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Kinship, Percentage and Behaviour498 Social structure varies with elevation in an Alpine ant

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Ecological Interactions508 Stable coexistence of incompatible Wolbachia along a

narrow contact zone in mosquito field populationsC. M. Atyame, P. Labbé, F. Rousset, M. Beji, P. Makoundou, O. Duron, E. Dumas, N. Pasteur, A. Bouattour, P. Fort & M. Weill

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259 Pushing the envelope in genetic analysis of species invasionS. A. Cushman

INVITED REVIEWS AND SYNTHESES263 Multicollinearity in spatial genetics: separating the wheat

from the chaff using commonality analysesJ. G. Prunier, M. Colyn, X. Legendre, K. F. Nimon & M. C. Flamand

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284 Human-aided and natural dispersal drive gene flow acrossthe range of an invasive mosquitoK. A. Medley, D. G. Jenkins & E. A. Hoffman

296 Life-stage differences in spatial genetic structure in anirruptive forest insect: implications for dispersal and spatialsynchronyP. M. A. James, B. Cooke, B. M. T. Brunet, L. M. Lumley, F. A. H. Sperling, M.- J. Fortin, V. S. Quinn & B. R. Sturtevant

310 Reconstructing the demographic history of orang-utans usingApproximate Bayesian ComputationA. Nater, M. P. Greminger, N. Arora, C. P. van Schaik, B. Goossens, I. Singleton, E. J. Verschoor, K. S. Warren & M. Krützen

328 Demographic inferences using short-read genomic data in anapproximate Bayesian computation framework: in silicoevaluation of power, biases and proof of concept in AtlanticwalrusA. B. A. Shafer, L. M. Gattepaille, R. E. A. Stewart & J. B. W. Wolf

346 High-stakes species delimitation in eyeless cave spiders(Cicurina, Dictynidae, Araneae) from central TexasM. Hedin

362 MHC variation reflects the bottleneck histories of NewZealand passerinesJ. T. Sutton, B. C. Robertson & I. G. Jamieson

374 Global invasion history of the tropical fire ant: a stowawayon the first global trade routesD. Gotzek, H. J. Axen, A. V. Suarez, S. H. Cahan & D. Shoemaker

Ecological Genomics389 Population genomics of natural and experimental

populations of guppies (Poecilia reticulata)B. A. Fraser, A. Künstner, D. N. Reznick, C. Dreyer & D. Weigel

mec_24_2_oc_Layout 1 13-01-2015 11:36

High-stakes species delimitation in eyeless cave spiders(Cicurina, Dictynidae, Araneae) from central Texas

MARSHAL HEDIN

Department of Biology, San Diego State University, San Diego, CA 92182, USA

Abstract

A remarkable radiation of completely eyeless, cave-obligate spider species (Cicurina)has been described from limestone caves of Texas. This radiation includes over 50

described species, with a large number of hypothesized single-cave endemics, and four

species listed as US Federally Endangered. Because of this conservation importance,

species delimitation in the group is ‘high-stakes’– it is imperative that species hypoth-

eses are data rich, objective, and robust. This study focuses on a complex of four cave-

dwelling Cicurina distributed on the northwestern edge of Austin, Texas. Several of

the existing species hypotheses in this complex are weak, based on morphological

comparisons of small samples of adult female specimens; one species description (for

C. wartoni) is based on a single adult specimen. Species limits in this group were

newly assessed using morphological, mitochondrial and nuclear DNA sequence data

evidence, analysed using a variety of approaches. All data support a clear lineage sepa-

ration between C. buwata versus the C. travisae complex (including C. travisae,C. wartoni and C. reddelli). Observed congruence across multiple analyses indicate that

the C. travisae complex represents a single species, and the formal species synonymy

presented here has important conservation implications. The integrative framework

utilized in this study serves as a potential model for other Texas cave Cicurina, includ-ing US Federally Endangered species. More generally, this study illustrates how and

why taxon-focused conservation efforts must prioritize modern species delimitation

research (if the existing taxonomy is weak), before devoting precious downstream

resources to conservation efforts. The study also highlights the issue of taxonomic type

II error that diversity biologists increasingly face as species delimitation moves into

the genomics era.

Keywords: Bayesian phylogenetics & phylogeography, conservation biology, endangered

species, population structure, species delimitation, species synonymy

Received 24 September 2014; revision received 3 December 2014; accepted 3 December 2014

Introduction

Species delimitation is the process whereby systematists

or evolutionary biologists combine specimens, specimen

data and data analyses to delimit species taxa while

working under an explicit species concept. Species

delimitation is an original and fundamental goal of sys-

tematic biology (Wiens 2007; Camargo & Sites 2013).

For some researchers, this process has changed dramati-

cally over the past 10–20 years, these changes paralleling

a flood of DNA sequence data and the development of

new analytical delimitation tools for both molecules

and morphology (Fujita et al. 2012; Camargo & Sites

2013; Carstens et al. 2013). However, these changes have

not been universally adopted, as the majority of new

species are still delimited and described under a quali-

tative ‘distinct morphological groups’ sorting process

(Pante et al. 2015).

In principle, it could be claimed that all species

delimitations are ‘high-stakes’ hypotheses, following

familiar arguments that species are fundamental units

of evolutionary biology, ecology, etc. More realistically,

however, most initial species hypotheses have neverCorrespondence: Marshal Hedin, Fax: (619) 594 5676; E-mail:

[email protected]

© 2014 John Wiley & Sons Ltd

Molecular Ecology (2015) 24, 346–361 doi: 10.1111/mec.13036

been subsequently tested, and in fact, species hypothe-

ses do not even exist for a majority of Earth’s biodiver-

sity (e.g. Mora et al. 2011). That said, certain species

delimitations are particularly important for political,

economic, and/or applied reasons. Examples include

species complexes that include disease vectors, venom-

ous taxa, and taxa of conservation concern. With respect

to taxon-focused conservation efforts, it is fundamen-

tally important to rigorously and accurately delimit con-

servation units (at or below the species level) before

dedicating finite conservation resources (Paquin et al.

2008; Weisrock et al. 2010; Niemiller et al. 2012, 2013;

Malaney & Cook 2013; McCormack & Maley 2015).

Taxa restricted to cave habitats present special chal-

lenges for species delimitation. Complexes of cave-

dwelling populations/taxa are often distributed in strict

allopatry, such that cases of sympatry (and natural tests

of reproductive isolation) are rare or nonexistent. Simi-

lar selective pressures across spatially isolated cave hab-

itats promote morphological stasis or homoplasy

(Derkarabetian et al. 2010; Bendik et al. 2013; Derkarabe-

tian & Hedin 2014), and many studies have now shown

that hypothesized widespread cave species often consist

of multiple, morphologically similar or cryptic species

(Juan et al. 2010; Niemiller et al. 2012, 2013; Derkarabe-

tian & Hedin 2014). In contrast, constrained gene flow

among isolated cave habitats often leads to extreme

population genetic structuring (e.g. Hedin 1997; Chiari

et al. 2012). If only single gene data are available (e.g.

mtDNA only), population genetic structure can be diffi-

cult (or impossible) to distinguish from population

divergence and speciation. For example, the single-locus

generalized mixed Yule coalescent method (Pons et al.

2006; Fujisawa & Barraclough 2013), commonly applied

in modern species delimitation, is susceptible to over-

splitting species diversity in the face of strong popula-

tion structure (Lohse 2009; Keith & Hedin 2012; Satler

et al. 2013; Hamilton et al. 2014). Likewise, population

structure in the nuclear genome presents potential prob-

lems for newer multispecies coalescent methods (O’Me-

ara 2010; Camargo & Sites 2013). An example is

Bayesian phylogenetics and phylogeography (BPP;

Yang & Rannala 2010; Rannala & Yang 2013), a method

that assumes panmixia within species, but has been

suggested to potentially oversplit diversity in dispersal-

limited taxa (e.g. Niemiller et al. 2012; Barley et al. 2013;

Carstens & Satler 2013; McKay et al. 2013).

A remarkable radiation of completely eyeless, cave-

obligate spider species has been described from lime-

stone cave habitats of Texas. The subgenus Cicurella

(genus Cicurina) includes about 80 species, over 50 of

which are eyeless and restricted to Texas caves (Gertsch

1992; Paquin & Dup�err�e 2009); available evidence sug-

gests monophyly of this group (Paquin & Hedin 2004;

Paquin & Dup�err�e 2009). Species delimitation in eyeless

Cicurina is challenging for several reasons beyond the

generalities discussed above. Adult specimens are rare,

with an estimated ratio of immature/adult female/

adult male specimens of 100/10/1 (Paquin & Dup�err�e

2009). Most araneomorph spider species are described

based on a combination of both male and female adult

morphology, but male-based evidence is essentially

lacking in eyeless Cicurina. Female somatic morphology

is highly conserved among eyeless Cicurina species,

while female genitalic morphology often varies within

species, blurring the distinction between geographic

variation and species-level divergence (Paquin &

Dup�err�e 2009). Finally, access to Texas caves can be

challenging, leading to geographic sampling gaps that

can impact genealogical interpretations (Lohse 2009;

Niemiller et al. 2012). Despite these several difficulties,

species delimitation in the group is ‘high-stakes,’ with

over 35 blind Texas Cicurina species known only from

their respective type localities (Paquin & Dup�err�e 2009),

and four cave taxa listed as US Federally Endangered

Species (Longacre 2000). Because of this obvious conser-

vation and political importance, it is imperative that

species hypotheses in the group are robust.

This study focuses on a complex of four eyeless spe-

cies (Cicurina wartoni, C. reddelli, C. travisae and C. buw-

ata) restricted to caves in Travis and Williamson

Counties, Texas. Existing species hypotheses in this

complex are not data rich; for example, the taxonomic

description of C. wartoni is based on a single adult

female specimen, which remains the only adult speci-

men known for this taxon (Gertsch 1992; Paquin &

Dup�err�e 2009). Cicurina reddelli is likewise known only

from the type locality. Cicurina travisae and C. buwata

are known from females for a larger number of caves

(>7 caves per taxon), but adult males have never been

described for these species. Taking into account intra-

specific variation, female genital morphologies for

members of this complex are highly similar (Paquin &

Dup�err�e 2009; fig. 131), suggesting the possibility that

these four species actually comprise a single taxon. Spe-

cies delimitation in this complex is particularly impor-

tant because of the rarity and apparent endemicity of

contained taxa in a region impacted by development on

the northwestern edge of Austin. For example, the only

known location for C. wartoni is a small, shallow cave

with many threats (e.g. fire ants, pollution, trash dump-

ing, etc.), and this species has been a candidate for list-

ing as a US Federally Endangered Species (U.S. Fish &

Wildlife Service 2010).

The research summarized here seeks to clarify the

evolutionary independence of species in this complex,

with an emphasis on the distinctiveness of C. wartoni.

Because any single data source or analytical method is


CAVE SPIDER SPECIES DELIMITATION 347

susceptible to error, most modern species delimitation

analyses now include both independent lines of evi-

dence (e.g. mtDNA data, nuclear data, morphology,

etc.) and comparisons of multiple analyses (Fujita et al.

2012; Carstens et al. 2013; Satler et al. 2013; Derkarabe-

tian & Hedin 2014). This integrative approach to the

species delimitation problem was used in this research.

Materials and methods

Specimen sampling

Specimens were available from 27 regional caves (Fig. 1,

Appendix 1), with one to three spiders sampled from

each cave. A priori specimen identification was based

on geographic location and/or genetic affiliation as fol-

lows: Cicurina buwata – sample including eyeless imma-

ture specimens from three caves (Buttercup Creek Cave,

Marigold Cave, and Testudo Tube) that are known loca-

tions for C. buwata (Paquin & Dup�err�e 2009). Eyeless

immature and/or adult specimens from eight additional

caves were identified as C. buwata based on consistent

placement into a C. buwata genetic clade (see Results);

C. wartoni – three eyeless immature specimens from the

type locality (Pickle Pit) were tentatively identified as

this species, as no other eyeless Cicurina have been

recorded from this cave; C. travisae – sample including

eyeless immature and/or adult specimens from five

caves (McDonald Cave, Amber Cave, Kretschmarr Dou-

ble Pit, North Root Cave, Tooth Cave = type locality)

that are known locations for C. travisae (Paquin &

Dup�err�e 2009); C. reddelli – an adult female and imma-

ture male specimen from Cotterell Cave, the type and

only known locality for C. reddelli; A priori unidentified –both immature and adult specimens from nine caves

geographically situated between Cotterell Cave and

McDonald Cave (Fig. 1, Appendix 1) are genetic mem-

bers of the clade of interest (see Results), but are from

Broken Arrow

Testudo

Marigold

ButtercupCreek

BabeLakeline

Weldon

NoRent

McNeilBat

AppleRiata

McDonald

Kretschmarr

Amber

TwoTrunks

Tooth

NorthRoot

Gallifer

Geode

Stovepipe

PicklePit

Spider

BeardRanch

KenButlerPit

JesterEstates

JestJohn

Cotterell

DiesRanchTreasure

Williamson County

Travis County

BB B

B

BB

B

B BB

BW

R

T

TT

T~ 5 km

T

(A) (B)

(C)

Fig. 1 (A) Map of Texas with counties,

Travis and Williamson counties high-

lighted. (B) Adult female Cicurina buwata

from Lakeline Cave (G2001). (C) Map

showing geographic distribution of

sampled cave populations – locations

approximate, to protect location anonym-

ity. Specimens allocated into five primary

a priori groups based on consideration of

morphology, geographic location and/or

genetic affiliation. Colours used to desig-

nate taxa (amber = C. buwata, blue =C. travisae, green = C. reddelli, red =C. wartoni, black boxes = ‘western undeter-

mined’, black circles = ‘eastern undeter-

mined’; see text for details).


348 M. HEDIN

caves without previous records for adult Cicurina. Adult

females are available from some of these caves, but

because C. travisae, C. reddelli and C. wartoni have very

similar female epigynal morphologies (Paquin &

Dup�err�e 2009, fig. 131), these specimens were not iden-

tified to species a priori.

Morphology

Genitalic structures (female epigyna, male pedipalps)

for all adult specimens used in genetic analyses were

digitally imaged. Specimens were imaged using a

Visionary Digital BK Plus system (http://www.vision-

arydigital.com), including a Canon 5D digital camera,

Infinity Optics Long Distance Microscope, P-51 camera

controller and FX2 lighting system. Individual images

were combined into a composite image using ZERENE

STACKER v1.04 (http://www.zerenesystems.com/); this

composite image was then edited using Adobe Photo-

shop CS6. Female epigyna were dissected from speci-

mens using fine forceps, immersed for 2–5 min in

BioQuip specimen clearing fluid (http://www.bio-

quip.com) on a depression slide and then imaged in

this fluid on slides.

Mitochondrial analyses

Mitochondrial cytochrome oxidase I (COI) data were

gathered for 46 specimens from 26 caves (Appendix 1),

using PCR and genomic DNA extracted from leg tis-

sues. Amplified PCR products were purified using Mil-

lipore plates and Sanger-sequenced in both directions at

Macrogen USA. DNA sequences were edited using GENE-

IOUS PRO R7 software (http://www.geneious.com/) and

trimmed to exclude primer sequences. Original

sequences were supplemented with transcriptome-

derived COI data for C. vibora (see below) and pub-

lished data for other Texas cave- and surface-dwelling

Cicurina (Paquin & Hedin 2004).

A COI gene tree was reconstructed using maximum

likelihood, using RAXML searches (Stamatakis 2006, 2014)

as implemented in RAXMLGUI 1.31 (Silvestro & Michalak

2012). This analysis included a thorough bootstrap

analysis (1000 bootstrap replicates) followed by multiple

inferences (100) on an alignment with redundant haplo-

types collapsed, using a GTR_Γ model for separate

codon partitions. A mitochondrial gene tree was also

reconstructed using Bayesian inference in the BEAST

v1.7.2 package (Drummond et al. 2012). BEAST analyses

were conducted using an uncorrelated lognormal

relaxed clock model (Drummond et al. 2006), imple-

menting best-fit models of molecular evolution chosen

using default parameters in PARTITIONFINDER v1.1.1 (Lan-

fear et al. 2012). Two replicate MCMC chains were run

for 50 million generations, with sampling every 1000

generations, using the ‘auto optimize’ operators option,

and a Speciation: Yule Process tree prior. The consis-

tency of parameter estimates across replicate runs was

assessed using Tracer (Drummond et al. 2012), and

results of replicate runs were combined such that ESS

values exceeded 200 for all parameters. LOGCOMBINER

was used to combine separate tree files

(burnin = 20 000), with a reduced resample frequency

of 25 000. From this reduced tree sample, TREEANNOTATOR

was used to reconstruct a maximum clade credibility

(mcc) tree.

The RAxML gene tree was used as input in a Bayes-

ian Poisson Tree Processes (bPTP) analysis, as imple-

mented on the bPTP server (http://species.h-its.org/

ptp/; Zhang et al. 2013). PTP is a single-locus species

delimitation method using only nucleotide substitution

information, implementing a model assuming gene tree

branch lengths generated by two independent Poisson

process classes (within- and among-species substitution

events). The bPTP analysis was run using 100 000

MCMC generations, with a thinning of 100 and burn-in

of 0.1. In addition, the relaxed clock BEAST mcc tree was

used as input in single- and multiple-threshold GMYC

analyses (http://species.h-its.org/gmyc/). Available

simulation studies suggest that PTP outperforms GMYC

(Pons et al. 2006; Fujisawa & Barraclough 2013) for sin-

gle-locus species delimitations (Zhang et al. 2013), with-

out requiring an ultrametric tree.

Nuclear marker development and analysis

Cicurina-specific nuclear phylogenetic markers were

developed from comparative transcriptome data (sum-

marized in Appendix S1, Supporting information). After

preliminary PCR primer testing and sequencing, eight

nuclear gene regions were chosen for comprehensive

specimen sampling (primers, PCR conditions and tran-

script annotations are provided in Appendix S2, Sup-

porting information). Collection of nuclear gene data

specifically targeted the four focal species from 26

regional caves (Appendix 1). This focused sampling fol-

lows from COI gene tree results, which indicate the

monophyly of this species complex (see below). Also,

although eyeless Cicurina occur in the karst landscapes

to the north and south of the focal region (e.g. C. vibora,

C. bandida), these taxa are both morphologically (Paquin

& Dup�err�e 2009) and genetically distinct (see Results)

from the focal species.

Nuclear PCR products were purified, Sanger-

sequenced and edited as summarized above for mito-

chondrial sequences. The C. travisae transcriptome

data were not used for downstream phylogenetic

analyses since heterozygosity could not be assessed



(transcriptomes derived from two individuals; Appendix

S1). Only one of the nuclear matrices included indels –for the B2_F10F11 matrix, a three-amino-acid indel

restricted to C. buwata specimens was recoded as a two-

state nucleotide transition. Heterozygous nuclear

sequences were bioinformatically phased to alleles. SEQ-

PHASE (Flot 2010) was used to convert matrices for input

into PHASE 2.1.1 (Stephens et al. 2001; Stephens & Donnel-

ly 2003) using default settings (phase threshold = 90%,

100 iterations, thinning interval = 1, burn-in = 100). Gene

trees for individual nuclear genes were reconstructed

using RAxML searches (as above), applying a single

GTR_Γ model to each gene region. Gene trees included

data for focal taxa, rooted with orthologous sequences

from the C. vibora transcriptome (Appendix S1).

Multigenic nuclear genetic distances among individu-

als were calculated using POFAD v1.03 (Joly & Bruneau

2006). Uncorrected p-distances for each locus were cal-

culated in PAUP v4.0b10 (Swofford 2002), and because all

nuclear matrices are similar in aligned length and levels

of variation, individual matrices were standardized to

have the same weight (standardized weights option).

Analyses using nonstandardized distances gave very

similar results (results not shown). POFAD distances

were used to reconstruct a NeighborNet network in

SPLITSTREE4 (Huson & Bryant 2006). To eliminate the

potentially confounding influence of female-based pop-

ulation structure, mitochondrial data were not included

in POFAD analyses. Also, more distant outgroups (i.e.

C. vibora) were excluded from this analysis. This

‘nuclear + ingroup only’ data set was also used for

Bayesian clustering and BPP analyses summarized

below.

Bayesian genetic clustering analyses were conducted

using STRUCTURAMA 2.0 (Huelsenbeck et al. 2011) and

STRUCTURE (Pritchard et al. 2000, 2010). Structurama treats

the number of populations (K = number of distinct

genetic clusters) as a random variable following a Di-

richlet process prior (Pella & Masuda 2006; Huelsen-

beck & Andolfatto 2007; Huelsenbeck et al. 2011), while

STRUCTURE requires multiple analyses at different fixed K

values, and the use of ad hoc statistics to choose an

optimal K value. For both analyses, SNAP Map (Price &

Carbone 2005; Aylor et al. 2006) was used to convert

phased nuclear DNA sequences to numbered unique

alleles (haplotypes). Both Structurama and STRUCTURE

have been used to discover the number of distinct

genetic clusters in other species delimitation studies

(e.g. Leach�e & Fujita 2010; Weisrock et al. 2010; Carstens

& Satler 2013; Satler et al. 2013).

Structurama settings were as follows: model num-

pops=rv, admixture=no, concparmprior=gamma(0.1,10),

mcmc ngen=1 000, 000, samplefreq=100, printfreq=1000,calcmarginal likelihood burnin=1000. Runs with differ-

ent alpha values (shape and scale values of gamma dis-

tribution) were conducted as follows: (1,1) (1,5) (1,10)

(0.1,1) (0.1,5) (0.1,10). STRUCTURE runs were conducted

assuming between 1 and 6 genetic clusters (K = 1–6),with analyses for each K value replicated three times.

Analyses used an admixture model with a burn-in of

100 000 (one million MCMC steps after burn-in), and

allele frequencies were considered independent among

populations. Both the maximum value of the log proba-

bility of the data given K (L(K)) and DK (= rate of

change in log probability between successive K values,

Evanno et al. 2005) were used to identify an optimal K

value. Estimates from multiple replicates for multiple K

values were calculated in STRUCTURE HARVESTER (Earl &

vonHoldt 2012), and data were summarized using the

FullSearch algorithm of CLUMPP (Jakobsson & Rosenberg

2007) and visualized with DISTRUCT (Rosenberg 2004).

The Bayesian phylogenetics and phylogeography

method (BPP, Yang & Rannala 2010; Rannala & Yang

2013) was used with the multilocus nuclear data to cal-

culate the posterior probabilities of different species

delimitation models. The rjMCMC species delimitation

method was used with algorithm 1 (a value = 2, m value

= 1), ambiguous data columns were not removed (clean-

data =0), and the analysis was set for automatic fine-tune

adjustments. Two different combinations for population

size (theta, h) gamma priors were used, including a large

theta prior G(alpha = 1, beta = 10) and a more diffuse

(uninformative) theta prior G(alpha = 2, beta = 100). For

both analyses, a diffuse tau (s) gamma prior of G(alpha

= 2, beta = 1000) was used for the age of the root (tau0),

while the other divergence time parameters were

assigned the Dirichlet prior (Yang & Rannala 2010). Each

prior combination was run twice to check for conver-

gence and proper mixing. Analyses were run for 100 000

generations, sampling every five generations with 10 000

burnin. Species tree nodes with posterior probability val-

ues >0.95 were considered supported species delimita-

tions; values below 0.95 were considered as evidence for

collapsing a species tree node.

Bayesian phylogenetics and phylogeography analyses

require a priori specimen allocation and an input guide

tree – two alternatives were considered. First, a conser-

vative analysis was conducted in which only specimens

from known cave locations were included (see Fig. 1).

The guide tree used was as follows: (C. buwata sister to

(C. travisae, (C. reddelli + C. wartoni)), which is a topol-

ogy consistent with POFAD, STRUCTURE (K = 3) and

Structurama results (see below). Second, following

results of POFAD, STRUCTURE (K = 3) and Structurama,

and geography (Fig. 1), four ‘undetermined’ cave popu-

lations in the vicinity of C. travisae (Two Trunks Cave,

Gallifer Cave, Geode Cave, Stovepipe Cave) were

allocated to C. travisae, and five eastern ‘undetermined’


350 M. HEDIN

populations (Spider Cave, Ken Butler Pit, Jester Estates

Cave, Jest John Cave, Beard Ranch Cave) were allocated

to C. reddelli. The same guide tree as above was used.

Results

Morphology

Although formal quantitative analyses were not con-

ducted because of small sample sizes, consideration of

patterns of qualitative variation provided important

information. The epigynal morphology of available

female specimens is consistent with that reported for

the focal species of interest (compare Fig. 2 to Paquin &

Dup�err�e 2009; fig. 131). However, defining ‘diagnostic’

epigynal features for different species in this complex is

very challenging, since epigynal variation within a spe-

cies (or cave population) sometimes exceeds variation

between hypothesized species (e.g. Tooth Cave speci-

mens G1958, G2014; Fig. 2; see also fig. 5b of Paquin

(K)

(L)

(M) (N)

(A) (B) (C)

(D)(E) (F)

(G) (H) (I)

(J)

(O)

Fig. 2 Epigynal (ventral view) and palpal

(left palp, ventral view) morphologies for

adult specimens used in this study. (A–E), Cicurina buwata females: (A) Broken

Arrow Cave, G1961; (B) Broken Arrow

Cave, G1959; (C) Babe Cave, G2011; (D)

Buttercup Creek Cave, G2007; (E) Apple

Riata, G1997; Specimen determinations

for C. buwata based primarily on phylo-

genetic placement in DNA analyses,

except for Buttercup Creek Cave (known

location for C. buwata). (F) C. reddelli

female, Cotterell Cave, G 1960 (type

locality for C. reddelli). (G–J) C. travisae

females: G) North Root Cave, G1970; (H)

Tooth Cave, G1958; (I) Tooth Cave,

G2014; (J) Kretschmarr Double Pit,

G1966; all known localities for C. travisae,

including type locality (Tooth Cave). (K)

‘eastern undetermined’ female, Jester

Estates Cave, G 1985. (L–O) adult males:

(L) Spider Cave, G1981; (M) Jester Estates

Cave, G 1986; (N) Stovepipe Cave,

G1977; (O) Amber Cave, G 1998; Amber

Cave is a known locality for C. travisae,

although adult males for this species

have never been described. Colours and

symbols used to designate populations as

in Fig. 1.



et al. 2008). Four adult male specimens were also avail-

able for study and imaged (Fig. 2). Because male speci-

mens have never been described for any of the focal

species (Paquin & Dup�err�e 2009), there is no basis for

formal species-level comparison. It is notable, however,

that all male specimens have essentially identical palp

morphologies.

Mitochondrial analyses

New mitochondrial sequences have been submitted to

GenBank (Appendix 1); GenBank numbers for previ-

ously published sequences are provided in Fig. 3.

Maximum-likelihood and Bayesian analyses (Fig. 3) of

mitochondrial sequences result in generally similar

gene tree topologies, with two minor differences.

First, the C. bandida/C. puentecilla clade (outside the

focal group) differs in position, but this position is

weakly supported in both analyses. Second, RAxML

analyses suggest a different root placement within

C. buwata, with eastern populations (No Rent, Apple

Riata, Weldon, McNeil Bat; Fig. 3) forming a grade

leading to western populations. Both Bayesian and

maximum-likelihood trees recover a well-supported

clade (bootstrap proportion values >70, posterior prob-

ability values >0.95) that includes the focal taxa, and

a primary separation between C. buwata versus

C. reddelli/C. travisae/C. wartoni (hereafter called the

C. travisae complex). The hypothesis of Gertsch (1992)

that C. buwata occurs in the southern Cotterell and

Gallifer Caves (see fig. 131 of Paquin & Dup�err�e

2009) is not supported by mitochondrial or nuclear

evidence (see below).

Mitochondrial species delimitation results (bPTP

and GMYC) are summarized on Fig. 3. Outside the

focal group, there is a high degree of congruence

between bPTP groups and morphologically described

species. Within the focal group, C. buwata forms a

single bPTP group, and the three members of the

C. travisae complex together form a single bPTP

group. This result is consistent with the hypothesis

that members of the C. travisae complex represent a

single species, rather than three distinct species. The

single-threshold GMYC model also recovers C. buwata

and members of the C. travisae complex as separate

individual groups, but seems overly conservative in

that other single GMYC clusters (outside the focal

group) include described species that are clearly mor-

phologically distinct and occupy highly disjunct geo-

graphic distributions (e.g. C. vibora, C. troglobia,

C. hoodensis). The multiple-threshold GMYC model

appears biologically unrealistic, fragmenting most

described species into several individual clusters

(Fig. 3).

Nuclear analyses

Data were collected and phased for eight nuclear genes;

unphased sequences have been submitted to GenBank

(Appendix 1), and alignments of phased data are avail-

able on Dryad (doi:10.5061/dryad.qc3s0). All nuclear

sequences correspond to exons, and PCR-amplified San-

ger data match transcriptome data. Final matrices

include very little missing data (8 nuclear gene matrices

X 34 individuals per matrix – 7 total missing

sequences), and no single specimen is missing data for

more than one nuclear gene (Appendix 1). Gene regions

are similar in aligned length, and all include phyloge-

netic information (Table 1).

Each nuclear gene tree is topologically unique

(Appendix S3, Supporting information), but general pat-

terns are apparent. Members of the C. travisae complex

are recovered as a clade separate from a C. buwata clade

in seven of eight gene trees, and these clades are

strongly supported (maximum-likelihood bootstrap

>70). Within the C. travisae complex, genetic relation-

ships vary from gene to gene, and sequences from indi-

vidual hypothesized species do not cluster together –instead, sequences from different hypothesized species

within the C. travisae complex are intermixed on nuclear

gene trees. Sequences from C. wartoni specimens never

form an exclusive group on nuclear gene trees, but

instead are always intermixed with other members of

the C. travisae complex (Appendix S3).

The POFAD network shows an obvious division

between C. buwata and members of the C. travisae com-

plex (Fig. 4A). Within the C. travisae complex, C. wartoni

specimens are not grouped together on the network.

Specimen (and population) placement on the network

coincides roughly with geographic position (western

populations basal, eastern populations derived; Fig. 4B,

C). A group of populations including C. reddelli from

Cotterell Cave, five eastern ‘undetermined’ cave popu-

lations (Beard Ranch Cave, Jest John Cave, Jester Estates

Cave, Ken Butler Pit, Spider Cave) and two specimens

of C. wartoni cluster together – this genetic association

mirrors that recovered by STRUCTURE K = 3 and Structu-

rama (see below).

STRUCTURE results suggest two genetic partitions, with

K = 2 including the largest DK (461.86) as estimated

using the Evanno method (Fig. 5A). The two genetic

clusters correspond to C. buwata and the C. travisae

complex, consistent with the hypothesis that members

of the C. travisae complex represent a single species.

Although K = 2 (mean LnP(K) = �1165.2) is optimal

under the Evanno method, other K values were consid-

ered, allowing for the possibility that the Evanno

method is underestimating species diversity in this

complex. In particular, a K = 3 (mean LnP


352 M. HEDIN

AY633093_Cpampa

AY633098_CbrunsiAY633012_013_Cloftini

AY633011_Cbullis

AY633008_Cbullis

AY633006_CbullisAY633007_CbullisAY633010_Cbullis

AY633009_Cbullis

AY633057_CmadlaAY633058_CmadlaAY633059_Cmadla

AY633056_CmadlaAY633061_Cmadla



AY633073_Cmadla

AY633055_CvesperaAY633068_Cmadla_AY633054_Cvespera

AY633069_Cmadla

AY633064_Cmadla

AY633072_CmadlaAY633052_070_Cmadla

AY633089_Cvibora_TemplesofThorAY633090_Cvibora_TemplesofThor

Cvibora_TemplesofThor_TranscriptomeAY633015_Ctroglobia

AY633014_Cmixmaster

AY633025_Choodensis

AY633024_ChoodensisAY633016_017_Ccaliga

AY633021_Choodensis_018_CcaligaAY633026_Choodensis

AY633020_023_ChoodensisAY633019_Choodensis

AY633082_083_Cbandida

AY633077_Cpuentecilla

AY633076_CpuentecillaAY633075_Cpuentecilla

AY633081_CpuentecillaAY633078_079_Cpuentecilla

AY633099_Creddelli_CotterellCave

AY633105_CplacidaAY633104_Cpallida

AY633030_Cvarians

8.0

Ctravisae_NorthRootCave_G1970

Creddelli_CotterellCave_G1960_G1989

StovepipeCave_G1977Cwartoni_PicklePit_G1979_G1980

Ctravisae_McDonaldCave_G1965Ctravisae_McDonaldCave_G1964GeodeCave_G1976

Ctravisae_ToothCave_G1958_G1972_G2014

KenButlerPit_G2009BeardRanchCave_G1984

SpiderCave_G1983SpiderCave_G1981SpiderCave_G1982JestJohnCave_G1988JesterEstatesCave_G1987JesterEstatesCave_G1985

Ctravisae_KretschmarrDPit_G1966Ctravisae_AmberCave_G1998_G1999

TwoTrunksCave_G1967_G1968

AY633084_085_Cbuwata_TestudoTube

Cbuwata_NoRentCave_G1994Cbuwata_AppleRiataTrace_G1997

Cbuwata_NoRentCave_G1995

Cbuwata_BrokenArrowCave_G1961Cbuwata_MarigoldCave_G2005_G2006

Cbuwata_NoRentCave_G1957_G1992

Cbuwata_McNeilBatCave_G1996

Cbuwata_LakelineCave_G2001Cbuwata_LakelineCave_G2000

Cbuwata_BabeCave_G2010_G2011Cbuwata_ButtercupCreekCave_G2007Cbuwata_DiesRanchTreasureCave_G2002

0.93/87

0.93/87

0.96 /79

0.99/99

0.84 /83

0.99/100

0.80/710.99/97

0.99/98

0.99 /93

0.99/90

0.98/82

0.99/100

0.99/98

RAxML

GMYC_Single

C. buwata

C. travisae Complex

bPTP

1.01.01.0

0.94

1.0

0.80

0.81

1.0

0.91

0.83

0.95

1.01.0

1.0

1.0

GMYC_Multiple

Fig. 3 Bayesian inference mitochondrial gene tree with posterior probability and RAxML bootstrap values (for primary lineages),

plus bPTP and GMYC results. Arrow denotes alternative RAxML phylogenetic placement for Cicurina puentecilla/C. bandida clade.

Redundant haplotypes not shown include CbuwataTestudoTube = CbuwataBroken ArrowCave_G1959_G1962 + CbuwataMarigold-

Cave_G2004, CbuwataWeldonCave_G1992 = CbuwataNoRentCave_G1994, TwoTrunksCave_G1967_G1968 = GalliferCave_G1975. Col-

ours and symbols used to designate focal populations as in Fig. 1.



(K) = �1046.4) hypothesis was considered – this genetic

clustering implies three separate genetic groups corre-

sponding to C. buwata, C. travisae (plus western unde-

termined) and C. reddelli + plus eastern undetermined +C. wartoni (Fig. 5B). Structurama analyses, using a no

admixture model with multiple prior alpha values, also

support this same K = 3 hypothesis (Table 2). Analyses

under certain alpha values include a low posterior

probability for K = 4 (e.g. alpha 1,1, pp = 0.21; Table 2),

but the fourth genetic cluster does not consistently

include the same set of individuals.

Bayesian phylogenetics and phylogeography analyses

provide strong support for a node separating C. buwata

versus the C. travisae complex, but fail to support fur-

ther subdivisions within the C. travisae complex (Fig. 6).

Again, these results are consistent with the hypothesis

that members of the C. travisae complex represent a sin-

gle species.

Discussion

The Cicurina travisae species complex

The approach to species delimitation used here relied

upon multiple lines of independent evidence, included

analyses with different analytical assumptions and inte-

grated results via observed congruence. When congru-

ence was not observed (e.g. K = 2 versus K = 3, K = 2

STRUCTURE versus STRUCTURAMA), additional analyses were

conducted to explore this incongruence. Taken together,

multiple lines of evidence support a clear distinction

between northern C. buwata and a southern C. travisae

complex that currently includes three described species.

As shown here and elsewhere (Paquin & Dup�err�e

2009), different members of the C. travisae complex have

extremely similar female epigynal morphologies

(Fig. 2). Male specimens are not available from all cave

populations in the complex, but those available

(spanning much of the range of the C. travisae complex)

have essentially indistinguishable pedipalps (Fig. 2).

Although mitochondrial data indicate high levels of

female-based genetic structuring (e.g. exclusive mito-

chondrial clades that correspond to single-cave popula-

tions), single-locus species delimitation analyses (bPTP,

single-threshold GMYC) recover members of this com-

plex as a single lineage. This result provides a general

illustration as to why simple phylogenetic patterns

observed on single gene trees (e.g. genealogical exclu-

sivity for single caves) should not be overinterpreted as

evidence for species status.

The multigenic nuclear perspective shows that

sequences from C. wartoni, C. travisae and C. reddelli are

intermixed on nuclear gene trees. There is some signal

for east to west geographic structuring in the nuclear-

only POFAD network, mirrored in K = 3 STRUCTURE and

STRUCTURAMA analyses, as might be expected in a system

where populations are restricted to caves. However, this

geographic structure is not supported as species-level

divergence in BPP validation analyses, which instead

support the C. travisae complex as a single genetic line-

age. This result is consistent with the K = 2 STRUCTURE

results, POFAD analyses and the above summarized

morphological and mitochondrial results.

It is important for researchers to complete taxonomic

actions implied by robust results, be this species synon-

ymy or new species description (e.g., Fujita & Leach�e

2010; Satler et al. 2013; Derkarabetian & Hedin 2014). To

this end, the species C. wartoni, C. travisae and C. reddel-

li are formally synonymized below. The species concept

used here corresponds to a general lineage concept (de

Queiroz 2007), with species viewed as separately evolv-

ing metapopulation lineages, thus allowing for some

internal genetic structuring (i.e. connected subpopula-

tions). Multiple lines of congruent evidence were used

to operationally recognize this metapopulation lineage,

with both population genetic and multispecies coales-

cent evidence from the nuclear genome emphasized.

Family DICTYNIDAE O. Pickard-Cambridge 1871

[urn:lsid:nmbe.ch:spiderfam:0042]

Genus Cicurina Menge 1871 [urn:lsid:nmbe.ch:spider-

gen:01972]

Subgenus Cicurella Chamberlin & Ivie 1940

Cicurina (Cicurella) travisae Gertsch 1992 [urn:lsid:

nmbe.ch:spidersp:022224]; FIGS 1-6

Cicurina travisae Gertsch 1992: 101, figs 63–70.Cicurina travisae Paquin et al. 2008: 147, fig. 5b.

Cicurina travisae Paquin & Dup�err�e 2009: 47, figs 106-

107, fig. 131.

Cicurina reddelli Gertsch 1992: 105, figs 77–78; new

synonymy.

Cicurina reddelli Paquin & Dup�err�e 2009: 40, figs 86–87, 131.

Table 1 Nuclear gene data summary

Primer

Names

Aligned

length

No.

Ingroup

Sequences

Parsimony

Informative

Sites

Nucleotide

Diversity

B1_B11B12 662 47 33 0.018

B1_C9C10 706 39 11 0.006

B1_E1E2 667 45 12 0.007

B1_G5G6 827 35 14 0.008

B1_G11G12 840 44 14 0.005

B2_D2D3 560 48 20 0.012

B2_F10F11 677 47 25 0.011

B2_H6H7 647 53 22 0.007

Diversity statistics calculated using MEGA 6.06 (Tamura et al.

2013), ingroup data only.


354 M. HEDIN

Cicurina wartoni Gertsch 1992: 101, figs. 75–76; new

synonymy.

Cicurina wartoni Paquin & Dup�err�e 2009:55, figs. 122–123, 131.

Taxonomic type II error and the ‘burden of proof’

Like all species hypotheses, the synonymy hypothesis

proposed above remains falsifiable. It is possible that

the combination of species criterion, data analyses and

data type utilized has failed to detect a species-level dif-

ference that actually exists, resulting in a so-called taxo-

nomic type II error (Padial et al. 2010; Miralles &

Vences 2013; McCormack & Maley 2015). For example,

until diversity biologists employ complete genomic data

for large, theoretically sufficient samples, it will always

be possible to speculate about the proverbial data nee-

dle (or needles) in the haystack. Although such argu-

ments are frankly difficult to counter, several are made

here for the case of Cicurina travisae.

First, there are clear indications that many genetic

species delimitation approaches are inflationist in natu-

rally fragmented systems – if population structure

exists, there is an apparent analytical bias towards

C_travisae Complex

0.1

CTRAVISAE_TOOTHCAVE_G1958

STOVEPIPECAVE_G1977

GALLIFERCAVE_G1975

CTRAVISAE_MCDONALDCAVE_G1964

CTRAVISAE_MCDONALDCAVE_G1965

CTRAVISAE_AMBERCAVE_G1998

GEODECAVE_G1976CWARTONI_PICKLEPIT_G1979

CREDDELLI_COTTERELLCAVE_G1960

CREDDELLI_COTTERELLCAVE_G1989

JESTJOHNCAVE_G1988

KENBUTLERPIT_G2009SPIDERCAVE_G1982

SPIDERCAVE_G1981

JESTERESTATESCAVE_G1985

BEARDRANCHCAVE_G1984

CWARTONI_PICKLEPIT_G1978CWARTONI_PICKLEPIT_G1980

CTRAVISAE_NORTHROOTCAVE_G1970TWOTRUNKSCAVE_G1968

CTRAVISAE_TOOTHCAVE_G2014

CTRAVISAE_KRETSCHMARRDOUBLEPIT_G1966 JESTERESTATES

CAVE_G1986

C_buwata(A)

(B)

(C)McDonald

Kretschmarr

Amber

TwoTrunks

Tooth

NorthRoot

Gallifer

Geode

Stovepipe

PicklePit

Spider

BeardRanch

KenButlerPit

JesterEstates

JestJohn

Cotterell

W

R

T

TT

TT

Fig. 4 NeighborNet network recon-

structed using standardized POFAD

nuclear distances. (A) Entire network,

showing primary division between Cic-

urina buwata and the C. travisae complex.

(B) POFAD network for C. travisae com-

plex. Colours and symbols as in Fig. 1.

(C) Map inset, colours and symbols used

to designate populations as in Fig. 1.



oversplitting (type I error; e.g. Hey 2009; Camargo &

Sites 2013). For example, both single-threshold GMYC

(Keith & Hedin 2012; Satler et al. 2013; Hamilton et al.

2014) and BPP (Barley et al. 2013; Carstens & Satler

2013; McKay et al. 2013; Miralles & Vences 2013; Satler

et al. 2013) have been suggested to oversplit in naturally

fragmented systems. These methods, however, support

a single species hypothesis for C. travisae. Second, for

nuclear analyses that support a more finely subdivided

taxonomy (e.g. STRUCTURE K = 3, Structurama), the smal-

ler units correspond to arrays of multiple adjacent pop-

ulations (e.g. eastern versus western cave populations,

Figs 4 and 5). None of the nuclear analyses conducted

support single cave populations as species. Third,

although genomic-scale data were not technically used

here (e.g. genome-wide SNP data, Leach�e et al. 2014),

considerable resources were devoted towards the devel-

opment of Cicurina-specific ‘rapidly evolving’ nuclear

markers. If speciation in Cicurina proceeds via an ‘isola-

tion plus drift’ model as expected, then nucleotide dif-

ferences observed in a subset of the nuclear genome

should reflect overall genomic divergence (Feder et al.

2012). If Cicurina speciation instead corresponds to a

‘genomic islands of divergence’ model, then speciation

would be potentially missed if selectively important loci

were not sampled, but this model of speciation is con-

sidered unlikely here. Finally, populations from Pickle

Pit (type for ‘C. wartoni’) and Cotterell Cave (type for

‘C. reddelli’) correspond to ‘evolutionary significant

units’, if monophyly for mitochondrial alleles is used as

the criteria for such units (Moritz 1994). As such, these

cave populations retain conservation importance, just

not as distinct species.

Genomics-scale species delimitation for all taxa on

Earth seems unrealistic and prohibitively expensive,

particularly given the enormity of the current task faced

by diversity biologists (i.e. most existing species

hypotheses never tested, millions of species remain

undescribed). However, for ‘high-stakes’ research on

conservation-relevant taxa (e.g., hypothesized single site

endemics), we might expect such data sets to become

the norm, or even the expectation (McCormack & Ma-

ley 2015). Such data sets, however, do not imply an

easy resolution to the species delimitation problem, as

both the genomic architecture of speciation and popula-

tion subdivision continue to present analytical chal-

lenges for most currently available methods.

Table 2 Structurama results

K Alpha 1,1 Alpha 1,5 Alpha 1,10 Alpha 0.1,1 Alpha 0.1,5 Alpha 0.1,10

Marginal likelihood �516.32 �517.41 �518.98 �517.31 �517.31 �516.67

Model Prob (# pops) 1 0 0 0 0 0 0

2 0 0 0 0 0 0

3 0.76 0.88 0.91 0.81 0.91 0.94

4 0.21 0.12 0.09 0.18 0.09 0.06

5 0.02 0.01 0 0.01 0 0

Appl

eRia

taBa

beCa

ve

Brok

enAr

row

Cave

Butte

rcup

Cree

kCav

eLa

kelin

eCav

eM

arig

oldC

ave

McN

eilB

atCa

ve

NoRen

tCav

e

Test

udoT

ube

Wel

donC

ave

Bear

dRan

chCa

veCo

ttere

llCav

e

Jest

John

Cave

Jest

erEs

tate

sCav

eKe

nBut

lerP

itSp

ider

Cave

Ambe

rCav

e

Gallif

erCa

veGeo

deCa

ve

Kret

schm

arrD

oubl

ePit

McD

onal

dCav

e

North

Root

Cave

Stov

epip

eCav

eTo

othC

ave

TwoT

runk

sCav

ePi

ckle

Pit

Appl

eRia

taBa

beCa

ve

Brok

enAr

row

Cave

Butte

rcup

Cree

kCav

eLa

kelin

eCav

eM

arig

oldC

ave

McN

eilB

atCa

ve

NoRen

tCav

e

Test

udoT

ube

Wel

donC

ave

Bear

dRan

chCa

veCo

ttere

llCav

e

Jest

John

Cave

Jest

erEs

tate

sCav

eKe

nBut

lerP

itSp

ider

Cave

Ambe

rCav

e

Gallif

erCa

veGeo

deCa

ve

Kret

schm

arrD

oubl

ePit

McD

onal

dCav

e

North

Root

Cave

Stov

epip

eCav

eTo

othC

ave

TwoT

runk

sCav

ePi

ckle

Pit

K = 2, mean LnP(K) = –1165.2

C buwata C travisae complex

C buwata

C. wartoni, C reddelli + “EASTERN undetermined”

C wartoni

G19

80

K = 3, mean LnP(K) = –1046.4

(A)

(B)

Fig. 5 STRUCTURE graphics (resulting from DISTRUCT) for K = 2

(A) and K = 3 (B). Each column represents a specimen,

grouped by cave population of origin. Different colours repre-

sent different genetic clusters (K); estimated membership coef-

ficients are proportional to bar colour height.


356 M. HEDIN

The importance or robust species hypotheses inconservation biology

This study has relevance to systematic studies of other

Texas cave Cicurina, including species currently listed

as US Federally Endangered (Longacre 2000; U.S. Fish

& Wildlife Service 2008). First, the mitochondrial delim-

itation results presented suggest the possible synonymy

of certain taxa (e.g. C. madla and C. vespera, C. mixmas-

ter/C. caliga/C. hoodensis, etc.; Fig. 3). This issue of syn-

onymy, and the idea that many Texas cave Cicurina

may not be as highly endemic as indicated by the cur-

rent taxonomy, has been mentioned elsewhere multiple

times (Paquin & Hedin 2004; Paquin et al. 2008). Indeed,

Paquin & Dup�err�e (2009) called species synonymy a

‘generalized problem’ in Texas cave Cicurina. The revi-

sionary work of Ledford et al. (2012) on Texas cave lep-

tonetid spiders suggests a similar pattern, with a new

integrative taxonomy indicating fewer, more wide-

spread species than a prior taxonomy, suggesting a

higher number of more narrowly endemic species.

More generally, the integrative framework applied here

(or something akin to this framework, e.g. Satler et al.

2013; Derkarabetian & Hedin 2014; etc.) should be

applied elsewhere in Texas cave Cicurina. Of the four

federally listed taxa (C. baronia, C. madla, C. venii, C. ves-

pera), three species are single site endemics, two species

hypotheses are based on single female specimens

(Paquin & Dup�err�e 2009), and only one species has

been studied using molecular evidence (mitochondrial

only, Paquin & Hedin 2004). A major hurdle in the

study of cave Cicurina species limits has been specimen

rarity and availability – new sequencing technologies

potentially allow for the collection of massive quantities

of DNA sequence data from museum specimens (Bi

et al. 2013), perhaps overcoming this hurdle.

The Draft Recovery Plan for listed Cicurina taxa (U.S.

Fish & Wildlife Service 2008) does not include ‘conduct

integrative taxonomy’ as a suggested research need.

Although the existing species delimitations may be

accurate, hypotheses with such obvious conservation,

political and economic importance need to be more data

rich [arguments echoed by Paquin & Dup�err�e (2009)],

and conducting modern integrative taxonomy must be

a fundamental and primary research need. If the con-

servation action is taxon focused, then this same argu-

ment applies to all taxa where the existing taxonomy is

arguably weak, whether these taxa are new candidates

for conservation action, or taxa that are already pro-

tected. Taxon-focused conservation biology is expensive

(e.g. McCarthy et al. 2012), financial resources are lim-

ited, and downstream resource allocations flow from

the most fundamental question – is the taxonomic unit

evolutionarily distinct? This question needs to be

answered first.

buwata

1.0

travisae

wartoni

reddelli

0.37/0.35

0.53/0.53

0.81/ 0.82 0.50/

0.51

(A) (B)

(C) (D)

Theta prior (2, 100)

Theta prior (2, 100)

Theta prior (1, 10)

Theta prior (1, 10)

buwata

1.0

travisae

wartoni

reddelli

buwata

1.0

travisae

wartoni

reddelli

buwata

1.0

travisae

wartoni

reddelli

Fig. 6 Summary of Bayesian phylogenet-

ics and phylogeography analyses. (A and

B) Undetermined cave populations not

included in analysis, guide tree = Cicuri-

na buwata sister to (C. travisae, (C. reddelli

+ C. wartoni)) – A) G (alpha = 2, beta =100) theta prior, (B) G (alpha = 1, beta =10) theta prior. (C and D) Western ‘unde-

termined’ cave populations allocated to

C. travisae, eastern ‘undetermined’ popu-

lations (Spider Cave, Ken Butler Pit, Jes-

ter Estates Cave, Jest John Cave, Beard

Ranch Cave) allocated to C. reddelli.

Guide tree = C. buwata sister to (C. travi-

sae, (C. reddelli + C. wartoni)) – (C) G

(alpha = 2, beta = 100) theta prior, (D) G

(alpha = 1, beta = 10) theta prior.



Acknowledgements

This research was funded by the US Fish & Wildlife Service,

contract #F13PX00770. Cyndee Watson provided support for

this project from inception to completion and deserves special

thanks. Specimens were obtained from Mark Sanders, Todd

Bayless, P. Fushille and Jet Larsen. Kristen Emata provided

expert laboratory assistance, with help from David Zezoff.

James Starrett extracted RNA, while Shahan Derkarabetian and

Dave Carlson assisted in the transcriptome assembly process.

Members of the Hedin laboratory, Jordan Satler, Bryan Car-

stens and two anonymous reviewers provided comments that

helped to improve the manuscript.

References

Aylor DL, Price EW, Carbone I (2006) SNAP: combine and map

modules for multilocus population genetic analysis. Bioinfor-

matics, 22, 1399–1401.Barley AJ, White J, Diesmos AC, Brown RM (2013) The chal-

lenge of species delimitation at the extremes: Diversification

without morphological change in Philippine sun skinks. Evo-

lution, 67, 3556–3572.Bendik NF, Meik JM, Gluesenkampo AG, Roelke CE, Chippin-

dale PT (2013) Biogeography, phylogeny, and morphological

evolution of central Texas cave and spring salamanders.

BMC Evolutionary Biology, 13, 201.

Bi K, Linderoth T, Vanderpool D, Good JM, Nielsen R, Moritz

C (2013) Unlocking the vault: next-generation museum pop-

ulation genomics. Molecular Ecology, 22, 6018–6032.Camargo A, Sites J Jr (2013) Species delimitation: a decade after

the renaissance. In: The Species Problem – Ongoing Issues (ed.

Pavlinov I), pp. 225–247. InTech, New York, New York.

Carstens BC, Satler JD (2013) The carnivorous plant described

as Sarracenia alata contains two cryptic species. Biological Jour-

nal of the Linnean Society, 4, 737–746.Carstens BC, Pelletier TA, Reid NM, Satler JD (2013) How to

fail at species delimitation. Molecular Ecology, 22, 4369–4383.Chiari Y, van der Meijden A, Mucedda M, Lourenco JM, Ho-

chkirch A, Veith M (2012) Phylogeography of Sardinian cave

salamanders (genus Hydromantes) is mainly determined by

geomorphology. PLoS ONE, 7, e32332.

Derkarabetian S, Hedin M (2014) Integrative taxonomy and

species delimitation in harvestmen: a revision of the western

North American genus Sclerobunus (Opiliones: Laniatores:

Travunioidea). PLoS ONE, 9, e104982.

Derkarabetian S, Steinmann DB, Hedin M (2010) Repeated and

time-correlated morphological convergence in cave-dwelling

harvestmen (Opiliones, Laniatores) from montane western

North America. PLoS ONE, 5, e10388.

Drummond AJ, Ho SYW, Phillips MJ, Rambaut A (2006)

Relaxed phylogenetics and dating with confidence. PLoS

Biology, 4, e88. doi:10.1371/journal.pbio.0040088.

Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayes-

ian phylogenetics with BEAUti and the BEAST 1.7. Molecular

Biology & Evolution, 29, 1969–1973.Earl DA, vonHoldt BM (2012) STRUCTURE HARVESTER: a website

and program for visualizing STRUCTURE output and imple-

menting the Evanno method. Conservation Genetic Resources,

4, 359–361.

Evanno G, Regnaut S, Goudet J (2005) Detecting the number of

clusters of individuals using the software STRUCTURE: a simu-

lation study. Molecular Ecology, 14, 2611–2620.Feder JL, Egan SP, Nosil P (2012) The genomic of speciation-

with-gene-flow. Trends in Genetics, 28, 342–349.Flot JF (2010) SEQPHASE: a web tool for interconverting PHASE

input/output files and FASTA sequence alignments. Molecu-

lar Ecology Resources, 10, 162–166.Fujisawa T, Barraclough TG (2013) Delimiting species using

single-locus data and the Generalized Mixed Yule Coalescent

approach: a revised method and evaluation on simulated

data sets. Systematic Biology, 62, 707–724.Fujita MK, Leach�e AD (2010) A coalescent perspective on

delimiting and naming species: a reply to Bauer et al. Pro-

ceedings of the Royal Society B: Biological Sciences, 278, 493–495.

Fujita MK, Leach�e AD, Burbrink FT, McGuire JA, Moritz C

(2012) Coalescent-based species delimitation in an integrative

taxonomy. Trends in Ecology & Evolution, 27, 480–488.Gertsch WJ (1992) Distribution patterns and speciation in

North American cave spiders with a list of the troglobites

and revision of the cicurinas of the subgenus Cicurella.. Texas

Memorial Museum Speleological Monographs, 3. Studies on the

Endogean fauna of North America, 2, 75–122.Hamilton CA, Hendrixson BE, Brewer MS, Bond JE (2014) An

evaluation of sampling effects on multiple DNA barcoding

methods leads to an integrative approach for delimiting spe-

cies: a case study of the North American tarantula genus

Aphonopelma (Araneae, Mygalomorphae, Theraphosidae).

Molecular Phylogenetics and Evolution, 71, 79–93.Hedin M (1997) Molecular phylogenetics at the population/

species interface in cave spiders of the southern Appala-

chians (Araneae: Nesticidae: Nesticus). Molecular Biology and

Evolution, 14, 309–324.Hey J (2009) On the arbitrary identification of real species. In:

Speciation and Patterns of Biodiversity (eds Butlin RK, Bridle

JR, Schlueter D), pp. 15–28. Cambridge University Press,

New York, New York.

Huelsenbeck JP, Andolfatto P (2007) Inference of population

structure under a Dirichlet process model. Genetics, 175,

1787–1802.Huelsenbeck JP, Huelsenbeck ET, Andolfatto P (2011) Structu-

rama: Bayesian inference of population structure. Evolution-

ary Bioinformatics Online, 7, 55–59.Huson DH, Bryant D (2006) Application of phylogenetic net-

works in evolutionary studies. Molecular Biology and Evolu-

tion, 23, 254–267.Jakobsson M, Rosenberg NA (2007) CLUMPP: a cluster matching

and permutation program for dealing with label switching

and multimodality in analysis of population structure. Bioin-

formatics, 23, 1801–1806.Joly S, Bruneau A (2006) Incorporating allelic variation for

reconstructing the evolutionary history of organisms from

multiple genes: an example from Rosa in North America.

Systematic Biology, 55, 623–636.Juan C, Guzik MT, Jaume D, Cooper SJB (2010) Evolution in

caves: Darwin’s ‘wrecks of ancient life’ in the molecular era.

Molecular Ecology, 19, 3865–3880.Keith RM, Hedin M (2012) Extreme mitochondrial population

subdivision in southern Appalachian paleoendemic spiders


358 M. HEDIN

(Araneae, Hypochilidae, Hypochilus), with implications for

species delimitation. Journal of Arachnology, 40, 167–181.Lanfear R, Calcott B, Ho SYW, Guindon S (2012) PARTITIONFIND-

ER: combined selection of partitioning schemes and substitu-

tion models for phylogenetic analyses. Molecular Biology and

Evolution, 29, 1695–1701.Leach�e AD, Fujita MK (2010) Bayesian species delimitation

in West African forest geckos (Hemidactylus fasciatus). Pro-

ceedings of the Royal Society B: Biological Sciences, 277, 3071–3077.

Leach�e AD, Fujita MK, Minin VN, Bouckaert RR (2014) Species

delimitation using genome-wide SNP data. Systematic Biol-

ogy, 63, 534–542.Ledford J, Paquin P, Cokendolpher J, Campbell J, Griswold C

(2012) Systematics, conservation and morphology of the spi-

der genus Tayshaneta (Araneae, Leptonetidae) in central

Texas caves. ZooKeys, 167, 1–102.Lohse K (2009) Can mtDNA barcodes be used to delimit spe-

cies? A response to Pons et al. (2006) Systematic Biology, 58,

439–442.Longacre C (2000) Department of the Interior, Fish and Wild-

life Service, 50 CFR Part 17, RIN 1018-AF33. Final rule to list

nine Bexar County, Texas invertebrate species as endan-

gered. Federal Register, 65, 81419–81433.Malaney JL, Cook JA (2013) Using biogeographical history to

inform conservation: the case of Preble’s meadow jumping

mouse. Molecular Ecology, 22, 6000–6017.McCarthy DP, Donald PF, Scharlemann JP et al. (2012) Finan-

cial costs of meeting global biodiversity conservation targets:

current spending and unmet needs. Science, 338, 946–949.McCormack JE, Maley JM (2015) Interpreting negative results

with conservation implications: another look at distinctness

of coastal California Gnatcatchers. The Auk, 132. doi:

10.1642/AUK-14-184.1.

McKay BD, Mays HL Jr, Wu Y et al. (2013) An empirical com-

parison of character-based and coalescent-based approaches

to species delimitation in a young avian complex. Molecular

Ecology, 22, 4943–4957.Miralles A, Vences M (2013) New metrics for comparison of

taxonomies reveal striking discrepancies among species

delimitation methods in Madascincus lizards. PLoS ONE, 8,

e68242.

Mora C, Tittensor DP, Adl S, Simpson AGB, Worm B (2011)

How many species are there on Earth and in the ocean?

PLoS Biology, 9, e1001127. doi:10.1371/journal.pbio.1001127.

Moritz C (1994) Defining “evolutionarily significant units” for

conservation. Trends in Ecology & Evolution, 9, 373–375.Niemiller ML, Near TJ, Fitzpatrick BM (2012) Delimiting spe-

cies using multilocus data: diagnosing cryptic diversity in

the southern cavefish, Typhlichthys subterraneus (Teleostei:

Amblyopsidae). Evolution, 66, 846–866.Niemiller ML, Graening GO, Fenolio DB et al. (2013) Doomed

before they are described? The need for conservation assess-

ments of cryptic species complexes using an amblyopsid

cavefish (Amblyopsidae, Typhlichthys) as a case study. Biodi-

versity & Conservation, 22, 1799–1820.O’Meara BC (2010) New heuristic methods for joint species

delimitation and species tree inference. Systematic Biology, 59,

59–73.Padial JM, Miralles A, De la Riva I, Vences M (2010) The inte-

grative future of taxonomy. Frontiers in Zoology, 7, 16.

Pante E, Schoelinck C, Puillandre N (2015) From integrative

taxonomy to species description: one step beyond. Systematic

Biology, 64, 152–160.Paquin P, Dup�err�e N (2009) A first step towards the revision

of Cicurina: redescription of type specimens of 60 troglobitic

species of the subgenus Cicurella (Araneae: Dictynidae), and

a first visual assessment of their distribution. Zootaxa, 2002,

1–67.Paquin P, Hedin M (2004) The power and perils of “molecular

taxonomy”: a case study of eyeless and endangered Cicurina

(Araneae: Dictynidae) from Texas caves. Molecular Ecology,

13, 3239–3255.Paquin P, Duperre N, Cokendolpher JC, White K, Hedin M

(2008) The fundamental importance of taxonomy in conser-

vation biology: the case of the eyeless Cicurina bandida (Ara-

neae: Dictynidae) of central Texas, including new synonyms

and the description of the male of the species. Invertebrate

Systematics, 22, 1–11.Pella J, Masuda M (2006) The Gibbs and split-merge sampler

for population mixture analysis from genetic data with

incomplete baselines. Canadian Journal Fish Aquatic Sciences,

63, 576–596.Pons J, Barraclough TG, Gomez-Zurita J et al. (2006) Sequence-

based species delimitation for DNA taxonomy of unde-

scribed insects. Systematic Biology, 55, 595–609.Price EW, Carbone I (2005) SNAP. Workbench management

tool for evolutionary population genetic analysis. Bioinformat-

ics Applications Note, 21, 402–404.Pritchard JK, Stephens M, Donnelly P (2000) Inference of popu-

lation structure using multilocus genotype data. Genetics,

155, 945–959.Pritchard JK, Wen X, Falush D (2010) Documentation for Struc-

ture software: Version 2.3. Available from http://

pritch.bsd.uchicago.edu/structure.html. 1-38.

de Queiroz K (2007) Species concepts and species delimitation.

Systematic Biology, 56, 879–886.Rannala B, Yang Z (2013) Improved reversible jump algo-

rithms for Bayesian species delimitation. Genetics, 194, 245–253.

Rosenberg NA (2004) DISTRUCT: a program for the graphical dis-

play of population structure. Molecular Ecology Notes, 4, 137–138.

Satler JD, Carstens BC, Hedin M (2013) Multilocus species

delimitation in a complex of morphologically conserved trap-

door spiders (Mygalomorphae, Antrodiaetidae, Aliatypus).

Systematic Biology, 62, 805–823.Silvestro D, Michalak I (2012) RAXMLGUI: a graphical front-end

for RAxML. Organisms Diversity & Evolution, 12, 335–337.

Stamatakis A (2006) RAxML-VI-HPC: maximum likelihood-

based phylogenetic analyses with thousands of taxa and

mixed models. Bioinformatics, 22, 2688–2690.Stamatakis A (2014) RAXML version 8: a tool for phylogenetic

analysis and post-analysis of large phylogenies. Bioinformat-

ics, 30, 1312–1313.Stephens M, Donnelly P (2003) A comparison of Bayesian

methods for haplotype reconstruction from population geno-

type data. American Journal of Human Genetics, 73, 1162–1169.Stephens M, Smith N, Donnelly P (2001) A new statistical

method for haplotype reconstruction from population data.

American Journal of Human Genetics, 68, 978–989.



Swofford DL (2002) PAUP*: Phylogenetic Analysis Using Parsi-

mony (*and Other Methods). Version 4. Sinauer Associates,

Sunderland, Massachusetts.

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013)

MEGA6: molecular evolutionary genetics analysis version 6.0.

Molecular Biology and Evolution, 30, 2725–2729.U.S. Fish and Wildlife Service (2008) Bexar County Karst Inverte-

brates Draft Recovery Plan. U.S. Fish and Wildlife Service,

Albuquerque, New Mexico.

U.S. Fish and Wildlife Service (2010) Department of the Inte-

rior, Fish and Wildlife Service, 50 CFR Part 17, Docket No.

FWS–R9–ES–2010–0065; MO–9221050083–B2. Endangered

and threatened wildlife and plants’ review of native species

that are candidates for listing as endangered or threatened;

annual notice of findings on resubmitted petitions; annual

description of progress on listing actions. Federal Register, 75,

69222–69294.Weisrock DW, Rasoloarison RM, Fiorentino I et al. (2010)

Delimiting species without nuclear monophyly in Madagas-

car’s mouse lemurs. PLoS ONE, 5, e9883. doi:10.1371/jour-

nal.pone.0009883.

Wiens JJ (2007) Species delimitation: new approaches for dis-

covering diversity. Systematic Biology, 56, 875–878.Yang Z, Rannala B (2010) Bayesian species delimitation using

multilocus sequence data. Proceedings of the National Academy

of Sciences, USA, 107, 9264–9269.Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general

species delimitation method with applications to phyloge-

netic placements. Bioinformatics, 29, 2869–2876.

M.H. implemented the study design, directed data col-

lection, conducted analyses and wrote the manuscript.

Data accessibility

DNA sequences: Genbank accessions KP221938-

KP222248.

DNA sequence alignments, RAxML and BEAST.tre

files, morphology JPG files: Dryad doi:10.5061/

dryad.qc3s0.

Illumina data submitted to NCBI Short Read Archive

– C. vibora (SRX652499), C. travisae (SRX761208).

PCR conditions and nuclear gene trees uploaded as

online Supporting Information.

Supporting information

Additional supporting information may be found in the online ver-

sion of this article.

Appendix S1 Development of nuclear markers.

Appendix S2 Nuclear primers, PCR conditions, transcript

annotations.

Appendix S3 RAxML maximum likelihood nuclear gene trees.


360 M. HEDIN

Appendix

1Vouch

ernumber,sp

eciesiden

tity

(original

taxonomy),sex,locationan

dGen

Ban

kinform

ation(unphased

sequen

ces)

LAB#

SPECIES

SEX

LOCALITY

COI

B1_G5G

6B1_B11B12

B2_D2D

3B1_C9C

10B2_F10F11

B2_H6H

7B1_E1E

2B1_G11G12

G1957

Cbuw

INoRen

tCav

eKP221938-40

KP221984

KP222016

KP222050

KP222084

KP222115

KP222148

KP222182

KP222216

G1992

Cbuw

IWeldonCav

eKP221941

KP221985

KP222017

KP222051

KP222085

KP222116

KP222149

KP222183

KP222217

G1996

Cbuw

IMcN

eilBat

Cav

eKP221942

KP221986

KP222018

KP222052

KP222086

KP222117

KP222150

KP222184

KP222218

G1997

Cbuw

FApple

Riata

Trace

KP221943

KP221987

KP222019

KP222053

KP222087

KP222118

KP222151

KP222185

KP222219

G2000

Cbuw

ILak

elineCav

eKP221944

KP221988

KP222020

KP222054

KP222088

KP222119

KP222152

KP222186

KP222220

G2001

Cbuw

ILak

elineCav

eKP221945

KP221989

KP222021

KP222055

KP222089

KP222120

KP222153

KP222187

KP222221

G1959

Cbuw

FBroken

Arrow

Cav

eKP221946-48

KP221990

KP222022

KP222056

KP222090

—KP222154

KP222188

KP222222

G1963

Cbuw

ITestudoTube

—KP221991

KP222023

KP222057

KP222091

KP222121

KP222155

KP222189

KP222223

G2004

Cbuw

IMarigold

Cav

eKP221949-51

KP221992

KP222024

KP222058

KP222092

KP222122

KP222156

KP222190

KP222224

G2007

Cbuw

FButtercu

pCreek

Cav

eKP221952

KP221993

KP222025

KP222059

KP222093

KP222123

KP222157

KP222191

KP222225

G2011

Cbuw

FBab

eCav

eKP221953,54

KP221994

KP222026

KP222060

KP222094

KP222124

KP222158

KP222192

KP222226

G2002

Cbuw

IDiesRan

chTreasure

Cav

eKP221955

——

——

——

——

G1964

Ctrav

IMcD

onaldCav

eKP221956

KP221995

KP222027

KP222061

KP222095

KP222125

KP222159

KP222193

KP222227

G1965

Ctrav

IMcD

onaldCav

eKP221957

KP221996

KP222028

KP222062

KP222096

KP222126

KP222160

KP222194

KP222228

G1966

Ctrav

FKretsch

marrDouble

Pit

KP221958

KP221997

KP222029

KP222063

KP222097

KP222127

KP222161

KP222195

KP2222229

G1968

APU

ITwoTrunksCav

eKP221959-61

KP221998

KP222030

KP222064

KP222098

KP222128

KP222162

KP222196

KP222230

G1970

Ctrav

FNorthRootCav

eKP221962

KP221999

KP222031

KP222065

KP222099

KP222129

KP222163

KP222197

KP222231

G1958

Ctrav

FTooth

Cav

eKP221963,64

KP222000

KP222032

KP222066

KP222100

KP222130

KP222164

KP222198

KP222232

G2014

Ctrav

FTooth

Cav

eKP221965

—KP222033

KP222067

KP222101

KP222131

KP222165

KP222199

KP222233

G1975

APU

IGalliferCav

eKP221966

KP222001

KP222034

KP222068

KP222102

KP222132

KP222166

KP222200

KP222234

G1976

APU

IGeo

deCav

eKP221967

KP222002

KP222035

KP222069

KP222103

KP222133

KP222167

KP222201

KP222235

G1998

Ctrav

MAmber

Cav

eKP221968,69

KP222003

KP222036

KP222070

KP222104

KP222134

KP222168

KP222202

KP222236

G1977

APU

MStovep

ipeCav

eKP221970

KP222004

KP222037

KP222071

KP222105

KP222135

KP222169

KP222203

KP222237

G1978

Cwar

IPickle

Pit

—KP222005

KP222038

KP222072

KP222106

KP222136

KP222170

KP222204

KP222238

G1979

Cwar

IPickle

Pit

KP221971

KP222006

KP222039

KP222073

—KP222137

KP222171

KP222205

KP222239

G1980

Cwar

IPickle

Pit

KP221972

KP222007

KP222040

KP222074

—KP222138

KP222172

KP222206

KP222240

G1981

APU

MSpider

Cav

eKP221973

KP222008

KP222041

KP222075

KP222107

KP222139

KP222173

KP222207

KP222241

G1982

APU

ISpider

Cav

eKP221974,75

KP222009

KP222042

KP222076

KP222108

KP222140

KP222174

KP222208

KP222242

G1984

APU

IBeard

Ran

chCav

eKP221976

—KP222043

KP222077

KP222109

KP222141

KP222175

KP222209

KP222243

G2009

APU

IKen

tButler

Pit

KP221977

KP222010

KP222044

KP222078

KP222110

KP222142

KP222176

KP222210

KP222244

G1985

APU

FJester

Estates

Cav

eKP221978

KP222011

KP222045

KP222079

KP222111

KP222143

KP222177

KP222211

KP222245

G1986

APU

MJester

Estates

Cav

eKP221979,80

KP222012

KP222046

KP222080

KP222112

KP222144

KP222178

KP222212

—G1988

APU

IJest

JohnCav

eKP221981

KP222013

KP222047

KP222081

KP222113

KP222145

KP222179

KP222213

KP222246

G1960

Cred

FCotterellCav

eKP221982

KP222014

KP222048

KP222082

KP222114

KP222146

KP222180

KP222214

KP222247

G1989

Cred

ICotterellCav

eKP221983

KP222015

KP222049

KP222083

—KP222147

KP222181

KP222215

KP222248

APU,apriori

uniden

tified

;I,im

mature.



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