ZOOLOGICAL RESEARCH Integrative taxonomy of Leptonetela spiders (Araneae, Leptonetidae), with descriptions of 46 new species Chun-Xia Wang1,2, Xin Xu3, Shu-Qiang Li1,4,* 1Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China 2Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin Nay Pyi Taw 05282, Myanmar 3College of Life Sciences, Hunan Normal University, Changsha Hunan 410006, China 4University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT palmata is a preoccupied name. Extreme environments, such as subterranean habitats, Keywords: DNA barcoding; Phylogeny; Phenotype; are suspected to be responsible for morphologically Species delineation inseparable cryptic or sibling species and can bias biodiversity assessment. A DNA barcode is a short, INTRODUCTION1 standardized DNA sequence used for taxonomic purposes and has the potential to lessen the challenges Subterranean ecosystems, such as caves and cracks, are presented by a biotic inventory. Here, we investigate evident mainly in karst areas, which represent nearly 4% of the the diversity of the genus Leptonetela Kratochvíl, rocky outcrops of the world. These environments are marked by 1978 that is endemic to karst systems in Eurasia permanent darkness, a lack of diurnal and annual rhythms, and using DNA barcoding. We analyzed six hundred and extremely scarce food sources (Culver & White, 2005; Howarth, twenty four specimens using one mitochondrial gene 1983; Poulson & White, 1969). Many studies show that despite fragment (COI). The results show that DNA barcoding stressful and unfavorable conditions, the subsurface habitat is an efficient and rapid species identification method harbors diverse animal communities (mainly invertebrates) in this genus. It indicated the existence of 90 species, (Amara-Zettler et al., 2002; Flot et al., 2010; López-García et al., a result consistent with previous taxonomic hypotheses 2001; Mathieu et al., 1997; Niemiller et al., 2012: Sket, 1999). and supported the existence of extreme male Troglobionts are expected to adopt strategies that are pedipalpal tibial spine and median apophysis characterized by significant geographic isolation and numerous polymorphism in Leptonetela species, with direct local endemics (Convey, 1997; Waterman, 2001). Because the implications for the taxonomy of the group and its diversity of possible adaptive responses decline with stress diversity, using DNA barcoding gap and automatic intensity (Nevo, 2001), evolution in harsh environments is also barcode gap discovery (ABGD) analyses. Based on expected to be influenced by convergence (Little & Vrijenhoek, the molecular and morphological evidence, we 2003: Rothschild & Mancinelli, 2001; Waterman, 2001). delimit and diagnose 90 Leptonetela species, including Therefore, in subterranean and more generally in extreme the type species Leptonetela kanellisi (Deeleman- environments, diversification and speciation processes should Reinhold, 1971); 46 of them are previously undescribed be largely influenced by island-like habitats, such as caves, species. Leptonetela tianxinensis (Tong & Li, 2008) allopatric speciation and vicariant events, and could be masked comb. nov. is transferred from the genus Leptoneta by morphological convergence. For these groups of organisms, Simon, 1872; The genus Guineta Lin & Li, 2010 syn. nov. is junior synonym of Leptonetela, Leptonetela Received: 22 October 2017; Accepted: 10 November 2017 gigachela (Lin & Li, 2010) comb. nov. is transferred Foundation items: This study was financially supported by the National from genus Guineta. The genus Sinoneta Lin & Li, 2010 syn. nov. is junior synonym of Leptonetela, Natural Sciences Foundation of China to Chunxia Wang (NSFC- Leptonetela notabilis (Lin & Li, 2010) comb. nov., 31471977) and Shuqiang Li (NSFC-31530067, 31471960). Part of the Leptonetela sexdigiti (Lin & Li, 2010) comb. nov. are lab work was supported by the Southeast Asia Biodiversity Research transferred from genus Sinoneta; Leptonetela sanchahe Institute, Chinese Academy of Sciences (2015CASEABRI005, Y4ZK111B01) nom. nov. is proposed as replace name for Sinoneta *Corresponding author, E-mail: [email protected] palmata (Chen et al., 2010) because Leptonetela DOI: 10.24272/j.issn.2095-8137.2017.076 Science Press Zoological Research 38(6): 1-11, 2017 1 morphology alone cannot determine species boundaries. So validation method, DNA barcoding gap analysis, (Hebert et al., identifying morphologically inseparable cryptic or sibling species 2003b) were both used, depending on whether the samples requires an integrative approach with a set of taxonomic tools, were partitioned prior to analysis. The main goals of our study including DNA analysis. were: (i) to test whether the COI barcoding fragment can DNA barcoding relies on the use of a standardized DNA region reliably resolve and identify subterranean Leptonetela species as a tag for accurate and rapid species identification (Hebert & by comparing the COI barcode fragment results with those from Gregory, 2005) and has been advanced to help overcome the morphological data; (ii) to test taxonomic value of morphological ‘taxonomic impediment’ (Herbert et al., 2003a; Tautz et al., 2003). characters used in traditional methods of classification. It aids in the identification of species in applied settings, the association of morphologically distinct life-cycle forms within a MATERIALS AND METHODS species, the detection of host-specific lineages and the detection Taxon sampling of morphologically cryptic species (Miller & Foottit, 2009). DNA We sampled 624 Leptonetela individuals from 122 populations barcoding has been used in a diverse range of vertebrate and (caves) (Table S1) in Eurasia (Insular and Peninsular Greece, invertebrate taxa (Clare et al., 2007; Ratnasingham & Hebert, and Southeast Asia; see inset in Fig. 1). Nine individuals from 2007) and has enabled an increasing number of taxa to be three other genera of the family Leptonetidae were chosen as identified. For example, a survey of crustacean stygofauna the out-group. All specimens were collected alive, fixed in suggests that there could be substantial levels of subterranean absolute ethanol, and the legs were removed for subsequent biodiversity hidden in Australia’s acquifer (Asmyhr & Cooper, DNA extraction. The remaining specimens were preserved in 2012). Nevertheless, the exclusive use of single-locus molecular 80% ethanol for identification and morphological examination. gene fragments is not without risks, for identical mitochondrial For small juveniles entire specimens were used for DNA extraction. DNA sequences can be present in unrelated species due to Voucher specimens and all type specimens were deposited in introgression, or incomplete lineage sorting (Ballard & Whitlock, the National Zoological Museum, Chinese Academy of Sciences 2004). Additionally, the use of a divergence threshold for (IZCAS), Beijing, China. distinguishing intra- versus interspecific sequence variation (Hebert et al., 2003a) can seriously compromise species Molecular protocols identification and suffers from severe statistical problems (Vences Total genomic DNA was extracted using the Animal Genomic et al., 2005). Furthermore, species misidentification has been DNA Isolation Kit (Dingguo, Beijing, China), following the observed when a reference database is not comprehensive; such manufacturer’s protocol. We amplified the cytochrome c that is does not contain all the species of the group under study oxidase subunit I (COI) barcode region using the primer pairs (Meyer & Paulay, 2005). LCOI490/HCO2198 (Folmer et al., 1994). PCR reaction The South China karst, a UNESCO World Heritage Site since conditions were: initial denaturation at 94 °C for 1 min; 35 2007, is noted for its karst features and landscapes as well as rich cycles of denaturation at 94 °C for 1 min, annealing at 45 °C for biodiversity. Numerous subterranean species have been reported 45 s, and elongation at 70 °C for 60 s; and final extension at 72 °C in this region, especially invertebrate fauna (Zhang, 1986). The for 5 min. The 25-L PCR reactions included 17.25 L of spider genus Leptonetela is discontinuously distributed in the double-distilled H2O, 2.5 L of 10× Taq buffer (mixed with MgCl2; South China karst and the Balkan Peninsula, a karstic region in TianGen Biotech, Beijing, China), 2.0 L of dNTP Mix (2.5 mM), Europe. The genus has 54 catalogued species (World Spider 1 L of each forward and reverse 10-M primer, 1 L of DNA Catalog, 2017), and with one exception (L. pungitia Wang & Li, template, and 0.25 L Taq DNA polymerase (2.5 U L1; 2011), nearly all Leptonetela species are endemic to either a TianGen Biotech, Beijing, China). Double-stranded PCR products single cave or a cave system. The spiders are cave adapted and were visualized by agarose gel electrophoresis (1% agarose). show morphological features, such as vestigial eyes and highly PCR products were purified and sequenced by Sunny Biotechnology reduced skin pigmentation. Over the past 9 years, we have Co., Ltd (Shanghai, China) using the ABI 3730XL DNA analyser. conducted extensive surveys of subterranean biodiversity in Sequences were aligned using ClustalW in Mega 6.0 (Tamura Eurasia. More than 1,500 caves were visited, and we ultimately et al., 2013), with visual inspection, translation, and manual sampled 122 Leptonetela populations (caves). Rapid and accurate adjustment to minimize alignment error. The most appropriate identification within this genus