Integrative Taxonomy of the Primitively Segmented Spider Genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA Barcoding Gap Agrees with Morphology

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

Integrative taxonomy of the primitively segmented spider genus Ganthela (Araneae: Mesothelae: Liphistiidae): DNA barcoding gap agrees with 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, China 2Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore 3Evolutionary Zoology Laboratory, Biological Institute ZRC SAZU, Novi trg 2, P. O. Box 306, SI-1001 Ljubljana, Slovenia 4Department 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 the rarity of adult morphs or sexes, and in cryptic species. In primitively segmented spiders, family Liphistiidae, males are often unknown, and female genital morphology – usually species-specific in spiders – exhibits considerable intraspecific 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 morphological species (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-delimitation methods: 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 agreed with the morphology, GMYC and SP indicate several additional species. We used the consensus results to delimit and 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 taxonomic delimitations, our results indicate that even single-locus analyses based on the COI barcodes, if integrated with morphological and geographical data, may provide sufficiently reliable species delimitation.

ADDITIONAL KEYWORDS: DNA barcoding – East China – new species – phylogeny – species delimitation – taxonomy – trapdoor spiders.

INTRODUCTION because accurate species boundaries are crucial for understanding speciation patterns and processes (Sites How a species is delimited has major implications, not & Marshall, 2004; Wiens, 2007; Camargo & Sites, 2013; only for taxonomy, but also for biology in general, Hedin, 2015), and will impact every subﬁeld of the life sciences. Species delimitation is far from a trivial task for taxa that are morphologically similar (cryptic species *Corresponding author. E-mail: [email protected]; or those lacking clear diagnostic features) or those rare [email protected] in collections (missing sexes or adult forms), however.

As traditional taxonomy usually makes qualitative de- ing on whether the samples are partitioned prior to cisions on species delimitation, it is a fairly subjec- analysis. Although it is unnecessary to partition the tive field (de Queiroz, 2007; Hausdorf, 2011). On the samples into putative species when discovering species other hand, modern species delimitation approaches through the use of ABGD (Puillandre et al., 2012), treat species as hypotheses in a statistical frame- GMYC (Pons et al., 2006), and SP (Templeton et al., work, and use objective tests to delineate evolution- 1992), species validation requires the a priori assign- arily independent lineages as species (for a review, see ment of putative species that can then be tested through Fujita et al., 2012; see also Agnarsson & Kuntner, 2007). DNA barcoding gap analysis (Hebert et al., 2003a, b) An integrative approach to taxonomy increasingly uses and P ID(Liberal) (Masters et al., 2011). Comparing the several lines of evidence to delimit and describe taxa results from various species-delimitation methods com- (Dayrat, 2005; Will, Mishler & Wheeler, 2005; Pante, bined with morphological-based species diagnoses should Schoelinck & Puillandre, 2015). Of these, modern in theory yield more accurate taxonomic units com- molecular-based species delimitation approaches are pared with classical approaches, especially for species now routinely used in a variety of taxa to comple- with high intraspecific variation. ment traditional taxonomy (Carstens et al., 2013; The primitively segmented spiders, family Liphistiidae, Fouquet et al., 2014; Shirley et al., 2014). Research on represent an ancient lineage in which members are species delimitation is somewhat geopolitically biased restricted to Southeast and East Asia (World Spider (e.g. Harris & Froufe, 2005), however, as the major- Catalog, 2015). The family has recently been re- ity of studies have been carried out using European viewed to contain eight genera, each endemic to a dif- and North American taxa (e.g. Hamilton, Formanowicz ferent geographical region (Xu et al., 2015a, b); however, & Bond, 2011; Derkarabetian & Hedin, 2014; Hamilton species delimitation in liphistiids is challenging for et al., 2014), with only very few studies performed in several reasons. Although a combination of both adult East and Southeast Asia (Huang et al., 2013). In spiders, male and female genital features is commonly used a phylogenetic bias is also apparent, as most prior to diagnose species of spiders, liphistiid females have studies that used molecular-based species delimita- simple genitals with extraordinarily intraspecific vari- tion have focused on derived spider groups to the ex- ation, blurring the distinction between geographic vari- clusion of the primitively segmented spiders, Liphistiidae ation and species-level divergence, and thus making (e.g. Hamilton et al., 2011, 2014; Cˇ andek & Kuntner, species delimitation based on female morphology ex- 2015; Derkarabetian & Hedin, 2014). tremely challenging (Haupt, 2003; Tanikawa, 2013; Whereas the use of single-locus data for delimiting Tanikawa & Miyashita, 2015). This problem is exac- species has been a source of some controversy (Hebert erbated by the fact that it is relatively easier to find et al., 2003a; Moritz & Cicero, 2004; Blaxter et al., 2005; adult females than adult males (our own data suggest Rubinoff & Holland, 2005), many species-delimitation that the ratio of immature : adult female : adult male methods do use single-locus data in order to balance specimens collected in the field is 13 : 16 : 1), with 23 costs and benefits. For example, DNA barcoding gap out of 89 liphistiid species only being known from a analysis routinely uses single-locus DNA data (Hebert single sex (20 from females and three from males only; et al., 2003a; Hebert, Ratnasingham & deWaard, 2003b), World Spider Catalog, 2015). In the acute absence of as do other recently proposed techniques, such as ‘auto- male diagnostic features, liphistiid taxonomy conse- matic barcode gap discovery’ (ABGD; Puillandre et al., quently suffers from common taxonomic errors such 2012), the ‘generalized mixed Yule-coalescent model’ as over-splitting, over-lumping, and incorrect species (GMYC; Pons et al., 2006), the species delimitation assignment of individuals or populations (Rittmeyer plugin [P ID(Liberal); Masters, Fan & Ross, 2011], the & Austin, 2012), with potentially erroneous implica- ‘statistics parsimony network analysis’ (SP; Templeton, tions regarding species diversity, intraspecific vari- Crandall & Sing, 1992), and the ‘W&P’ method (Wiens ation, and gene flow (Funk & Omland, 2003; Bickford & Penkrot, 2002). An increasing body of literature uses et al., 2007). Over-lumping, in particular, is a major these approaches and confirms their usefulness in de- concern to biodiversity research as the units of diver- limiting species to complement traditional (morpho- sity, and therefore conservation decisions, are under- logical) taxonomy (Hart & Sunday, 2007; Longhorn et al., estimated (Bickford et al., 2007). This is particularly 2007; Hamilton et al., 2011, 2014; Kuntner & Agnarsson, relevant to liphistiids, in which the species-level tax- 2011; Weigand et al., 2011, 2013; Jörger et al., 2012; onomy has been nearly devoid of molecular analyses Paz & Crawford, 2012; Fujisawa & Barraclough, 2013; (but, see Tanikawa, 2013; Tanikawa & Miyashita, 2015), Hendrixson et al., 2013; Kekkonen & Hebert, 2014; yet the known species are often island endemics or Opatova & Arnedo, 2014). otherwise restricted in ranges (Xu et al., 2015a, b). Approaches to species delimitation using single- Here, we report on a species delimitation study that locus data can be broadly categorised into two groups, integrated morphological data with DNA nucleotide data species discovery versus species validation, depend- in a group of primitively segmented spiders. We used

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 288–306 290 X. XU ET AL. the standard DNA barcoding region of the mitochondrial cols. Following the standard polymerase chain reac- cytochrome c oxidase subunit I (COI; Hebert et al., 2003a, tion (PCR) settings (Bond & Hedin, 2006; Agnarsson, b), which has been shown to represent a rapid and ac- 2010; Kuntner et al., 2013; Zhang & Maddison, 2013), curate marker in testing species hypotheses in animals we amplified cytochrome c oxidase subunit I (COI) using (Hebert et al., 2003a, b; Barrett & Hebert, 2005; the primer pairs LCOI490/HCO2198 (Folmer et al., 1994) Robinson et al., 2009; Paz & Crawford, 2012; Kekkonen with the following PCR reaction protocol: initial de- & Hebert, 2014), and in particular in spiders (e.g. naturation at 95 °C for 5 min; 35 cycles of denatura- Hamilton et al., 2011, 2014; Cˇ andek & Kuntner, 2015). tion at 95 °C for 1 min, annealing at 40 °C for 1 min, We studied Ganthela Xu & Kuntner, 2015, a genus re- and elongation at 72 °C for 90 s; and final extension stricted to east China (Fujian and Jiangxi Provinces) at 72 °C for 7 min. The 25-μL PCR reactions includ- that contains seven morphological species. In addi- ing 16.7 μL of double-distilled H2O, 2.5 μL of 10× Taq tion to the recently described type species, Ganthela buffer (mixed with MgCl2; TianGen Biotech, Beijing, yundingensis Xu, 2015, there is only one other known China), 2.5 μL of dNTP Mix (2.5 mM), 1 μL of each species, Ganthela cipingensis (Wang, 1989), and judging forward and reverse 10-μM primer, 1 μL of DNA tem- from female morphology five undescribed species (Xu plate, and 0.3 μL Taq DNA polymerase (2.5 U μL−1; et al., 2015a). As in other liphistiids, Ganthela female TianGen Biotech, Beijing, China). The double-stranded genital morphology is highly variable and the males PCR products were visualized by agarose gel electro- of five new (morphological) species are not available phoresis (1% agarose). PCR products were purified and from collections. We therefore generated DNA barcode sequenced by Sunny Biotechnology Co., Ltd (Shang- data to devise a specimen phylogeny, and used the five hai, China) using the ABI 3730XL DNA analyser. species-delimitation methods listed above to test the We manually edited the sequences using morphology-based species identification. We then used GENEIOUS 5.6.6 (Biomatters Ltd, 2012), translated the consensus results to delimit and diagnose the species nucleotide reads to amino acids to check for stop codons diversity in Ganthela. and to ensure the proper configuration of codon posi- tions, and aligned the sequences in GENEIOUS with gap opening/extension penalties set to 24/3. MATERIAL AND METHODS TAXON SAMPLING PHYLOGENETIC ANALYSES We sampled 51 individuals from five localities of one described and five new species of Ganthela in East China The COI data were treated as a single partition, and (Fujian and Jiangxi Provinces; see inset in Fig. 1), and the best COI substitution model, chosen based on the used a specimen of Sinothela sinensis (Bishop & Crosby, Akaike information criterion (AIC) in jMODELTEST 2.1.3 1932) as the out-group (Table 1). We collected adults (Darriba et al., 2012), was GTR+I+G.Weperformed and immature spiders by excavating them from their maximum-likelihood (ML) analyses in GARLI 2.01 subterranean burrows. We found a single species per (Zwickl, 2006), and assessed branch supports by 100 locality except for two species at Mount Wangjiang, bootstrap replicates. We used SumTrees in the DendroPy Fujian Province. All specimens were collected alive and phylogenetic Python library (Sukumaran & Holder, 2010) fixed in absolute ethanol if they were adults, and then to generate a majority-rule bootstrap consensus tree. the right legs were removed to be stored at −80 °C for We conducted Bayesian-inference (BI) analyses in subsequent DNA extraction. The remainder of the speci- MrBayes 3.2.1 (Ronquist et al., 2012) by running Markov mens was preserved in 80% ethanol for identification chain Monte Carlo (MCMC) with one independent chain and morphological examination. Small juveniles were for 50 million generations. We monitored stationarity used whole for DNA extraction, but large juveniles and in TRACER 1.6 (Rambaut et al., 2014) and discarded subadults were collected alive and reared in the la- as ‘burn-in’ the first quarter of cold-chain samples with boratory until maturation. Voucher specimens are de- the remaining trees used to build a consensus. We used posited at the Centre for Behavioural Ecology and FigTree 1.4.0 (Rambaut, 2012) to visualize and ma- Evolution (CBEE), College of Life Sciences, Hubei Uni- nipulate trees, and manually summarized the results versity, Wuhan, China, and all type specimens are de- from different approaches using vector graphics in Adobe posited in the National Zoological Museum, Chinese ILLUSTRATOR. Academy of Sciences, Beijing, China.

SPECIES DELIMITATION MOLECULAR PROTOCOLS We analysed the COI data set (see Appendix S1) using Total genomic DNA was extracted from spider legs using ﬁve different species-delimitation methods. As both DNA the Animal Genomic DNA Isolation Kit (Dingguo, barcoding gap (Hebert et al., 2003a) and P ID(Liberal) Beijing, China), following the manufacturer’s proto- require a priori species designation, we divided 51

Figure 1. Bayesian COI gene tree for 51 terminals of Ganthela, with the results of five different species delimitation approaches, in addition to morphology (see legend). Numbers above branches show posterior probability and bootstrap supports, and values below branches show mean intraspecific (black) and interspecific genetic distances (red), calculated as 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.

Ganthela individuals into six putative species based The next three methods that we used do not require on a combination of phylogenetic topology from gene assigning terminals to putative species. The ABGD trees, morphological characters, and geographic infor- procedure (Puillandre et al., 2012) calculates all pairwise mation. In the DNA barcoding gap analysis, we ex- distances in the data set, evaluates intraspecific di- amined the overlap between the mean intraspecific and vergences, and then sorts the terminals into candi- interspecific Kimura two-parameter (K2P) and uncor- date species with calculated P values. We performed rected p-distance for each candidate species calculat- the ABGD analyses online (http://wwwabi.snv.jussieu.fr/ ed in MEGA 5.2.2 (Tamura et al., 2011). The species- public/abgd/), using three different distance metrics: delimitation plug-in (Masters et al., 2011) in Jukes–Cantor (JC69; Jukes & Cantor, 1969), K2P GENEIOUS 5.6.6 (Biomatters Ltd, 2012) was used to (Kimura, 1980), and simple distance (p-distance; obtain P ID(Liberal) statistical values. P ID(Liberal) is Nei & Kumar, 2000). We analysed the data using two based on the putative species groups to calculate the different values for the parameters Pmin (0.0001 and mean probability of intra-/intergenetic distance ratios 0.001), Pmax (0.1 and 0.2), and relative gap width for these candidate species groups. The BI tree was (X = 1 or 1.5), with the other parameters set to default used as a guide tree to test species hypothesis. values.

© 2015 The Linnean Society of London, Zoological Journal of the Linnean Society, 2015, 175, 288–306 Table 1. Samples used in species delimitation: specimen label, taxon name, number of haplotypes, sample collection locality with coordinates, and GenBank 292 accession numbers .XU X. Elevation COI GenBank Specimen code Genus Species Haplotype Locality Country Coordinates (m a.s.l.) accession TAL ET JiAn_3530 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05865°N, 115.04759°E 104 KP875486 JiAn_3531 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05865°N, 115.04759°E 104 KP875514 JiAn_3533 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05865°N, 115.04759°E 104 KP875481 . 05TeLnenSceyo London, of Society Linnean The 2015 © JiAn_3534 Ganthela jianensis sp. nov. H_2 Ji’an City, Jiangxi Province China 27.05865°N, 115.04759°E 104 KP875503 JiAn_3535 Ganthela jianensis sp. nov. H_3 Ji’an City, Jiangxi Province China 27.05865°N, 115.04759°E 104 KP875495 JiAn_3536 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05869°N, 115.04761°E 107 KP875522 JiAn_3538 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05869°N, 115.04761°E 107 KP875521 JiAn_3539 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05869°N, 115.04761°E 107 KP875497 JiAn_3540 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05869°N, 115.04761°E 107 KP229909 JiAn_3541 Ganthela jianensis sp. nov. H_1 Ji’an City, Jiangxi Province China 27.05869°N, 115.04761°E 107 KP875517 Jinggangshan_3503 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.57916°N, 114.15894°E 822 KP875500 Jinggangshan_3504 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.57888°N, 114.15890°E 836 KP875496 Jinggangshan_3508 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.58004°N, 114.15881°E 888 KP875499 Jinggangshan_3510 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.58004°N, 114.15881°E 888 KP875516 Jinggangshan_3511 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.58004°N, 114.15881°E 888 KP875519 Jinggangshan_3512 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.58004°N, 114.15881°E 888 KP875502 Jinggangshan_3514 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.57976°N, 114.15991°E 901 KP875491 Jinggangshan_3516 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.56902°N, 114.16351°E 862 KP875509 Jinggangshan_3517 Ganthela cipingensis H_4 Ciping Town, Jinggangshan City, Jiangxi Pro. China 26.56970°N, 114.16383°E 849 KP229886 Qingyuanshan_2287 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60315°E 475 KP229807 Qingyuanshan_2288 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60315°E 475 KP875525 Qingyuanshan_2290 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60315°E 475 KP875511 Qingyuanshan_2292 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60315°E 475 KP875523 olgclJunlo h ina Society Linnean the of Journal Zoological Qingyuanshan_2294 Ganthela qingyuanensis sp. nov. H_7 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60318°E 484 KP875505 Qingyuanshan_2295 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60318°E 484 KP875482 Qingyuanshan_2296 Ganthela qingyuanensis sp. nov. H_7 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95773°N, 118.60318°E 484 KP875501 Qingyuanshan_2298 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95777°N, 118.60329°E 482 KP875506 Qingyuanshan_3138 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95783°N, 118.60336°E 480 KP875515 Qingyuanshan_3139 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95783°N, 118.60336°E 480 KP875485 Qingyuanshan_3140 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95783°N, 118.60336°E 480 KP875493 Qingyuanshan_3141 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95783°N, 118.60336°E 480 KP875484 Qingyuanshan_3142 Ganthela qingyuanensis sp. nov. H_5 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95783°N, 118.60336°E 480 KP875518 Qingyuanshan_3146 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95779°N, 118.60328°E 485 KP875489 Qingyuanshan_3147 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95779°N, 118.60328°E 485 KP875513 Qingyuanshan_3148 Ganthela qingyuanensis sp. nov. H_6 Mount Qingyuan, Quanzhou City, Fujian Pro. China 24.95779°N, 118.60328°E 485 KP875498 Wangjiangshan_2275 Ganthela wangjiangensis sp. nov. H_8 Mount Wangjiang, Putian City, Fujian Pro. China 25.66220°N, 118.87235°E 680 KP875510 Wangjiangshan_2278 Ganthela wangjiangensis sp. nov. H_9 Mount Wangjiang, Putian City, Fujian Pro. China 25.66224°N, 118.87240°E 667 KP875508 Wangjiangshan_2279 Ganthela wangjiangensis sp. nov. H_9 Mount Wangjiang, Putian City, Fujian Pro. China 25.66224°N, 118.87240°E 667 KP875504 Wangjiangshan_3157 Ganthela wangjiangensis sp. nov. H_9 Mount Wangjiang, Putian City, Fujian Pro. China 25.66230°N, 118.87235°E 675 KP875494 Wangjiangshan_3159 Ganthela wangjiangensis sp. nov. H_9 Mount Wangjiang, Putian City, Fujian Pro. China 25.66233°N, 118.87186°E 698 KP229899 Wangjiangshan_3160 Ganthela venus sp. nov. H_10 Mount Wangjiang, Putian City, Fujian Pro. China 25.65730°N, 118.87946°E 459 KP875483 Wangjiangshan_3160A Ganthela venus sp. nov. H_11 Mount Wangjiang, Putian City, Fujian Pro. China 25.65730°N, 118.87946°E 459 KP875492

2015, , Wangjiangshan_3161 Ganthela venus sp. nov. H_10 Mount Wangjiang, Putian City, Fujian Pro. China 25.65711°N, 118.87975°E 446 KP875524 Xianyou_2283 Ganthela xianyouensis sp. nov. H_12 Xianyou County, Putian City, Fujian Pro. China 25.24953°N, 118.68851°E 556 KP875488 Xianyou_2284 Ganthela xianyouensis sp. nov. H_12 Xianyou County, Putian City, Fujian Pro. China 25.24953°N, 118.68851°E 556 KP875512 Xianyou_3151 Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24944°N, 118.70219°E 336 KP229880 175 Xianyou_3152 Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24944°N, 118.70219°E 336 KP875520 Xianyou_3153 Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24940°N, 118.70224°E 337 KP875526 288–306 , Xianyou_3153A Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24940°N, 118.70224°E 337 KP875490 Xianyou_3154 Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24928°N, 118.70213°E 336 KP875487 Xianyou_3155 Ganthela xianyouensis sp. nov. H_13 Xianyou County, Putian City, Fujian Pro. China 25.24980°N, 118.70172°E 335 KP875507 Zhangqiu_2034 Sinothela sinensis Puji Town, Zhangqiu City, Shandong Pro. China 36.72757°N, 117.61106°E 103 KP229908 INTEGRATIVE TAXONOMY OF GANTHELA 293

The GMYC methodology (Pons et al., 2006) uses like- or lateral eyes; PME, posterior median eyes; RC, lihood to test for species boundaries by detecting the receptacular cluster; T, tegulum. transition point of interspecific versus intraspecific rates of lineage coalescence. We performed GMYC analyses RESULTS in the ‘splits’ package for R (Ezard, Fujisawa & Barraclough, 2009). We used the single-threshold model PHYLOGENETIC INFERENCE (Pons et al., 2006) because prior studies have shown The COI matrix of 51 individuals of Ganthela of 656 bp that the output of the multiple-threshold model had 210 variable and 209 parsimony-informative sites, (Monaghan et al., 2009) is no better than the single- and contained 13 haplotypes (Table 1; Appendix S1). threshold model (Hamilton et al., 2011, 2014; Paz & The phylogenetic topologies from BI and ML analy- Crawford, 2012; Fujisawa & Barraclough, 2013; ses agreed on Ganthela monophyly, with the node being Hendrixson et al., 2013; Talavera, Dinca & Vila, 2013; highly supported (Fig. 1; posterior probability, PP = 1; Kekkonen & Hebert, 2014). We used BEAST 1.8.0 bootstrap value, BS = 1). The two highly supported (Drummond et al., 2012) to obtain an ultrametric gene (PP > 0.95, BS > 75%) sister clades within Ganthela cor- tree that GMYC requires as a guide tree, in combi- respond to two geographical regions: Jiangxi and Fujian nation with a strict molecular clock (Zuckerkandl & provinces (Fig. 1). Within the Jiangxi and the Fujian Pauling, 1962) and Yule speciation model (Yule, 1924; clades, the individuals from each separate locality Gernhard, 2008). We used standard arthropod substi- grouped into solidly supported clades (PP = 1, BS > 75%). tution rates (Brower, 1994) by setting the COI sub- These locality clades mostly comprise closely related stitution rate parameter as a normal prior with a mean individuals, except in the case of Mount Wangjiang, value of 0.0115, and ran 50 million generations, sam- where a single locality harbours two distinct and highly pling every 5000 generations. We used TRACER 1.6 supported species clades (Fig. 1). (Rambaut et al., 2014) to assess the chain convergence and correct mixing of each MCMC chain, then discarded as ‘burn-in’ 10% of the trees in each chain to SPECIES DELIMITATION settle on an ultrametric tree using TreeAnnotator DNA barcoding gap (Drummond et al., 2012). Based on our prior species hypotheses, the lowest mean Finally, we employed SP haplotype network analy- interspecific distance was 12/11% (K2P/uncorrected sis (Templeton et al., 1992) to investigate intraspecific p-distance) found between G. cipingensis and Ganthela relationships among the terminals, and thus delimit jianensis sp. nov., which was about eight times the species (Jörger et al., 2012; Weigand et al., 2013). We highest mean intraspecific distance (1.62/1.57% for K2P/ generated haplotype networks using TCS 1.21 (Clement, uncorrected p-distance) estimated for Ganthela Posada & Crandall, 2000) with a 95% parsimony xianyouensis sp. nov. (Table 2). Histograms with pairwise criterion. distances visualize a barcoding gap in Ganthela of 4–12% (K2P)/4–11% (uncorrected p-distance; Fig. 2). Six TAXONOMY species were identified by the barcoding gap range, of Within a liphistiid genus-level revision, we recently de- which one is known and five are new (Fig. 1). scribed (Xu et al., 2015a) the type species of Ganthela: G. yundingensis Xu, 2015. Additional Ganthela speci- P ID(Liberal) mens were examined with an Olympus SZX16 ster- The results based on the Bayesian inference tree found eomicroscope, and anatomical details were studied with high P ID(Liberal) values of ≥0.97 (0.88–1.0) and low a Leica M205C stereomicroscope. Male palps and female intra-/interspecific ratios of ≤0.1 for all species hy- genitalia were examined and photographed with a Leica pothesized (Table 2), thereby also supporting the tax- M205C stereomicroscope and Olympus BX51 com- onomy of six putative species (Fig. 1). pound microscope after being dissected from the spider bodies. Genitalia were cleared in boiling 10% KOH for ABGD analysis a few minutes to dissolve soft tissues. Unless other- The ABGD results using different parameter combi- wise noted, left palps were depicted. All measure- nations and the initial partition of six species (Table 3) ments are in millimetres. Leg and palp measurements agreed on those six species, whereas those under a re- are given in the following order: total length cursive partition regime yielded more species (Table 3).

(femur + patella + tibia + metatarsus + tarsus). The settings Pmin/Pmax = 0.0001/0.2 yielded the most sig- Abbreviations used are: ALE, anterior lateral eyes; niﬁcant P values. AME, anterior median eyes; BL, body length; CL, cara- All analyses reported so far support six morphologi- pace length; Co, conductor; CT, contrategulum; CW, cara- cal species, with syntopic terminals being conspeciﬁc, pace width; E, embolus; OL, opisthosoma length; OW, except in the case of Mount Wangjiang, where two opisthosoma width; PC, paracymbium; PLE, posteri- species coexist within an absolute distance of 1 km.

In contrast, the following two analyses suggest more species. The single-threshold model GMYC resulted in Intra/ inter 0.06 0.01 0.03 0.1 0.02 0.02 nine clusters and entities with different conﬁdence in- tervals of 7–9 and 7–11, respectively (Table 4). These results indicate two species in each of the Ji’an, Xianyou, and Mount Qingyuan clades (Fig. 1). The SP haplotype sp. nov.

sp. nov. networks also show similar results, but with a single sp. nov. species within the Mount Qingyuan clade (Figs 1, 3). sp. nov.

sp. nov. DISCUSSION In this study, under an integrative framework, we analysed original DNA barcoding data with five different Closest P ID(Liberal) species G. jianensis G. cipingensis G. wangjiangensis G. xianyouensis G. venus G. qingyuanensis species-delimitation methods to test the validity of one described and five undescribed morphological species of the liphistiid genus Ganthela known to us, in addition to the type species G. yundingensis Xu, 2015. Our results show that three molecular species- delimitation methods – DNA barcoding gap, P ID(Liberal), 1.00 (0.95–1.00) 0.99 (0.94–1.00) 1.00 (0.86–1.00) 1.00 (0.97–1.00) 0.98 (0.88–1.00) 0.97 (0.91–1.00) and ABGD – all fully confirm the morphological understanding of Ganthela species diversity, and thus el- egantly complement traditional taxonomy (Fig. 1); however, GMYC and SP found more species of Ganthela, sp. nov.

sp. nov. and thus disagree with the morphology, a result that

sp. nov. reinforces conclusions from previous studies (Esselstyn et al., 2012; Paz & Crawford, 2012; Miralles & Vences, 2013; Talavera et al., 2013; Hamilton et al., 2014). We sp. nov.

sp. nov. use the consensus from our analyses for species delimitation and diagnoses to conclude the formal revision of Ganthela (see Taxonomy). G. wangjiangensis G. xianyouensis G. venus G. qingyuanensis The traditional DNA barcoding gap and ABGD methods are both based on the identification of a sig- G. jianensis G. cipingensis nificant difference between inter- and intraspecific genetic distances in samples of terminal taxa, in order to assign organisms into putative species. For the DNA

Closest interspeciﬁc K2P/P-distance P ID(Liberal) barcoding gap analysis, we assigned Ganthela specimens into putative species considering the phylogenetic, morphological, and geographic information available. Our results are roughly comparable with those from the mygalomorph spider literature, which found a barcode gap of 5–6% (Hamilton et al., 2011, 2014); however, the gap is wider in Ganthela, ranging from Intraspeciﬁc K2P/p-distance 0/0 0.12/0.11; 0.0071/0.0069 0.12/0.11; 0.0010/0.00100.0033/0.0033 0.1265/0.1151; 0.1321/0.1191; 0.0010/0.0006 0.1265/0.1151; 0.0162/0.0157 0.1321/0.1191; 4 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).

sp. nov. In this study, however, all analyses under different as- sp. nov.

sp. nov. sumptions conﬁrmed the six species, thereby lending further support to the utility of the barcoding gap in sp. nov. delimiting species (Weigand et al., 2013; Cˇ andek &

sp. nov. Kuntner, 2015; Hamilton et al., 2014). P ID(Liberal) is usually used to test species hypotheses on phylogenetic trees (Masters et al., 2011; Hamilton Summary of the mean intraspecific and closest interspecific genetic distance, the mean probability with 95% confidence interval, and the intra/ et al., 2014). According to our findings, a P ID(Liberal) value > 95% would be reasonable to distinguish Ganthela species, and thus the cut-off of 95% can be used to Table 2. Putative species interspecific ratio for the six putative species Ganthela cipingensis Ganthela jianensis Ganthela venus Ganthela qingyuanensis Ganthela wangjiangensis Ganthela xianyouensis delimit Ganthela and probably other liphisitiid species.

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

Prior intraspeciﬁc divergence (P) Substitution model Pmin/Pmax X Partition 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 6666666 Recursive 11 9 9 9987766 K2P 0.001/0.1 1.5 Initial 6 6 6 6666666 Recursive 11 9 9 9987766 Simple 0.001/0.1 1.5 Initial 6 6 6 6666666 Recursive 11 9 9 9987766 JC 0.001/0.1 1 Initial 6 6 6 6666666 Recursive 11 9 9 9987766 K2P 0.001/0.1 1 Initial 6 6 6 6666666 Recursive 11 9 9 9987766 Simple 0.001/0.1 1 Initial 6 6 6 6666666 Recursive 11 9 9 9987766 0.0001 0.0002 0.0005 0.0013 0.0029 0.0068 0.0159 0.0369 0.086 JC 0.0001/0.2 1.5 Initial 6 6 6 666666 Recursive 11 11 11 11 99876 K2P 0.0001/0.2 1.5 Initial 6 6 6 666666 Recursive 11 11 11 11 99876 Simple 0.0001/0.2 1.5 Initial 6 6 6 666666 Recursive 11 11 11 11 99866 JC 0.0001/0.2 1 Initial 6 6 6 666666 Recursive 11 11 11 11 99876

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

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, singleton OTUs 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.

As a species delimitation technique, the often used indicate that even single-locus analyses based on the GMYC is known to overestimate the number of species COI barcodes, if integrated with morphological and geo- (Esselstyn et al., 2012; Paz & Crawford, 2012; Miralles graphical data, may provide sufficiently reliable species & Vences, 2013; Talavera et al., 2013; Hamilton et al., delimitation, and that multi-locus phylogenetic ap- 2014; but see, Jörger et al., 2012). In our case GMYC proaches may not be necessary for reliable taxonomic indeed seemingly overestimates the Ganthela species, decisions. suggesting that there are nine. Because GMYC requires an input tree, a differently obtained ultramatric TAXONOMY gene tree, particularly one resulting from several genes, GENUS GANTHELA XU &KUNTNER, 2015 would probably change these results (Talavera et al., For genus diagnosis and description, see Xu et al., 2015a. 2013; Kekkonen & Hebert, 2014). Our starting tree was Type species by original designation, G. yundingensis derived from a single mitochondrial gene, but the ad- Xu, 2015. For diagnosis and description, see Xu et al., dition of nuclear genes would be useful (Jörger et al., 2015a. 2012; Dellicour & Flot, 2015). Whereas SP has been successfully used in delimiting species in some taxa (Pons et al., 2006; Sauer & GANTHELA CIPINGENSIS (WANG, 1989) (FIG.4) Hausdorf, 2012), it is often used to investigate Liphistius cipingensis Wang, 1989: 30, figs 1–6 (de- intraspecific relationships. In our study, it oversplits scription of female, type may be lost from Hunan Ganthela species and thus may not be helpful in de- Normal University, not available for examination). limiting Ganthela species. Heptathela cipingensis Platnick, 1993: 77 (trans- The peril of employing single-locus species delimi- ferred from Liphistius). tation techniques is the possibility of over-splitting taxa Songthela cipingensis Ono, 2000: 150. as a result of co-amplified nuclear mitochondrial Material examined pseudogenes (Song et al., 2008), incomplete lineage Five females [XUX-2013-(508/510/511/512/514)] col- sorting and introgression (Hausdorf & Hennig, 2010; lected at Cemetery of Mount Jinggang Revolutionary Edwards & Knowles, 2014), and particularly the deep Martyrs, Ciping Town, Jinggangshan City, Jiangxi Prov- genetic structuring of mtDNA sequences (Bond et al., ince, China, 26.58°N, 114.16°E, 900 m a.s.l., 2001; Arnedo & Ferrández, 2007). To avoid spurious 21 October 2013; two females [XUX-2013-(515–518)] col- results, taxonomic studies ideally incorporate multilocus lected at Nanshan, Ciping Town, Jinggangshan City, data and analyse it in a phylogenetic framework to Jiangxi Province, China, 26.57°N, 114.16°E, 850 m a.s.l., test the validity of putative species (e.g. Satler, Carstens 22 October 2013, collected by F.X. Liu, C. Xu, and X. Xu. & Hedin, 2013; Edwards & Knowles, 2014; Opatova No male found. & Arnedo, 2014). Multi-locus approaches may be particularly useful to understand lineages with seden- Diagnosis tary females and vagile males (Opatova & Arnedo, 2014; Females of G. cipingensis resemble G. xianyouen- Hedin, 2015), where maternally inherited mtDNA may sis sp. nov., but differ by the details in genitalia, with show deep genetic structuring, even in the absence of relatively longer genital stalks (Fig. 4B, C). Ganthela geographical barriers (Bond et al., 2001). cipingensis differs from all other Ganthela species by Showing poor dispersal abilities, primitively seg- the following unique nucleotide substitutions in the mented spiders are strongly endemic and range- standard DNA barcode alignment: G (20), G (26), C restricted, and the high interspecific genetic distances (77), C (326), and C (413). in our study support the hypothesized low levels of gene flow among populations, which is typical of mygalomorphs Description (Hamilton et al., 2011, 2014; Hendrixson et al., 2013; Female (Fig. 4A). Carapace and opisthosoma dark Opatova & Arnedo, 2014). Nevertheless, our results brown; with clear fovea; chelicerae robust, with

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

Figure 4. Ganthela cipingensis (Wang, 1989). A, female (XUX-2013-516). B, C, female genitalia (XUX-2013-517): B, dorsal view; C, ventral view; RC, receptacular cluster. Scale bar: 0.5 mm. promargin of cheliceral groove with between ten and Paratypes 13 strong denticles of variable size; legs with strong Ten females (XUX-2013-530-531/533–536/538-541A) col- hairs and spines; opisthosoma with 12 tergites, with lected at the same locality, 23 October 2013, collected tergite 5 being largest; seven spinnerets. Measure- by F.X. Liu, X. Xu, and Z.T. Zhang. No male found. 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 > Etymology AME; palp 9.72 (3.47 + 1.70 + 2.15 + 2.40), leg I 12.00 ‘Jian’ refers to the type locality of this species. (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 Diagnosis (3.60 + 2.20 + 2.25 + 3.35 + 1.90), and leg IV 19.07 Females of G. jianensis sp. nov. differ from other species (5.30 + 2.67 + 3.45 + 5.20 + 2.44). of Ganthela by details in genitalia, with short and thick genital stalks (Fig. 5B, C), and by the slightly curved posterior part of the genital area (Fig. 5B, C). Ganthela Female genitalia jianensis sp. nov. differs from all other Ganthela species The posterior part of the genital area wide, rectan- by the following unique nucleotide substitutions in gular (Fig. 4B, C), a pair of receptacular clusters close the standard DNA barcode alignment: G (59), A (131), to each other and more or less parallel, with short A (155), C (245), T (249), G (317), C (398), C (528), C genital stalks (Fig. 4B, C). (581), and C (638).

Distribution Description Jiangxi (Jinggangshan) Province, China. Female (holotype) (Fig. 5A). Carapace and opisthosoma, dark brown; chelicerae robust, with promargin of cheliceral groove with between ten and 13 strong denticles of variable size; legs with strong hairs GANTHELA JIANENSIS XU,KUNTNER & and spines; opisthosoma with 12 tergites, with tergite 5 CHEN SP. NOV. (FIG.5) being largest; seven spinnerets. Measurements: Holotype BL 11.80–15.50, CL 5.60–7.40, CW 5.30–6.50, OL 5.95– Female (XUX-2013-534), Mount Qingyuan, Ji’an City, 9.10, and OW 4.46–6.70; ALE > PLE > PME > AME; Jiangxi Province, China, 27.06°N, 115.05°E, 100 m a.s.l., palp 11.55 (4.00 + 2.10 + 2.45 + 3.00), leg I 13.25 23 October 2013, collected by F.X. Liu, X. Xu, and (4.15 + 2.50 + 2.35 + 2.65 + 1.60), leg II 12.88 Z.T. Zhang. (3.73 + 2.37 + 2.25 + 2.75 + 1.78), leg III 13.82

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

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

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

Diagnosis Males of G. qingyuanensis sp. nov. differ from G. yundingensis by anatomical details in the palps, which have a small posterior apophysis on the conductor, in addition to the spiniform apex (Fig. 6G). Females of G. qingyuanensis sp. nov. can be distinguished from all other Ganthela species by details in the genitalia, with longer genital stalks (Fig. 6B, C). Ganthela qingyuanensis sp. nov. differs from all other Ganthela species by the following unique nucleotide substitutions in the standard DNA barcode alignment: 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).

Figure 5. Ganthela jianensis Xu, Kuntner & Chen Description sp. nov. A, female (XUX-2013-536). B, C, female genitalia Male (holotype). Carapace brown; opisthosoma light (XUX-2013-534): B, dorsal view; C, ventral view; RC, brown, with dark brown tergites; sternum narrow, much receptacular cluster. Scale bar 0.5 mm. longer than wide; a few long pointed hairs running over ocular mound in a longitudinal row; chelicerae robust, with promargin of cheliceral groove with 11 denticles (4.00 + 2.42 + 2.17 + 3.23 + 2.00), and leg IV 20.37 of variable size; legs with strong hairs and spines; (5.61 + 3.00 + 3.45 + 5.53 + 2.78). opisthosoma with 12 tergites, with tergites 2–6 larger than others, and with tergite 4 being largest; seven Female genitalia spinnerets. Measurements: BL 10.55, CL 5.00, CW 4.85, > > > Posterior part of genital area slightly curved (Fig. 5B, OL 5.60, and OW 3.63; ALE PLE PME AME; leg I C), with pair of receptacular clusters close to each 14.50 (4.11 + 1.80 + 2.89 + 3.70 + 2.00), leg II 15.14 other, and with very short and thick genital stalks (3.87 + 1.82 + 3.00 + 4.15 + 2.30), leg III 16.47 (Fig. 5B, C). (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).

Distribution Palp Jiangxi (Ji’an) Province, China Cymbium with a projection; prolateral side of paracymbium unpigmented and unsclerotized, numer- ous setae and spines at the tip of paracymbium (Fig. 6F, GANTHELA QINGYUANENSIS XU,KUNTNER & G). Contrategulum has two marginal apophyses with LIU SP. NOV. (FIG.6) smooth margin and blunt distal end (Fig. 6H). Tegulum Holotype with a dentate, wide edge (Fig. 6F, G). Conductor with Male (XUX-2012-288, matured 18 July 2013 at CBEE, a spiniform apex and a small apophysis at posterior College of Life Sciences, Hubei University), around part, parallel with embolus (Fig. 6G). Embolus largely TV Tower, Mount Qingyuan, Quanzhou City, Fujian sclerotized, with a wide and ﬂat opening, and distal Province, China, 24.95°N, 118.60°E, 475 m a.s.l., embolus slightly sclerotized (Fig. 6G).

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.

Female (Fig. 6A). Carapace and opisthosoma of female (3.40 + 1.95 + 1.96 + 2.45 + 1.32), leg III 11.81 similar to male, except coloration lighter than male; (3.46 + 2.05 + 1.92 + 2.70 + 1.68), and leg IV 17.46 chelicerae robust, with promargin of cheliceral groove (4.95 + 2.48 + 3.05 + 4.68 + 2.30). with between ten and 12 strong denticles of variable size; legs with strong hairs and spines; opisthosoma with 12 tergites, similar to male; seven spinnerets. Female genitalia Measurements: BL 10.68–14.48, CL 5.48–7.05, CW 4.52– Posterior part of genital area rectangular (Fig. 6B– 6.00, OL 5.77–7.62, and OW 4.13–5.68; ALE > PLE > E), with pair of receptacular clusters with genital stalks PME > AME; palp 9.51 (3.40 + 1.70 + 2.00 + 2.41), leg I (Fig. 6B, C), or with paired receptacular clusters fused 11.43 (3.85 + 1.95 + 2.08 + 2.25 + 1.30), leg II 11.08 into a big cluster (Fig. 6D, E).

Variation tergites 2–6 larger than others, and the with tergite 4 Female receptacular clusters show considerable being largest; seven spinnerets. Measurements: BL 11.50, intraspeciﬁc variation, with the pair of receptacular CL 4.90, CW 4.30, OL 6.40, OW 5.55; ALE > PLE > clusters fused. PME > AME; palp 7.94 (2.78 + 1.50 + 1.48 + 2.18), leg I 9.10 (3.05 + 1.35 + 1.70 + 1.80 + 1.20), leg II 9.50 Distribution (2.80 + 1.67 + 1.71 + 2.02 + 1.30), leg III 10.00 Fujian (Quanzhou) Province, China. (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).

GANTHELA WANGJIANGENSIS XU,KUNTNER &LIU Female genitalia SP. NOV. (FIG.7) The posterior part of genital area W-shaped (Fig. 7A, Holotype B), with a pair of receptacular clusters separated from Female (XUX-2013-159), Mount Wangjiang, Huangyang each other, and with the basal parts of short genital Village, Zhuangbian Town, Hanjiang District, Putian stalks fused (Fig. 7A, B). City, Fujian Province, China, 25.66°N, 118.87°E, 680 m a.s.l., 10 July 2013, collected by F.X. Liu, X. Xu, Distribution and Z.T. Zhang. No male found. Fujian (Putian) Province, China.

Etymology Remarks ‘Wangjiang’ refers to the type locality of this species, Ganthela wangjiangensis sp. nov. was collected within Mount Wangjiang. 1kmofG. venus sp. nov., but at a higher altitude.

Diagnosis GANTHELA XIANYOUENSIS XU,KUNTNER &CHEN Females of G. wangjiangensis sp. nov. differ from all SP. NOV. (FIG.8) other Ganthela species by the W-shaped posterior part of the genital area, and can be further distinguished Holotype from G. venus sp. nov. by the connected basal genital Female (XUX-2013-153), Qingfengge, Dongshi Village, stalks (Fig. 7A, B). Ganthela wangjiangensis sp. nov. Yuanzhuang Town, Xianyou County, Putian City, Fujian differs from all other Ganthela species by the follow- Province, China; 25.24°N, 118.70°E, 336 m a.s.l., ing unique nucleotide substitutions in the standard DNA 10 July 2013; collected by F.X. Liu, X. Xu, and Z.T. Zhang. barcode alignment: G (54), C (101), C (179), C (320), A (336), T (372), C (386), C (422), G (476), T (512), Paratypes and C (632). Two females (XUX-2013-153/154) collected at the same locality, 10 July 2013; collected by F.X. Liu, X. Xu, and Description Z.T. Zhang. No male found. Female (holotype). Carapace and opisthosoma light brown; tergites slightly dark brown; sternum narrow, Etymology length more or less twice of width; a few long pointed ‘Xianyou’ refers to the type locality of this species. hairs running over ocular mound in a longitudinal row; chelicerae robust, with promargin of cheliceral groove Diagnosis with 12 strong denticles of variable size; legs with strong Females of G. xianyouensis sp. nov. can be distin- hairs and spines; opisthosoma with 12 tergites, with 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.

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. the genitalia, with very short but slender genital stalks Distribution (Fig. 8B, C). Ganthela xianyouensis sp. nov. further differs Fujian (Putian) Province, China. from all other Ganthela species by the following unique nucleotide substitutions in the standard DNA barcode GANTHELA VENUS XU SP. NOV. (FIG.9) alignment: G (96), G (146), C (277), C (314), T (338), C (393), C (428), and T (572). Holotype Female (XUX-2013-160), Mount Wangjiang, Huangyang Description Village, Zhuangbian Town, Hanjiang District, Putian Female (holotype) (Fig. 8A). Carapace and opisthosoma City, Fujian Province, China, 25.66°N, 118.88°E, light brown; tergites dark brown; sternum narrow, length 459 m a.s.l., 11 July 2013, collected by F.X. Liu, X. Xu, more or less twice of width; a few long pointed hairs and Z.T. Zhang. No male found. running over ocular mound in a longitudinal row; chelicerae robust, with promargin of cheliceral groove Etymology with 11 or 12 strong denticles of variable size; legs with Named after the Greek goddess of love, Venus. strong hairs and spines; opisthosoma with 12 tergites, with tergites 2–6 larger than others, and with tergite 4 Diagnosis being largest; seven spinnerets. Measurements: Females of G. venus sp. nov. can be distinguished from BL 13.60, CL 6.10, CW 4.96, OL 6.56, and OW 5.45; G. wangjiangensis sp. nov. by details in the genitalia, ALE > PLE > which have separated stalks on the anterior part PME > AME; palp 9.48 (3.26 + 1.66 + 2.06 + 2.50), leg I (Fig. 9A, B). Ganthela venus sp. nov. furthermore differs 11.58 (3.72 + 1.92 + 2.15 + 2.32 + 1.47), leg II 11.45 from all other Ganthela species by the following unique (3.64 + 1.93 + 2.05 + 2.25 + 1.58), leg III 12.58 nucleotide substitutions in the standard DNA barcode (3.55 + 2.10 + 2.10 + 3.00 + 1.83), and leg IV 17.53 alignment: C (36), A (80), T (227), C (333), C (341), G (4.88 + 2.25 + 3.15 + 4.55 + 2.70). (347), T (354), C (431), and T (539).

Female genitalia Description The posterior part of genital area rectangular (Fig. 8B, Female (holotype). Carapace and opisthosoma light C), with a pair of receptacular clusters close to each brown; tergites slightly dark brown; sternum narrow, other, and with very short but slender genital stalks length more or less twice of width; a few long pointed (Fig. 8B, C). hairs running over ocular mound in a longitudinal row;

Figure 9. Ganthela venus Xu sp. nov. A, B, female genitalia (XUX-2013-160): A, dorsal view; B, ventral view. RC, receptacular cluster. Scale bar: 0.5 mm. chelicerae robust, with promargin of cheliceral groove MK). We thank Ingi Agnarsson and two anonymous with ten strong denticles of variable size; legs with referees for their valuable input. strong hairs and spines; opisthosoma with 12 tergites, with tergites 2–6 larger than others, and with tergite 4 being largest; seven spinnerets. Measurements: BL 9.80, REFERENCES > > CL 4.38, CW 4.20, OL 5.6, and OW 5.00; ALE PLE Agnarsson I. 2010. The utility of ITS2 in spider phylogenetics?: > PME AME; palp 7.63 (2.50 + 1.31 + 1.65 + 2.17), leg I notes on prior work and an example from Anelosimus. Journal missing, leg II 8.49 (2.71 + 1.50 + 1.68 + 1.50 + 1.10), of Arachnology 38: 377–382. leg III 9.34 (2.52 + 1.58 + 1.56 + 2.30 + 1.38), and leg IV Agnarsson I, Kuntner M. 2007. Taxonomy in a changing world: 13.70 (3.75 + 1.60 + 2.55 + 3.68 + 2.12). seeking solutions for a science in crisis. Systematic Biology 56: 531–539. Arnedo MA, Ferrández MA. 2007. Mitochondrial markers Female genitalia reveal deep population subdivision in the European protect- Posterior part of genital area W-shaped (Fig. 9A, B), ed spider Macrothele calpeiana (Walckenaer, 1805) (Araneae, with pair of receptacular clusters with short stalks on Hexathelidae). Conservation Genetics 8: 1147–1162. the anterior part of genital separated from one another Barrett RDH, Hebert PDN. 2005. Identifying spiders through (Fig. 9A, B). DNA barcodes. Canadian Journal of Zoology 491: 481– 491. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Distribution Winker K, Ingram KK, Das I. 2007. Cryptic species as a Fujian (Putian) Province, China. window on diversity and conservation. Trends in Ecology and Evolution 22: 148–155. Bishop SC, Crosby CR. 1932. A new species of the spider Remarks family Liphistiidae from China. Peking Natural History Bul- See comments under G. wangjiangensis sp. nov. letin 6: 5–8. Blaxter ML, Mann J, Chapman T, Thomas F, Whitton C, Floyd R, Abebe E. 2005. Defining operational taxonomic ACKNOWLEDGEMENTS units using DNA barcode data. Philosophical Transactions of the Royal Society of London B: Biological Sciences 360: We thank Chen Xu and Zengtao Zhang for assis- 1935–1943. tance with fieldwork, Matjaž Gregoricˇ, Nina Vidergar, Bond JE, Hedin M. 2006. A total evidence assessment of the and Ren-Chung Cheng for help and/or advice on mo- phylogeny of North American euctenizine trapdoor spiders lecular resources and procedures, and the staff of the (Araneae, Mygalomorphae, Cyrtaucheniidae) using Bayes- Centre for Behavioural Ecology and Evolution (CBEE, ian inference. Molecular Phylogenetics and Evolution 41: 70– Hubei University) for all their help and support through- 85. out this study, in particularly Bo Chen, Xiaoguo Jiao, Bond JE, Hedin MC, Ramirez MG, Opell BD. 2001. Deep Jie Liu, Yu Peng, Chen Xu, Long Yu, and Zengtao molecular divergence in the absence of morphological and Zhang. This study was supported in part by funds from ecological change in the Californian coastal dune endemic China [NSFC grant (31272324)], the Singapore Min- trapdoor spider Aptostichus simus. Molecular Ecology 10: 899– istry of Education (MOE) [AcRF Tier 1 grant (R-154- 910. 000-591-112)], and by the Slovenian Research Agency Brower AVZ. 1994. Rapid morphological radiation and con- (grants P1-10236, MU-PROM/12-001 and J1-6729 to vergence among races of the butterfly Heliconius erato

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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).