1981), making
them unpalatable to most cursorial spiders (Nentwig 1986). Although
some tropical thomisid species have been reported to feed exclusively
on ants (Lubin 1983; Castanho & Oliveira 1997), such a high rate of
ant capture (83.3%) has never been recorded for crab spiders from
temperate regions. However, worker ants were found in lower
proportions in the diets of Xysticus cristatus (Clerck 1757) and
Xysticus loeffleri Roewer 1955 (Nyffeler & Benz 1979; Guseinov
2006). These data suggest that myrmecophagy is a widespread
phenomenon within the genus Xysticus.

Does X. marmoratus prefer ants to other prey? Such a conclusion
cannot be derived from dietary data alone. The prevalence of ants in
the diet of X. marmoratus could be related to their abundance in its
habitat. Indeed, at least in the second half of the day, ants were by far
the most abundant visitors to Eryngium. In any case, the present field
and laboratory findings unambiguously indicate that X. marmoratus
is a quite competent ant-feeder.

Experimental studies of prey size preference in spiders have shown
that while most cursorial spiders prefer prey not exceeding their own
size, the crab spider Xysticus cristatus readily accepted insects two
times larger than itself (Nentwig & Wissel 1986). Although X.
marmoratus sometimes captured very large prey, most of its prey
consisted of arthropods smaller than itself. This is in contrast with
observations of two other thomisid species, Thomisus onustus
Walckenaer 1805 and Riincinia grammica (C.L. Koch 1837) that also
inhabit Eryngium plants in the same localities. Over 90% of the prey
of these spiders consisted of large insects exceeding the size of their
captors (Huseynov 2007 a, b). However, both of these thomisids fed
primarily on non-ant prey and did not accept ants in the laboratory as
readily as did X. marmoratus (Huseynov unpubl. data). Thus the bias
toward smaller prey in the diet of X. marmoratus is apparently due to
the prevalence of small ants in its microhabitat.

LITERATURE CITED

Agnew, C.W. & J.W. Smith. 1989. Ecology of spiders (Araneae) in a
peanut agroecosystem. Environmental Entomology 18:30-42.
Blum, M.S. 1981. Chemical Defenses of Arthropods. Academic Press,
New York.

Broekhuysen, G.J. 1948. The behaviour and the life history of a
Javanese spider, Thomisus sp. Journal of the Entomological
Society of South Africa 10:135-164.

Castanho, L.M. & P.S. Oliveira. 1997. Biology and behaviour of the
neotropical ant-mimicking spider Aphantochilus rogersi (Araneae:
Aphantochilidae): nesting, maternal care and ontogeny of ant-
hunting techniques. Journal of Zoology, London 242:643-650.
Dean, D.A., W.L. Sterling, M. Nyffeler & R.G. Breene. 1987.
Foraging by selected spider predators on the cotton fieahopper and
other prey. Southwestern Entomologist 12:263-270.

Foelix, R.F. 1996. Biology of Spiders. Harvard University Press,
Cambridge, Massachusetts, USA.

Guseinov, E.F. 2006. The prey of a lithophilous crab spider Xysticus
loeffleri (Araneae, Thomisidae). Journal of Arachnology 34:37^5.
Huseynov, E.F. 2007a. Natural prey of the crab spider Riincinia
grammica (Araneae: Thomisidae) on Eryngium plants. Bulletin of
the British Arachnological Society 14:93-96.

Huseynov, E.F. 2()07b. Natural prey of the crab spider Thomisus
onustus (Araneae: Thomisidae), an extremely powerful predator of
insects. Journal of Natural History 41:2341-2349.

132

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Lubin, Y.D. 1983. An ant eating crab spider from the Galapagos.
Noticias de Galapagos 37:18-19.

Marusik, Y.M. & D.V. Logunov. 1994. The crab spiders of Middle
Asia (Aranei, Thomisidae), 2. Beitrage zur Araneologie 4:133-175.

Morse, D.H. 1981. Prey capture by the crab spider Misumena valid
(Clerck) (Thomisidae) on three common native flowers. American
Midland Naturalist 105:358-367.

Morse, D.H. 1983. Foraging patterns and time budgets of the crab
spiders Xyslicus emerfoni Keyserling and Misumena vatia (Clerck)
(Araneae: Thomisidae) on flowers. Journal of Arachnology 11:
87-94.

Nentwig, W. 1986. Non-webbuilding spiders: prey specialists or
generalists? Oecologia 69:571-576.

Nentwig, W. & C. Wissel. 1986. A comparison of prey lengths among
spiders. Oecologia 68:595-600.

Nyffeler, M. & G. Benz. 1979. Nischeniiberlappung beziiglich der
Raum-und Nahrungskomponenten bei Krabbenspinnen (Araneae:
Thomisidae) und Wolfspinnen (Araneae: Lycosidae) in Mahwie-
sen. Revue Suisse de Zoologie 86:855-865.

Nyffeler, M. & R.G. Breene. 1990. Evidence of low daily food
consumption by wolf spiders in meadowland and comparison with

other cursorial hunters. Journal of Applied Entomology 110:
73-81.

Platnick, N.I. 2013. The World Spider Catalog, Version 14.0.
American Museum of Natural History. Online at http://research.
amnh.org/entomology/spiders/catalog/

Ricek, E.W. 1982. Die Lauerposten der Krabbenspinne Xysticus
hifasciatus C. L. Koch. Linzer biologische Beitrage 14:15-22.

Romero, Q.R. & J. Vasconcellos-Neto. 2003. Natural history of
Misumenops argenteus (Thomisidae): seasonality and diet on
Trichogoniopsis adenantha (Asteraceae). Journal of Arachnology
31:297-304.

Schmalhofer, V.R. 2001. Tritrophic interactions in a pollination
system: impacts of species composition and size of flower patches
on the hunting success of a flower-dwelling spider. Oecologia
129:292-303.

Tarabaev, C.K. 1979. Peculiarities of morphology and biology of a
crab spider Diaea dorsata Fabr. (Aranei, Thomisidae) in the
southeast of Kazakhstan. Entomological Review 58:200-210. (in
Russian).

Manuscript received 8 October 2013, revised 2 January 2014,

2014. The Journal of Arachnology 42:133-134

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Carico, J.E. 1993. Trechaleidae: a “new” American spider
family. Pp. 305. lu Proceedings of the Ninth International
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Y.D. Liibin & B.C. Robinson, eds.). Smithsonian Institu-
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Pholcidae). Canadian Journal of Zoology 74:905-918.
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Platnick, N.I. 2011. The World Spider Catalog, Version 12.0.
American Museum of Natural History, New York. Online at
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Roewer, C.F. 1954. Katalog der Araneae, Volume 2a. Institut
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The following individuals contributed to the quality of i\\Q Journal of Arachnology in 2013 by serving as
reviewers. Their labors are gratefully acknowledged.

Agnarsson, I.

Femandez-Montraveta, C.

Kasumovic, M.

Selden, P.

Arnedo, M.

Fet, V.

Kloock, C.

Shaw, E.

Baker, M.

Fiser, C.

Korenko, S.

Shear, W.

Bonaldo, A.

Franke, 0.

Louren^o, W.

Shillington, C.

Bowden, J.

Gaffin, D.

Lowe, G

Shultz, J.

Brescovit, A.

Godwin, R.

Mahnert, V.

Sissom, D.

Cabra, J.

Gregoric, M.

Marshall, S.

Spagna, J.

Cady, A.

Planner, A.

Mattoni, C.

Stephen, C.

Camilo, G.

Harwood, J.

Mendes, A.

Stratton, G.

Cardoso, P.

Hedin, M.

Miller, J.

Styrsky, J.

Carrel, J.

Hendrixson, B.

Moya-Larano, J.

Taylor, C.

Carvalho, L.

Henningsen, J.

Nelson, X.

Toft, S.

Chatzaki, M.

Herberstein, M.

Patrick, B.

Tourinho, A.

Costa, F.

Hsieh, S

Perez, A.

Ubick, D.

Cramer, K.

Hu, D.

Persons, M.

Uetz, G.

Cross, F.

Huber, B.

Petillon, J.

Valdez-Mondragon, A.

Cushing, P.

Huseynov, E.

Prendini, L.

Vetter, R.

De Busschere, C.

Hyzny, M.

Rauch, J.

Viera, C.

Dunlop, J.

Jackson, R.

Reichling, S.

Vink, C.

Eason, P.

Jimenez, M.-L.

Rodriguez, R.

Weiss, M.

Eberhard, W.

Johnson, C.

Rypstra, A.

Wilder, S.

Edwards, G.B.

Jones, T.C.

Sanchez Pinero, F.

Yip, E.

Ending, M.

Kaltsas, D.

Schmalhofer, V.

Zaragoza, J.

CONTENTS

Journal of Arachnology

Volume 42

Number 1

Invited Review

New sequencing technologies, the development of genomics tools, and their applications in evolutionary

arachnology by Michael S. Brewer, Darko D. Cotoras, Peter J. P. Crowcher & Rosemary G. Gillespie .... 1

Featured Articles

A new spider (Araneae: Haplogynae: Plectreuridae) from the Cretaceous Fossil-Lagerstatte of El Montsec, Spain

by Paul A. Seldeu 16

Sex ratio bias caused by endosymbiont infection in the dwarf spider Oedothorax retusus

by Bram VaHthournout, Viki Vandomme & Frederik Hendrickx 24

Vertical stratification of spider assemblages in tvv'o conifer plantations in central Japan

by HiroM Oguri, Tomohiro Yoshida, Akihiro Nakamura, Masashi Soga & Naolci Hijii. 34

Assessing spider diversity on the forest floor: expert knowledge beats systematic design

by Elvira Sereda, Theo Blick, Wolfgang H. O, Dorow, Volkmar Wolters & Klaus Birkhofer 44

The conservation value of secondary forests in the southern Brazilian Mata Atlantica from a spider perspective

by Florian Raub, Hubert Hofer, Ludger Scheuermann & Roland Brandi 52

Trophic niche and predatory behavior of the goblin spider Triaeris stenaspis (Oonopidae): a springtail specialist?

by Stanislav Korenko, Katerina Hamouzova & Stano Pekar 74

Use of locomotor performance capacities reflects the risk level associated with specific cue types in two cursorial

spider species by Joseph A. Dillon & Jonathan N. Pruitt 79

A review and redescription of the cosmopolitan pseudoscorpion Chelifer cancroides (Pseudoscorpiones:

Cheliferidae) by Mark S. Harvey 86

A new troglobitic ideoroncid pseudoscorpion (Pseudoscorpiones: Ideoroncidae) from southern Africa

by Mark S. Harvey & Gerhard Du Preez 105

Comparison of scorpion behavioral responses to UV under sunset and nighttime irradiances

by Douglas D. Gaffin & Tristan N. Barker Ill

Short Communications

Fine structure of the stinger (aculeus) in Euscorpius

by Rainer Foelix, Bruno Erb & Matt Braunwalder 119

Intense leg tapping behavior by the harvestman Mischonyx cuspidatus (Gonyleptidae): an undescribed defensive
behavior in Opiliones?

by Barbara Crespo Dias, Elene da Silva Souza, Mareos Ryotaro Kara & Rodrigo Hirata Wiliemart 123

Seasonal patterns of microhabitat selection by a sub-tropical whip spider, Phrynus longipes, in the Luquiilo
Experimental Forest, Puerto Rico

by Caroline A. Curtis & Christopher P. Bloch 126

Natural prey of the crab spider marmoratus (Araneae: Thomisidae) on Eryngium plants

by Elchin Fizuli oglu Huseynov 13§

Instructions to Authors 133

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