integrative taxonomy of the primitively segmented...

Integrative taxonomy of the primitively segmentedspider genus Ganthela (Araneae: Mesothelae:Liphistiidae): DNA barcoding gap agreeswith morphology

XIN XU1, FENGXIANG LIU1, JIAN CHEN1, DAIQIN LI1,2* and MATJAŽ KUNTNER1,3,4*

1Centre for Behavioural Ecology and Evolution, College of Life Sciences, Hubei University, Wuhan,China2Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543,Singapore3Evolutionary Zoology Laboratory, Biological Institute ZRC SAZU, Novi trg 2, P. O. Box 306, SI-1001Ljubljana, Slovenia4Department of Entomology, National Museum of Natural History, Smithsonian Institution,Washington, DC, USA

Received 23 January 2015; revised 25 March 2015; accepted for publication 25 March 2015

Species delimitation is difficult for taxa in which the morphological characters are poorly known because of therarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, malesare often unknown, and female genital morphology – usually species-specific in spiders – exhibits considerableintraspecific variation. Here, we report on an integrative taxonomic study of the liphistiid genus Ganthela Xu &Kuntner, 2015, endemic to south-east China, where males are only available for two of the seven morphologicalspecies (two known and five undescribed). We obtained DNA barcodes (cytochrome c oxidase subunit I gene, COI)for 51 newly collected specimens of six morphological species and analysed them using five species-delimitationmethods: DNA barcoding gap, species delimitation plugin [P ID(Liberal)], automatic barcode gap discovery (ABGD),generalized mixed Yule-coalescent model (GMYC), and statistical parsimony (SP). Whereas the first three agreedwith the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimitand diagnose six Ganthela species, which in addition to the type species Ganthela yundingensis Xu, 2015, com-pletes the revision of the genus. Although multi-locus phylogenetic approaches may be needed for complex taxo-nomic delimitations, our results indicate that even single-locus analyses based on the COI barcodes, if integratedwith morphological and geographical data, may provide sufficiently reliable species delimitation.

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 288–306.doi: 10.1111/zoj.12280

ADDITIONAL KEYWORDS: DNA barcoding – East China – new species – phylogeny – species delimita-tion – taxonomy – trapdoor spiders.

INTRODUCTION

How a species is delimited has major implications, notonly for taxonomy, but also for biology in general,

because accurate species boundaries are crucial forunderstanding speciation patterns and processes (Sites& Marshall, 2004; Wiens, 2007; Camargo & Sites, 2013;Hedin, 2015), and will impact every subfield of the lifesciences. Species delimitation is far from a trivial taskfor taxa that are morphologically similar (cryptic speciesor those lacking clear diagnostic features) or those rarein collections (missing sexes or adult forms), however.

*Corresponding author. E-mail: [email protected];[email protected]

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Zoological Journal of the Linnean Society, 2015, 175, 288–306. With 9 figures

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 288–306288

mailto:[email protected]

mailto:[email protected]

As traditional taxonomy usually makes qualitative de-cisions on species delimitation, it is a fairly subjec-tive field (de Queiroz, 2007; Hausdorf, 2011). On theother hand, modern species delimitation approachestreat species as hypotheses in a statistical frame-work, and use objective tests to delineate evolution-arily independent lineages as species (for a review, seeFujita et al., 2012; see also Agnarsson & Kuntner, 2007).An integrative approach to taxonomy increasingly usesseveral lines of evidence to delimit and describe taxa(Dayrat, 2005; Will, Mishler & Wheeler, 2005; Pante,Schoelinck & Puillandre, 2015). Of these, modernmolecular-based species delimitation approaches arenow routinely used in a variety of taxa to comple-ment traditional taxonomy (Carstens et al., 2013;Fouquet et al., 2014; Shirley et al., 2014). Research onspecies delimitation is somewhat geopolitically biased(e.g. Harris & Froufe, 2005), however, as the major-ity of studies have been carried out using Europeanand North American taxa (e.g. Hamilton, Formanowicz& Bond, 2011; Derkarabetian & Hedin, 2014; Hamiltonet al., 2014), with only very few studies performed inEast and Southeast Asia (Huang et al., 2013). In spiders,a phylogenetic bias is also apparent, as most priorstudies that used molecular-based species delimita-tion have focused on derived spider groups to the ex-clusion of the primitively segmented spiders, Liphistiidae(e.g. Hamilton et al., 2011, 2014; Candek & Kuntner,2015; Derkarabetian & Hedin, 2014).

Whereas the use of single-locus data for delimitingspecies has been a source of some controversy (Hebertet al., 2003a; Moritz & Cicero, 2004; Blaxter et al., 2005;Rubinoff & Holland, 2005), many species-delimitationmethods do use single-locus data in order to balancecosts and benefits. For example, DNA barcoding gapanalysis routinely uses single-locus DNA data (Hebertet al., 2003a; Hebert, Ratnasingham & deWaard, 2003b),as do other recently proposed techniques, such as ‘auto-matic barcode gap discovery’ (ABGD; Puillandre et al.,2012), the ‘generalized mixed Yule-coalescent model’(GMYC; Pons et al., 2006), the species delimitationplugin [P ID(Liberal); Masters, Fan & Ross, 2011], the‘statistics parsimony network analysis’ (SP; Templeton,Crandall & Sing, 1992), and the ‘W&P’ method (Wiens& Penkrot, 2002). An increasing body of literature usesthese approaches and confirms their usefulness in de-limiting species to complement traditional (morpho-logical) taxonomy (Hart & Sunday, 2007; Longhorn et al.,2007; Hamilton et al., 2011, 2014; Kuntner & Agnarsson,2011; Weigand et al., 2011, 2013; Jörger et al., 2012;Paz & Crawford, 2012; Fujisawa & Barraclough, 2013;Hendrixson et al., 2013; Kekkonen & Hebert, 2014;Opatova & Arnedo, 2014).

Approaches to species delimitation using single-locus data can be broadly categorised into two groups,species discovery versus species validation, depend-

ing on whether the samples are partitioned prior toanalysis. Although it is unnecessary to partition thesamples into putative species when discovering speciesthrough the use of ABGD (Puillandre et al., 2012),GMYC (Pons et al., 2006), and SP (Templeton et al.,1992), species validation requires the a priori assign-ment of putative species that can then be tested throughDNA barcoding gap analysis (Hebert et al., 2003a, b)and P ID(Liberal) (Masters et al., 2011). Comparing theresults from various species-delimitation methods com-bined with morphological-based species diagnoses shouldin theory yield more accurate taxonomic units com-pared with classical approaches, especially for specieswith high intraspecific variation.

The primitively segmented spiders, family Liphistiidae,represent an ancient lineage in which members arerestricted to Southeast and East Asia (World SpiderCatalog, 2015). The family has recently been re-viewed to contain eight genera, each endemic to a dif-ferent geographical region (Xu et al., 2015a, b); however,species delimitation in liphistiids is challenging forseveral reasons. Although a combination of both adultmale and female genital features is commonly usedto diagnose species of spiders, liphistiid females havesimple genitals with extraordinarily intraspecific vari-ation, blurring the distinction between geographic vari-ation and species-level divergence, and thus makingspecies delimitation based on female morphology ex-tremely challenging (Haupt, 2003; Tanikawa, 2013;Tanikawa & Miyashita, 2015). This problem is exac-erbated by the fact that it is relatively easier to findadult females than adult males (our own data suggestthat the ratio of immature : adult female : adult malespecimens collected in the field is 13 : 16 : 1), with 23out of 89 liphistiid species only being known from asingle sex (20 from females and three from males only;World Spider Catalog, 2015). In the acute absence ofmale diagnostic features, liphistiid taxonomy conse-quently suffers from common taxonomic errors suchas over-splitting, over-lumping, and incorrect speciesassignment of individuals or populations (Rittmeyer& Austin, 2012), with potentially erroneous implica-tions regarding species diversity, intraspecific vari-ation, and gene flow (Funk & Omland, 2003; Bickfordet al., 2007). Over-lumping, in particular, is a majorconcern to biodiversity research as the units of diver-sity, and therefore conservation decisions, are under-estimated (Bickford et al., 2007). This is particularlyrelevant to liphistiids, in which the species-level tax-onomy has been nearly devoid of molecular analyses(but, see Tanikawa, 2013; Tanikawa & Miyashita, 2015),yet the known species are often island endemics orotherwise restricted in ranges (Xu et al., 2015a, b).

Here, we report on a species delimitation study thatintegrated morphological data with DNA nucleotide datain a group of primitively segmented spiders. We used

INTEGRATIVE TAXONOMY OF GANTHELA 289


the standard DNA barcoding region of the mitochondrialcytochrome c oxidase subunit I (COI; Hebert et al., 2003a,b), which has been shown to represent a rapid and ac-curate marker in testing species hypotheses in animals(Hebert et al., 2003a, b; Barrett & Hebert, 2005;Robinson et al., 2009; Paz & Crawford, 2012; Kekkonen& Hebert, 2014), and in particular in spiders (e.g.Hamilton et al., 2011, 2014; Candek & Kuntner, 2015).We studied Ganthela Xu & Kuntner, 2015, a genus re-stricted to east China (Fujian and Jiangxi Provinces)that contains seven morphological species. In addi-tion to the recently described type species, Ganthelayundingensis Xu, 2015, there is only one other knownspecies, Ganthela cipingensis (Wang, 1989), and judgingfrom female morphology five undescribed species (Xuet al., 2015a). As in other liphistiids, Ganthela femalegenital morphology is highly variable and the malesof five new (morphological) species are not availablefrom collections. We therefore generated DNA barcodedata to devise a specimen phylogeny, and used the fivespecies-delimitation methods listed above to test themorphology-based species identification. We then usedthe consensus results to delimit and diagnose the speciesdiversity in Ganthela.

MATERIAL AND METHODSTAXON SAMPLING

We sampled 51 individuals from five localities of onedescribed and five new species of Ganthela in East China(Fujian and Jiangxi Provinces; see inset in Fig. 1), andused a specimen of Sinothela sinensis (Bishop & Crosby,1932) as the out-group (Table 1). We collected adultsand immature spiders by excavating them from theirsubterranean burrows. We found a single species perlocality except for two species at Mount Wangjiang,Fujian Province. All specimens were collected alive andfixed in absolute ethanol if they were adults, and thenthe right legs were removed to be stored at −80 °C forsubsequent DNA extraction. The remainder of the speci-mens was preserved in 80% ethanol for identificationand morphological examination. Small juveniles wereused whole for DNA extraction, but large juveniles andsubadults were collected alive and reared in the la-boratory until maturation. Voucher specimens are de-posited at the Centre for Behavioural Ecology andEvolution (CBEE), College of Life Sciences, Hubei Uni-versity, Wuhan, China, and all type specimens are de-posited in the National Zoological Museum, ChineseAcademy of Sciences, Beijing, China.

MOLECULAR PROTOCOLS

Total genomic DNA was extracted from spider legs usingthe Animal Genomic DNA Isolation Kit (Dingguo,Beijing, China), following the manufacturer’s proto-

cols. Following the standard polymerase chain reac-tion (PCR) settings (Bond & Hedin, 2006; Agnarsson,2010; Kuntner et al., 2013; Zhang & Maddison, 2013),we amplified cytochrome c oxidase subunit I (COI) usingthe primer pairs LCOI490/HCO2198 (Folmer et al., 1994)with the following PCR reaction protocol: initial de-naturation at 95 °C for 5 min; 35 cycles of denatura-tion at 95 °C for 1 min, annealing at 40 °C for 1 min,and elongation at 72 °C for 90 s; and final extensionat 72 °C for 7 min. The 25-μL PCR reactions includ-ing 16.7 μL of double-distilled H2O, 2.5 μL of 10× Taqbuffer (mixed with MgCl2; TianGen Biotech, Beijing,China), 2.5 μL of dNTP Mix (2.5 mM), 1 μL of eachforward and reverse 10-μM primer, 1 μL of DNA tem-plate, and 0.3 μL Taq DNA polymerase (2.5 U μL−1;TianGen Biotech, Beijing, China). The double-strandedPCR products were visualized by agarose gel electro-phoresis (1% agarose). PCR products were purified andsequenced by Sunny Biotechnology Co., Ltd (Shang-hai, China) using the ABI 3730XL DNA analyser.

We manually edited the sequences usingGENEIOUS 5.6.6 (Biomatters Ltd, 2012), translatednucleotide reads to amino acids to check for stop codonsand to ensure the proper configuration of codon posi-tions, and aligned the sequences in GENEIOUS withgap opening/extension penalties set to 24/3.

PHYLOGENETIC ANALYSES

The COI data were treated as a single partition, andthe best COI substitution model, chosen based on theAkaike information criterion (AIC) in jMODELTEST 2.1.3(Darriba et al., 2012), was GTR + I + G. We performedmaximum-likelihood (ML) analyses in GARLI 2.01(Zwickl, 2006), and assessed branch supports by 100bootstrap replicates. We used SumTrees in the DendroPyphylogenetic Python library (Sukumaran & Holder, 2010)to generate a majority-rule bootstrap consensus tree.We conducted Bayesian-inference (BI) analyses inMrBayes 3.2.1 (Ronquist et al., 2012) by running Markovchain Monte Carlo (MCMC) with one independent chainfor 50 million generations. We monitored stationarityin TRACER 1.6 (Rambaut et al., 2014) and discardedas ‘burn-in’ the first quarter of cold-chain samples withthe remaining trees used to build a consensus. We usedFigTree 1.4.0 (Rambaut, 2012) to visualize and ma-nipulate trees, and manually summarized the resultsfrom different approaches using vector graphics in AdobeILLUSTRATOR.

SPECIES DELIMITATION

We analysed the COI data set (see Appendix S1) usingfive different species-delimitation methods. As both DNAbarcoding gap (Hebert et al., 2003a) and P ID(Liberal)require a priori species designation, we divided 51

290 X. XU ET AL.


Ganthela individuals into six putative species basedon a combination of phylogenetic topology from genetrees, morphological characters, and geographic infor-mation. In the DNA barcoding gap analysis, we ex-amined the overlap between the mean intraspecific andinterspecific Kimura two-parameter (K2P) and uncor-rected p-distance for each candidate species calculat-ed in MEGA 5.2.2 (Tamura et al., 2011). The species-delimitation plug-in (Masters et al., 2011) inGENEIOUS 5.6.6 (Biomatters Ltd, 2012) was used toobtain P ID(Liberal) statistical values. P ID(Liberal) isbased on the putative species groups to calculate themean probability of intra-/intergenetic distance ratiosfor these candidate species groups. The BI tree wasused as a guide tree to test species hypothesis.

The next three methods that we used do not requireassigning terminals to putative species. The ABGDprocedure (Puillandre et al., 2012) calculates all pairwisedistances in the data set, evaluates intraspecific di-vergences, and then sorts the terminals into candi-date species with calculated P values. We performedthe ABGD analyses online (http://wwwabi.snv.jussieu.fr/public/abgd/), using three different distance metrics:Jukes–Cantor (JC69; Jukes & Cantor, 1969), K2P(Kimura, 1980), and simple distance (p-distance;Nei & Kumar, 2000). We analysed the data using twodifferent values for the parameters Pmin (0.0001 and0.001), Pmax (0.1 and 0.2), and relative gap width(X = 1 or 1.5), with the other parameters set to defaultvalues.

Figure 1. Bayesian COI gene tree for 51 terminals of Ganthela, with the results of five different species delimitationapproaches, in addition to morphology (see legend). Numbers above branches show posterior probability and bootstrapsupports, and values below branches show mean intraspecific (black) and interspecific genetic distances (red), calculatedas Kimura two-parameter (K2P)/p-distance. Species names and locality group terminals (for specimen codes, see Table 1)according to consensus results of species delimitation approaches.



http://wwwabi.snv.jussieu.fr/public/abgd/

http://wwwabi.snv.jussieu.fr/public/abgd/

Tab

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36.7

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103

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292 X. XU ET AL.


The GMYC methodology (Pons et al., 2006) uses like-lihood to test for species boundaries by detecting thetransition point of interspecific versus intraspecific ratesof lineage coalescence. We performed GMYC analysesin the ‘splits’ package for R (Ezard, Fujisawa &Barraclough, 2009). We used the single-threshold model(Pons et al., 2006) because prior studies have shownthat the output of the multiple-threshold model(Monaghan et al., 2009) is no better than the single-threshold model (Hamilton et al., 2011, 2014; Paz &Crawford, 2012; Fujisawa & Barraclough, 2013;Hendrixson et al., 2013; Talavera, Dinca & Vila, 2013;Kekkonen & Hebert, 2014). We used BEAST 1.8.0(Drummond et al., 2012) to obtain an ultrametric genetree that GMYC requires as a guide tree, in combi-nation with a strict molecular clock (Zuckerkandl &Pauling, 1962) and Yule speciation model (Yule, 1924;Gernhard, 2008). We used standard arthropod substi-tution rates (Brower, 1994) by setting the COI sub-stitution rate parameter as a normal prior with a meanvalue of 0.0115, and ran 50 million generations, sam-pling every 5000 generations. We used TRACER 1.6(Rambaut et al., 2014) to assess the chain convergenceand correct mixing of each MCMC chain, then dis-carded as ‘burn-in’ 10% of the trees in each chain tosettle on an ultrametric tree using TreeAnnotator(Drummond et al., 2012).

Finally, we employed SP haplotype network analy-sis (Templeton et al., 1992) to investigate intraspecificrelationships among the terminals, and thus delimitspecies (Jörger et al., 2012; Weigand et al., 2013). Wegenerated haplotype networks using TCS 1.21 (Clement,Posada & Crandall, 2000) with a 95% parsimonycriterion.

TAXONOMY

Within a liphistiid genus-level revision, we recently de-scribed (Xu et al., 2015a) the type species of Ganthela:G. yundingensis Xu, 2015. Additional Ganthela speci-mens were examined with an Olympus SZX16 ster-eomicroscope, and anatomical details were studied witha Leica M205C stereomicroscope. Male palps and femalegenitalia were examined and photographed with a LeicaM205C stereomicroscope and Olympus BX51 com-pound microscope after being dissected from the spiderbodies. Genitalia were cleared in boiling 10% KOH fora few minutes to dissolve soft tissues. Unless other-wise noted, left palps were depicted. All measure-ments are in millimetres. Leg and palp measurementsare given in the following order: total length(femur + patella + tibia + metatarsus + tarsus).

Abbreviations used are: ALE, anterior lateral eyes;AME, anterior median eyes; BL, body length; CL, cara-pace length; Co, conductor; CT, contrategulum; CW, cara-pace width; E, embolus; OL, opisthosoma length; OW,opisthosoma width; PC, paracymbium; PLE, posteri-

or lateral eyes; PME, posterior median eyes; RC,receptacular cluster; T, tegulum.

RESULTSPHYLOGENETIC INFERENCE

The COI matrix of 51 individuals of Ganthela of 656 bphad 210 variable and 209 parsimony-informative sites,and contained 13 haplotypes (Table 1; Appendix S1).The phylogenetic topologies from BI and ML analy-ses agreed on Ganthela monophyly, with the node beinghighly supported (Fig. 1; posterior probability, PP = 1;bootstrap value, BS = 1). The two highly supported(PP > 0.95, BS > 75%) sister clades within Ganthela cor-respond to two geographical regions: Jiangxi and Fujianprovinces (Fig. 1). Within the Jiangxi and the Fujianclades, the individuals from each separate localitygrouped into solidly supported clades (PP = 1, BS > 75%).These locality clades mostly comprise closely relatedindividuals, except in the case of Mount Wangjiang,where a single locality harbours two distinct and highlysupported species clades (Fig. 1).

SPECIES DELIMITATION

DNA barcoding gapBased on our prior species hypotheses, the lowest meaninterspecific distance was 12/11% (K2P/uncorrectedp-distance) found between G. cipingensis and Ganthelajianensis sp. nov., which was about eight times thehighest mean intraspecific distance (1.62/1.57% for K2P/uncorrected p-distance) estimated for Ganthelaxianyouensis sp. nov. (Table 2). Histograms with pairwisedistances visualize a barcoding gap in Ganthela of4–12% (K2P)/4–11% (uncorrected p-distance; Fig. 2). Sixspecies were identified by the barcoding gap range, ofwhich one is known and five are new (Fig. 1).

P ID(Liberal)The results based on the Bayesian inference tree foundhigh P ID(Liberal) values of ≥0.97 (0.88–1.0) and lowintra-/interspecific ratios of ≤0.1 for all species hy-pothesized (Table 2), thereby also supporting the tax-onomy of six putative species (Fig. 1).

ABGD analysisThe ABGD results using different parameter combi-nations and the initial partition of six species (Table 3)agreed on those six species, whereas those under a re-cursive partition regime yielded more species (Table 3).The settings Pmin/Pmax = 0.0001/0.2 yielded the most sig-nificant P values.

All analyses reported so far support six morphologi-cal species, with syntopic terminals being conspecific,except in the case of Mount Wangjiang, where twospecies coexist within an absolute distance of 1 km.



In contrast, the following two analyses suggest morespecies. The single-threshold model GMYC resulted innine clusters and entities with different confidence in-tervals of 7–9 and 7–11, respectively (Table 4). Theseresults indicate two species in each of the Ji’an, Xianyou,and Mount Qingyuan clades (Fig. 1). The SP haplotypenetworks also show similar results, but with a singlespecies within the Mount Qingyuan clade (Figs 1, 3).

DISCUSSION

In this study, under an integrative framework, we ana-lysed original DNA barcoding data with five differentspecies-delimitation methods to test the validity of onedescribed and five undescribed morphological speciesof the liphistiid genus Ganthela known to us, in ad-dition to the type species G. yundingensis Xu, 2015.Our results show that three molecular species-delimitation methods – DNAbarcoding gap, P ID(Liberal),and ABGD – all fully confirm the morphological under-standing of Ganthela species diversity, and thus el-egantly complement traditional taxonomy (Fig. 1);however, GMYC and SP found more species of Ganthela,and thus disagree with the morphology, a result thatreinforces conclusions from previous studies (Esselstynet al., 2012; Paz & Crawford, 2012; Miralles & Vences,2013; Talavera et al., 2013; Hamilton et al., 2014). Weuse the consensus from our analyses for species de-limitation and diagnoses to conclude the formal revi-sion of Ganthela (see Taxonomy).

The traditional DNA barcoding gap and ABGDmethods are both based on the identification of a sig-nificant difference between inter- and intraspecificgenetic distances in samples of terminal taxa, in orderto assign organisms into putative species. For the DNAbarcoding gap analysis, we assigned Ganthela speci-mens into putative species considering the phylogenetic,morphological, and geographic information available.Our results are roughly comparable with those fromthe mygalomorph spider literature, which found abarcode gap of 5–6% (Hamilton et al., 2011, 2014);however, the gap is wider in Ganthela, ranging from4 to 12% (K2P)/4 to 11% (uncorrected p-distance; Fig. 2).ABGD usually generates diverse outcomes (Jörger et al.,2012; Puillandre et al., 2012; Kekkonen & Hebert, 2014).In this study, however, all analyses under different as-sumptions confirmed the six species, thereby lendingfurther support to the utility of the barcoding gap indelimiting species (Weigand et al., 2013; Candek &Kuntner, 2015; Hamilton et al., 2014).

P ID(Liberal) is usually used to test species hypoth-eses on phylogenetic trees (Masters et al., 2011; Hamiltonet al., 2014). According to our findings, a P ID(Liberal)value > 95% would be reasonable to distinguish Ganthelaspecies, and thus the cut-off of 95% can be used todelimit Ganthela and probably other liphisitiid species.T

able

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00)

G.q

ingy

ua

nen

sis

sp.n

ov.

0.02

294 X. XU ET AL.


Figure 2. DNA barcoding gap for Ganthela. Histograms show division of intraspecific (grey) and interspecific (black)COI sequence variation based on Kimura two-parameter (K2P, A) and uncorrected p-distance (B).

Table 3. Results of the automatic barcode gap discovery (ABGD) analyses

Substitutionmodel Pmin/Pmax X Partition

Prior intraspecific divergence (P)

0.001 0.0017 0.0028 0.0046 0.0077 0.0129 0.0215 0.0359 0.0599 0.1

JC 0.001/0.1 1.5 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

K2P 0.001/0.1 1.5 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

Simple 0.001/0.1 1.5 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

JC 0.001/0.1 1 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

K2P 0.001/0.1 1 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

Simple 0.001/0.1 1 Initial 6 6 6 6 6 6 6 6 6 6Recursive 11 9 9 9 9 8 7 7 6 6

0.0001 0.0002 0.0005 0.0013 0.0029 0.0068 0.0159 0.0369 0.086JC 0.0001/0.2 1.5 Initial 6 6 6 6 6 6 6 6 6

Recursive 11 11 11 11 9 9 8 7 6K2P 0.0001/0.2 1.5 Initial 6 6 6 6 6 6 6 6 6

Recursive 11 11 11 11 9 9 8 7 6Simple 0.0001/0.2 1.5 Initial 6 6 6 6 6 6 6 6 6

Recursive 11 11 11 11 9 9 8 6 6JC 0.0001/0.2 1 Initial 6 6 6 6 6 6 6 6 6

Recursive 11 11 11 11 9 9 8 7 6

JC, Jukes–Cantor subsitution model; K2P, Kimura two-parameter substitution model; Simple, p-distance; X, relative gap width.



As a species delimitation technique, the often usedGMYC is known to overestimate the number of species(Esselstyn et al., 2012; Paz & Crawford, 2012; Miralles& Vences, 2013; Talavera et al., 2013; Hamilton et al.,2014; but see, Jörger et al., 2012). In our case GMYCindeed seemingly overestimates the Ganthela species,suggesting that there are nine. Because GMYC re-quires an input tree, a differently obtained ultramatricgene tree, particularly one resulting from several genes,would probably change these results (Talavera et al.,2013; Kekkonen & Hebert, 2014). Our starting tree wasderived from a single mitochondrial gene, but the ad-dition of nuclear genes would be useful (Jörger et al.,2012; Dellicour & Flot, 2015).

Whereas SP has been successfully used in delimit-ing species in some taxa (Pons et al., 2006; Sauer &Hausdorf, 2012), it is often used to investigateintraspecific relationships. In our study, it oversplitsGanthela species and thus may not be helpful in de-limiting Ganthela species.

The peril of employing single-locus species delimi-tation techniques is the possibility of over-splitting taxaas a result of co-amplified nuclear mitochondrialpseudogenes (Song et al., 2008), incomplete lineagesorting and introgression (Hausdorf & Hennig, 2010;Edwards & Knowles, 2014), and particularly the deepgenetic structuring of mtDNA sequences (Bond et al.,2001; Arnedo & Ferrández, 2007). To avoid spuriousresults, taxonomic studies ideally incorporate multilocusdata and analyse it in a phylogenetic framework totest the validity of putative species (e.g. Satler, Carstens& Hedin, 2013; Edwards & Knowles, 2014; Opatova& Arnedo, 2014). Multi-locus approaches may be par-ticularly useful to understand lineages with seden-tary females and vagile males (Opatova & Arnedo, 2014;Hedin, 2015), where maternally inherited mtDNA mayshow deep genetic structuring, even in the absence ofgeographical barriers (Bond et al., 2001).

Showing poor dispersal abilities, primitively seg-mented spiders are strongly endemic and range-restricted, and the high interspecific genetic distancesin our study support the hypothesized low levels of geneflow among populations, which is typical of mygalomorphs(Hamilton et al., 2011, 2014; Hendrixson et al., 2013;Opatova & Arnedo, 2014). Nevertheless, our results

indicate that even single-locus analyses based on theCOI barcodes, if integrated with morphological and geo-graphical data, may provide sufficiently reliable speciesdelimitation, and that multi-locus phylogenetic ap-proaches may not be necessary for reliable taxonomicdecisions.

TAXONOMYGENUS GANTHELA XU & KUNTNER, 2015

For genus diagnosis and description, see Xu et al., 2015a.Type species by original designation, G. yundingensis

Xu, 2015. For diagnosis and description, see Xu et al.,2015a.

GANTHELA CIPINGENSIS (WANG, 1989) (FIG. 4)

Liphistius cipingensis Wang, 1989: 30, figs 1–6 (de-scription of female, type may be lost from HunanNormal University, not available for examination).

Heptathela cipingensis Platnick, 1993: 77 (trans-ferred from Liphistius).

Songthela cipingensis Ono, 2000: 150.

Material examinedFive females [XUX-2013-(508/510/511/512/514)] col-lected at Cemetery of Mount Jinggang RevolutionaryMartyrs, Ciping Town, Jinggangshan City, Jiangxi Prov-ince, China, 26.58°N, 114.16°E, 900 m a.s.l.,21 October 2013; two females [XUX-2013-(515–518)] col-lected at Nanshan, Ciping Town, Jinggangshan City,Jiangxi Province, China, 26.57°N, 114.16°E, 850 m a.s.l.,22 October 2013, collected by F.X. Liu, C. Xu, and X. Xu.No male found.

DiagnosisFemales of G. cipingensis resemble G. xianyouen-sis sp. nov., but differ by the details in genitalia, withrelatively longer genital stalks (Fig. 4B, C). Ganthelacipingensis differs from all other Ganthela species bythe following unique nucleotide substitutions in thestandard DNA barcode alignment: G (20), G (26), C(77), C (326), and C (413).

DescriptionFemale (Fig. 4A). Carapace and opisthosoma darkbrown; with clear fovea; chelicerae robust, with

Table 4. Results of the general mixed Yule-coalescent (GMYC) analyses

Analysis Clusters (CI) Entities (CI) Likelihood (null) Likelihood (GMYC) Likelihood ratio Threshold

Single 9 (7–9) 9 (7–11) 153.0361 166.2342 26.39622*** −0.3631672

Clusters, operational taxonomic units (OTUs) delineated by GMYC with more than one specimen; Entities, singletonOTUs delineated by GMYC; CI, confidence interval; Likelihood (null), likelihood of the null model; Likelihood (GMYC),likelihood of the GMYC model; Threshold, the threshold between speciation and coalescence processes; Single, single-threshold model; ***P < 0.001.

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Figure 3. Haplotype networks of Ganthela under a 95% parsimony criterion. The size of each open circle indicates haplotypefrequency, numbers preceded by ‘H’ indicate haplotype number, and numbers in brackets indicate population sizes. Opendots on lines connecting haplotypes indicate a substitution. Dashed lines enclosing haplotype networks correspond tomorphological and consensus species.



promargin of cheliceral groove with between ten and13 strong denticles of variable size; legs with stronghairs and spines; opisthosoma with 12 tergites, withtergite 5 being largest; seven spinnerets. Measure-ments: BL 8.70–14.70, CL 4.13–6.40, CW 3.70–5.00,OL 4.65–8.00, and OW 3.25–5.40; ALE > PLE > PME >AME; palp 9.72 (3.47 + 1.70 + 2.15 + 2.40), leg I 12.00(3.75 + 2.15 + 2.40 + 2.30 + 1.40), leg II 12.06(3.70 + 2.13 + 2.23 + 2.50 + 1.50), leg III 13.30(3.60 + 2.20 + 2.25 + 3.35 + 1.90), and leg IV 19.07(5.30 + 2.67 + 3.45 + 5.20 + 2.44).

Female genitaliaThe posterior part of the genital area wide, rectan-gular (Fig. 4B, C), a pair of receptacular clusters closeto each other and more or less parallel, with shortgenital stalks (Fig. 4B, C).

DistributionJiangxi (Jinggangshan) Province, China.

GANTHELA JIANENSIS XU, KUNTNER &CHEN SP. NOV. (FIG. 5)

HolotypeFemale (XUX-2013-534), Mount Qingyuan, Ji’an City,Jiangxi Province, China, 27.06°N, 115.05°E, 100 m a.s.l.,23 October 2013, collected by F.X. Liu, X. Xu, andZ.T. Zhang.

ParatypesTen females (XUX-2013-530-531/533–536/538-541A) col-lected at the same locality, 23 October 2013, collectedby F.X. Liu, X. Xu, and Z.T. Zhang. No male found.

Etymology‘Jian’ refers to the type locality of this species.

DiagnosisFemales of G. jianensis sp. nov. differ from other speciesof Ganthela by details in genitalia, with short and thickgenital stalks (Fig. 5B, C), and by the slightly curvedposterior part of the genital area (Fig. 5B, C). Ganthelajianensis sp. nov. differs from all other Ganthela speciesby the following unique nucleotide substitutions inthe standard DNA barcode alignment: G (59), A (131),A (155), C (245), T (249), G (317), C (398), C (528), C(581), and C (638).

DescriptionFemale (holotype) (Fig. 5A). Carapace and opisthosoma,dark brown; chelicerae robust, with promargin ofcheliceral groove with between ten and 13 strongdenticles of variable size; legs with strong hairsand spines; opisthosoma with 12 tergites, with tergite 5being largest; seven spinnerets. Measurements:BL 11.80–15.50, CL 5.60–7.40, CW 5.30–6.50, OL 5.95–9.10, and OW 4.46–6.70; ALE > PLE > PME > AME;palp 11.55 (4.00 + 2.10 + 2.45 + 3.00), leg I 13.25(4.15 + 2.50 + 2.35 + 2.65 + 1.60), leg II 12.88(3.73 + 2.37 + 2.25 + 2.75 + 1.78), leg III 13.82

Figure 4. Ganthela cipingensis (Wang, 1989). A, female (XUX-2013-516). B, C, female genitalia (XUX-2013-517): B, dorsalview; C, ventral view; RC, receptacular cluster. Scale bar: 0.5 mm.

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(4.00 + 2.42 + 2.17 + 3.23 + 2.00), and leg IV 20.37(5.61 + 3.00 + 3.45 + 5.53 + 2.78).

Female genitaliaPosterior part of genital area slightly curved (Fig. 5B,C), with pair of receptacular clusters close to eachother, and with very short and thick genital stalks(Fig. 5B, C).

DistributionJiangxi (Ji’an) Province, China

GANTHELA QINGYUANENSIS XU, KUNTNER &LIU SP. NOV. (FIG. 6)

HolotypeMale (XUX-2012-288, matured 18 July 2013 at CBEE,College of Life Sciences, Hubei University), aroundTV Tower, Mount Qingyuan, Quanzhou City, FujianProvince, China, 24.95°N, 118.60°E, 475 m a.s.l.,

11 December 2012, collected by D. Li, F.X. Liu,M. Kuntner, and X. Xu.

ParatypesThree females [XUX-2012-(287/290/297)] collected atthe same locality, 11 December 2012; eight females [XUX-2013-(138–142/146A-148)] collected at the same local-ity, 9 July 2013; collected by F.X. Liu, X. Xu, andZ.T. Zhang.

Etymology‘Qingyuan’ refers to the type locality of this species,Mount Qingyuan.

DiagnosisMales of G. qingyuanensis sp. nov. differ fromG. yundingensis by anatomical details in the palps,which have a small posterior apophysis on the con-ductor, in addition to the spiniform apex (Fig. 6G).Females of G. qingyuanensis sp. nov. can be distin-guished from all other Ganthela species by details inthe genitalia, with longer genital stalks (Fig. 6B, C).Ganthela qingyuanensis sp. nov. differs from all otherGanthela species by the following unique nucleotidesubstitutions in the standard DNA barcode align-ment: C (42), C (50), C (104), G (108), C (158), G (197),C (218), A (269), C (380), G (339), C (353), C (383), C(449), C (504), C (575), and C (625).

DescriptionMale (holotype). Carapace brown; opisthosoma lightbrown, with dark brown tergites; sternum narrow, muchlonger than wide; a few long pointed hairs running overocular mound in a longitudinal row; chelicerae robust,with promargin of cheliceral groove with 11 denticlesof variable size; legs with strong hairs and spines;opisthosoma with 12 tergites, with tergites 2–6 largerthan others, and with tergite 4 being largest; sevenspinnerets. Measurements: BL 10.55, CL 5.00, CW 4.85,OL 5.60, and OW 3.63; ALE > PLE > PME > AME; leg I14.50 (4.11 + 1.80 + 2.89 + 3.70 + 2.00), leg II 15.14(3.87 + 1.82 + 3.00 + 4.15 + 2.30), leg III 16.47(4.00 + 1.85 + 2.92 + 5.05 + 2.65), and leg IV 21.53(5.18 + 2.00 + 4.10 + 6.85 + 3.40).

PalpCymbium with a projection; prolateral side ofparacymbium unpigmented and unsclerotized, numer-ous setae and spines at the tip of paracymbium (Fig. 6F,G). Contrategulum has two marginal apophyses withsmooth margin and blunt distal end (Fig. 6H). Tegulumwith a dentate, wide edge (Fig. 6F, G). Conductor witha spiniform apex and a small apophysis at posteriorpart, parallel with embolus (Fig. 6G). Embolus largelysclerotized, with a wide and flat opening, and distalembolus slightly sclerotized (Fig. 6G).

Figure 5. Ganthela jianensis Xu, Kuntner & Chensp. nov. A, female (XUX-2013-536). B, C, female genitalia(XUX-2013-534): B, dorsal view; C, ventral view; RC,receptacular cluster. Scale bar 0.5 mm.



Female (Fig. 6A). Carapace and opisthosoma of femalesimilar to male, except coloration lighter than male;chelicerae robust, with promargin of cheliceral groovewith between ten and 12 strong denticles of variablesize; legs with strong hairs and spines; opisthosomawith 12 tergites, similar to male; seven spinnerets.Measurements: BL 10.68–14.48, CL 5.48–7.05, CW 4.52–6.00, OL 5.77–7.62, and OW 4.13–5.68; ALE > PLE >PME > AME; palp 9.51 (3.40 + 1.70 + 2.00 + 2.41), leg I11.43 (3.85 + 1.95 + 2.08 + 2.25 + 1.30), leg II 11.08

(3.40 + 1.95 + 1.96 + 2.45 + 1.32), leg III 11.81(3.46 + 2.05 + 1.92 + 2.70 + 1.68), and leg IV 17.46(4.95 + 2.48 + 3.05 + 4.68 + 2.30).

Female genitaliaPosterior part of genital area rectangular (Fig. 6B–E), with pair of receptacular clusters with genital stalks(Fig. 6B, C), or with paired receptacular clusters fusedinto a big cluster (Fig. 6D, E).

Figure 6. Ganthela qingyuanensis Xu, Kuntner & Liu sp. nov. A, female (XUX-2013-139). B, C, female genitalia (XUX-2013-142). D, E, female genitalia (XUX-2013-148). B, D, dorsal view; C, E, ventral view. F–H, male (XUX-2012-228) palp:F, prolateral view; G, ventral view; H, retrolateral view. Abbreviations: Co, conductor; CT, contrategulum; E, embolus;PC, paracymbium; T, tegulum; Scale bars: B–E, 0.5 mm; F–H, 1 mm.

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VariationFemale receptacular clusters show considerableintraspecific variation, with the pair of receptacularclusters fused.

DistributionFujian (Quanzhou) Province, China.

GANTHELA WANGJIANGENSIS XU, KUNTNER & LIU

SP. NOV. (FIG. 7)

HolotypeFemale (XUX-2013-159), Mount Wangjiang, HuangyangVillage, Zhuangbian Town, Hanjiang District, PutianCity, Fujian Province, China, 25.66°N, 118.87°E,680 m a.s.l., 10 July 2013, collected by F.X. Liu, X. Xu,and Z.T. Zhang. No male found.

Etymology‘Wangjiang’ refers to the type locality of this species,Mount Wangjiang.

DiagnosisFemales of G. wangjiangensis sp. nov. differ from allother Ganthela species by the W-shaped posterior partof the genital area, and can be further distinguishedfrom G. venus sp. nov. by the connected basal genitalstalks (Fig. 7A, B). Ganthela wangjiangensis sp. nov.differs from all other Ganthela species by the follow-ing unique nucleotide substitutions in the standard DNAbarcode alignment: G (54), C (101), C (179), C (320),A (336), T (372), C (386), C (422), G (476), T (512),and C (632).

DescriptionFemale (holotype). Carapace and opisthosoma lightbrown; tergites slightly dark brown; sternum narrow,length more or less twice of width; a few long pointedhairs running over ocular mound in a longitudinal row;chelicerae robust, with promargin of cheliceral groovewith 12 strong denticles of variable size; legs with stronghairs and spines; opisthosoma with 12 tergites, with

tergites 2–6 larger than others, and the with tergite 4being largest; seven spinnerets. Measurements: BL 11.50,CL 4.90, CW 4.30, OL 6.40, OW 5.55; ALE > PLE >PME > AME; palp 7.94 (2.78 + 1.50 + 1.48 + 2.18), leg I9.10 (3.05 + 1.35 + 1.70 + 1.80 + 1.20), leg II 9.50(2.80 + 1.67 + 1.71 + 2.02 + 1.30), leg III 10.00(2.75 + 1.76 + 1.57 + 2.45 + 1.47), and leg IV 13.62(3.83 + 1.92 + 2.28 + 3.52 + 2.07).

Female genitaliaThe posterior part of genital area W-shaped (Fig. 7A,B), with a pair of receptacular clusters separated fromeach other, and with the basal parts of short genitalstalks fused (Fig. 7A, B).

DistributionFujian (Putian) Province, China.

RemarksGanthela wangjiangensis sp. nov. was collected within1 km of G. venus sp. nov., but at a higher altitude.

GANTHELA XIANYOUENSIS XU, KUNTNER & CHEN

SP. NOV. (FIG. 8)

HolotypeFemale (XUX-2013-153), Qingfengge, Dongshi Village,Yuanzhuang Town, Xianyou County, Putian City, FujianProvince, China; 25.24°N, 118.70°E, 336 m a.s.l.,10 July 2013; collected by F.X. Liu, X. Xu, and Z.T. Zhang.

ParatypesTwo females (XUX-2013-153/154) collected at the samelocality, 10 July 2013; collected by F.X. Liu, X. Xu, andZ.T. Zhang. No male found.

Etymology‘Xianyou’ refers to the type locality of this species.

DiagnosisFemales of G. xianyouensis sp. nov. can be distin-guished from all other Ganthela species by details of

Figure 7. Ganthela wangjiangensis Xu, Kuntner & Liu sp. nov. A, B, female genitalia (XUX-2013-159). A, dorsal view;B, ventral view. RC, receptacular cluster. Scale bar: 0.5 mm.



the genitalia, with very short but slender genital stalks(Fig. 8B, C). Ganthela xianyouensis sp. nov. further differsfrom all other Ganthela species by the following uniquenucleotide substitutions in the standard DNA barcodealignment: G (96), G (146), C (277), C (314), T (338),C (393), C (428), and T (572).

DescriptionFemale (holotype) (Fig. 8A). Carapace and opisthosomalight brown; tergites dark brown; sternum narrow, lengthmore or less twice of width; a few long pointed hairsrunning over ocular mound in a longitudinal row;chelicerae robust, with promargin of cheliceral groovewith 11 or 12 strong denticles of variable size; legs withstrong hairs and spines; opisthosoma with 12 tergites,with tergites 2–6 larger than others, and with tergite 4being largest; seven spinnerets. Measurements:BL 13.60, CL 6.10, CW 4.96, OL 6.56, and OW 5.45;ALE > PLE >PME > AME; palp 9.48 (3.26 + 1.66 + 2.06 + 2.50), leg I11.58 (3.72 + 1.92 + 2.15 + 2.32 + 1.47), leg II 11.45(3.64 + 1.93 + 2.05 + 2.25 + 1.58), leg III 12.58(3.55 + 2.10 + 2.10 + 3.00 + 1.83), and leg IV 17.53(4.88 + 2.25 + 3.15 + 4.55 + 2.70).

Female genitaliaThe posterior part of genital area rectangular (Fig. 8B,C), with a pair of receptacular clusters close to eachother, and with very short but slender genital stalks(Fig. 8B, C).


GANTHELA VENUS XU SP. NOV. (FIG. 9)

HolotypeFemale (XUX-2013-160), Mount Wangjiang, HuangyangVillage, Zhuangbian Town, Hanjiang District, PutianCity, Fujian Province, China, 25.66°N, 118.88°E,459 m a.s.l., 11 July 2013, collected by F.X. Liu, X. Xu,and Z.T. Zhang. No male found.

EtymologyNamed after the Greek goddess of love, Venus.

DiagnosisFemales of G. venus sp. nov. can be distinguished fromG. wangjiangensis sp. nov. by details in the genitalia,which have separated stalks on the anterior part(Fig. 9A, B). Ganthela venus sp. nov. furthermore differsfrom all other Ganthela species by the following uniquenucleotide substitutions in the standard DNA barcodealignment: C (36), A (80), T (227), C (333), C (341), G(347), T (354), C (431), and T (539).

DescriptionFemale (holotype). Carapace and opisthosoma lightbrown; tergites slightly dark brown; sternum narrow,length more or less twice of width; a few long pointedhairs running over ocular mound in a longitudinal row;

Figure 8. Ganthela xianyouensis Xu, Kuntner & Chen sp. nov. A, female (XUX-2013-151). B, C, female genitalia (XUX-2013-153): B, dorsal view; C, ventral view. RC, receptacular cluster. Scale bar: 0.5 mm.

302 X. XU ET AL.


chelicerae robust, with promargin of cheliceral groovewith ten strong denticles of variable size; legs withstrong hairs and spines; opisthosoma with 12 tergites,with tergites 2–6 larger than others, and with tergite 4being largest; seven spinnerets. Measurements: BL 9.80,CL 4.38, CW 4.20, OL 5.6, and OW 5.00; ALE > PLE >PME > AME; palp 7.63 (2.50 + 1.31 + 1.65 + 2.17), leg Imissing, leg II 8.49 (2.71 + 1.50 + 1.68 + 1.50 + 1.10),leg III 9.34 (2.52 + 1.58 + 1.56 + 2.30 + 1.38), and leg IV13.70 (3.75 + 1.60 + 2.55 + 3.68 + 2.12).

Female genitaliaPosterior part of genital area W-shaped (Fig. 9A, B),with pair of receptacular clusters with short stalks onthe anterior part of genital separated from one another(Fig. 9A, B).


RemarksSee comments under G. wangjiangensis sp. nov.

ACKNOWLEDGEMENTS

We thank Chen Xu and Zengtao Zhang for assis-tance with fieldwork, Matjaž Gregoric, Nina Vidergar,and Ren-Chung Cheng for help and/or advice on mo-lecular resources and procedures, and the staff of theCentre for Behavioural Ecology and Evolution (CBEE,Hubei University) for all their help and support through-out this study, in particularly Bo Chen, Xiaoguo Jiao,Jie Liu, Yu Peng, Chen Xu, Long Yu, and ZengtaoZhang. This study was supported in part by funds fromChina [NSFC grant (31272324)], the Singapore Min-istry of Education (MOE) [AcRF Tier 1 grant (R-154-000-591-112)], and by the Slovenian Research Agency(grants P1-10236, MU-PROM/12-001 and J1-6729 to

MK). We thank Ingi Agnarsson and two anonymousreferees for their valuable input.

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SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article at the publisher’s web-site:

Appendix S1. Aligned COI sequences used for species delimitation (separate nexus file).

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