Integrative taxonomy of Leptonetela spiders (Araneae, Leptonetidae), with descriptions of 46 new species

Extreme environments, such as subterranean habitats, are suspected to be responsible for morphologically inseparable cryptic or sibling species and can bias biodiversity assessment. A DNA barcode is a short, standardized DNA sequence used for taxonomic purposes and has the potential to lessen the challenges presented by a biotic inventory. Here, we investigate the diversity of the genus Leptonetela Kratochvíl, 1978 that is endemic to karst systems in Eurasia using DNA barcoding. We analyzed 624 specimens using one mitochondrial gene fragment (COI). The results show that DNA barcoding is an efficient and rapid species identification method in this genus. DNA barcoding gap and automatic barcode gap discovery (ABGD) analyses indicated the existence of 90 species, a result consistent with previous taxonomic hypotheses, and supported the existence of extreme male pedipalpal tibial spine and median apophysis polymorphism in Leptonetela species, with direct implications for the taxonomy of the group and its diversity. Based on the molecular and morphological evidence, we delimit and diagnose 90 Leptonetela species, including the type species Leptonetela kanellisi(Deeleman-Reinhold, 1971). Forty-six of them are previously undescribed. The female of Leptonetela zhai Wang & Li, 2011 is reported for the first time. Leptonetela tianxinensis (Tong & Li, 2008) comb. nov. is transferred from the genus Leptoneta Simon, 1872;the genus Guineta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela; Leptonetela gigachela(Lin & Li, 2010) comb. nov. is transferred from Guineta. The genus Sinoneta Lin & Li, 2010 syn. nov. is a junior synonym of Leptonetela; Leptonetela notabilis(Lin & Li, 2010) comb. nov. and Leptonetela sexdigiti(Lin & Li, 2010) comb. nov. are transferred from Sinoneta; Leptonetela sanchahe Wang & Li nom. nov. is proposed as a replacement name for Sinoneta palmata(Chen et al, 2010) because Leptonetela palmata is preoccupied.


INTRODUCTION 1
Subterranean ecosystems, such as caves and cracks, are evident mainly in karst areas, which represent nearly 4% of the rocky outcrops of the world. These environments are marked by permanent darkness, a lack of diurnal and annual rhythms, and extremely scarce food sources (Culver & White, 2005;Howarth, 1983;Poulson & White, 1969). Many studies show that despite stressful and unfavorable conditions, the subsurface habitat harbors diverse animal communities (mainly invertebrates) (Amara-Zettler et al., 2002;Flot et al., 2010;López-García et al., 2001;Mathieu et al., 1997;Niemiller et al., 2012;Sket, 1999). Troglobionts are expected to adopt strategies that are characterized by significant geographic isolation and numerous local endemics (Convey, 1997;Waterman, 2001). Because the diversity of possible adaptive responses declines with stress intensity (Nevo, 2001), evolution in harsh environments is also expected to be influenced by convergence (Little & Vrijenhoek, 2003;Rothschild & Mancinelli, 2001;Waterman, 2001). Therefore, in subterranean, and more generally in extreme environments, diversification and speciation processes should be largely influenced by island-like habitats, such as caves, allopatric speciation and vicariant events, and could be masked by morphological convergence. For these groups of organisms, morphology alone cannot determine species boundaries, so identifying morphologically inseparable cryptic or sibling species requires an integrative approach that often includes DNA analysis.
DNA barcoding relies on the use of a standardized DNA region as a tag for accurate and rapid species identification (Hebert & Gregory, 2005) and has been used to help overcome the 'taxonomic impediment ' (Herbert et al., 2003a;Tautz et al., 2003). It aids in the identification of species in applied settings, the association of morphologically distinct life-cycle forms within a species, the detection of host-specific lineages and the detection of morphologically cryptic species (Miller & Foottit, 2009). DNA barcoding has been used in a diverse range of vertebrate and invertebrate taxa (Clare et al., 2007;Ratnasingham & Hebert, 2007) and has enabled an increasing number of taxa to be identified. For example, a survey of crustacean stygofauna suggests that there could be substantial levels of subterranean biodiversity hidden in Australia's acquifer (Asmyhr & Cooper, 2012). Nevertheless, the exclusive use of single-locus molecular gene fragments is not without risks, for identical mitochondrial DNA sequences can be present in unrelated species due to introgression, or incomplete lineage sorting (Ballard & Whitlock, 2004). Additionally, the use of a divergence threshold for distinguishing intra-vs. interspecific sequence variation (Hebert et al., 2003a) can seriously compromise species identification and suffers from severe statistical problems (Vences et al., 2005). Furthermore, species misidentification has been observed when a reference database is not comprehensive; such that is does not contain all the species of the group under study (Meyer & Paulay, 2005).
The South China karst, a United Nations Educational, Scientific and Cultural Organization (UNESCO) World Heritage Site since 2007, is noted for its karst features and landscapes as well as rich biodiversity. Numerous subterraean species have been reported in this region, especially invertebrate fauna (Zhang, 1986). The spider genus Leptonetela is discontinuously distributed in the South China karst and the Balkan Peninsula, a karstic region in Europe. The genus has 54 catalogued species (World Spider Catalog, 2017), and with one exception (L. pungitia , nearly all Leptonetela species are endemic to either a single cave or a cave system. The spiders are cave adapted as shown by morphological features, such as vestigial eyes and highly reduced skin pigmentation. Over the past nine years, we have conducted extensive surveys of subterranean biodiversity in Eurasia. More than 1 500 caves were visited, and we ultimately sampled 122 Leptonetela populations (caves). Rapid and accurate identification within this genus is difficult due to congeneric species sharing similar morphological traits, a lack of obvious morphological differences between closely related species and some species only differ in one or a few quantitative differences, such as the location, length ratio or thickness of the male pedipalpal tibial spines and the number of teeth on the median apophysis.
In this study, we test the usefulness of DNA barcoding for species identification in the subterranean genus Leptonetela and investigate the diversity of the genus. The standard molecular barcode, cytochrome c oxidase subunit I (COI) was used. A species discovery method, automatic barcode gap discovery (ABGD) (Puillandre et al., 2012), and a species validation method, DNA barcoding gap analysis, (Hebert et al., 2003b) were both used, depending on whether the samples were partitioned prior to analysis. The main goals of our study were: (i) to test whether the COI barcoding fragment can reliably resolve and identify subterranean Leptonetela species by comparing the COI barcode fragment results with those from morphological data; (ii) to test taxonomic value of morphological characters used in traditional methods of classification.

Taxon sampling
We sampled 624 Leptonetela individuals from 122 populations (caves) (Supplementary Table S1) in Eurasia (Insular and Peninsular Greece, and Southeast Asia; see inset in Figure 1). Nine individuals from three other genera of the family Leptonetidae were chosen as outgroups. All specimens were collected alive, fixed in absolute ethanol, and the legs were removed for subsequent DNA extraction. The remaining specimens were preserved in 80% ethanol for identification and morphological examination. Voucher specimens and all type specimens were deposited in the Institute of Zoology, Chinese Academy of Sciences (IZCAS), Beijing, China.

Molecular protocols
Total genomic DNA was extracted using the Animal Genomic DNA Isolation Kit (Dingguo, Beijing, China) following the manufacturer's protocol. We amplified the cytochrome c oxidase subunit I (COI) barcode region using the primer pairs LCOI490/HCO2198 (Folmer et al., 1994). PCR reaction conditions were: initial denaturation at 94 °C for 1 min; 35 cycles of denaturation at 94 °C for 1 min, annealing at 45 °C for 45 s, and elongation at 70 °C for 60 s; and a final extension at 72 °C for 5 min. The 25 μL PCR reactions included 17.25 μL of double-distilled H 2 O, 2.5 μL of 10× Taq buffer (mixed with MgCl 2 ; TianGen Biotech, Beijing, China), 2.0 μL of dNTP Mix (2.5 mmol/L), 1 μL of each forward and reverse 10 μmol/L primer, 1 μL of DNA template, and 0.25 μL Taq DNA polymerase (2.5 U/μL; TianGen Biotech, Beijing, China). Double-stranded PCR products were visualized by agarose gel electrophoresis (1% agarose). PCR products were purified and sequenced by Sunny Biotechnology Co., Ltd (Shanghai, China) using the ABI 3730XL DNA analyser. Sequences were aligned using ClustalW in Mega 6.0 (Tamura et al., 2013), with visual inspection, translation, and manual adjustment to minimize alignment error. The most appropriate phylogenetic model for the sequence alignment was selected using jModelTest2 (Darriba et al., 2012) under the Akaike Information Criterion (Posada & Crandall, 1998).

Phylogenetic analyses
Phylogenetic analyses were performed using maximum likelihood (ML) in RAXML v. 7.0.3 with the GTRCAT model (Stamatakis, 2006). One hundred replicate ML inferences were performed in the search for an optimal ML tree, each initiated with a

Species delineation
We analyzed the COI barcode dataset (see Supplementary Table  S1) using two species delineation methods. DNA barcoding gap analyses require an a priori species designation. Therefore, we divided the 624 Leptonetela individuals of 122 populations (caves) into 90 putative species based on morphological characters and geographic information. In our DNA barcoding gap analysis, we examined the overlap between the mean intraspecific and interspecific Kimura 2-parameter (K2P) (Kimura, 1980) and uncorrected p-distance (Nei & Kumar, 2000) for each candidate species, as calculated by Mega v. 6.0 (Tamura et al., 2013).

Taxonomy
The terminology and the measurements in this paper generally follow  and Ledford et al. (2011). All measurements were taken in millimetres (mm). The left palpi of male spiders are illustrated, except where otherwise indicated. Abbreviations used in text include: PL: prolateral lobe; E: embolus; C: conductor; MA: median apophysis; At: atrium; SS: spermathecae stalk; SH: spermathecae.

Nomenclatural acts
This article conforms to the requirements of the amended International Code of Zoological Nomenclature. All nomenclatural acts contained within this published work have been registered in ZooBank. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:7ECB1BDC-8893-4D0F-8BEA-17ECE327FC47

RESULTS
In total, 624 DNA barcodes were analyzed. A full list of the analyzed specimens can be found in Supplementary Table S1. Fragment lengths of the analyzed DNA barcodes ranged from 107 (0.005%) to 617 bp (89%). For all populations, except L. kanellisi and L. robustispina, four or more DNA barcodes were generated. All nucleotides were translated into functional protein sequences in the correct reading frame, with no stop codons or indels observed. Similar to other arthropod studies, our data indicated a high AT-content for this mitochondrial gene fragment: the mean sequence compositions were A=20.5%, C=12.6%, G=24.4%, T=41.4%.

Phylogenetic inference
The ML gene tree topology suggests that Leptonetela is monophyletic, with the node highly supported (Figure 2; bootstrap value, BS=92). Our analyses revealed all Leptonetela species formed non-overlapping clusters, with bootstrap support   values of 100. In contrast, relationships among putative species were largely unresolved, usually with low bootstrap support on the ML gene tree, particularly at deeper phylogenetic levels.

Figure 3 DNA barcoding for Leptonetela
Histograms show division of intraspecific (grey) and interspecific (black) COI sequence variation based on Kimira two-parameter (K2P, A) and uncorrected p-distance (B).

DISCUSSION
DNA barcoding is widely recognized as a useful tool for species identification across the animal kingdom (Chesters et al., 2012;. Our research represents an important step towards the application of DNA barcodes for identification of Leptonetela taxa, and for 119 taxa (97%), our data represent the first published DNA barcodes.
Classically, geographic isolation is considered a primary feature of troglobitic taxa (Hedin, 1997;Hedin & Thomas, 2010). Our DNA barcoding result is consistent with this view and similar to other DNA barcoding studies, in which COI showed high genetic structure between populations within species (Tavares et al., 2001).
Choosing appropriate thresholds that can delimit species is one of the main challenges and concerns for DNA barcoding research (Ferguson, 2002). Our DNA barcoding gap analysis shows an overlap in the range of intra-and interspecific COI sequence divergence. The interspecific genetic divergences between L. chuan Wang & Li sp. nov. and L. changtu Wang & Li  Etymology. The specific name refers to the type locality; noun.    Figure 4C-D): tibia with 2 large spines prolaterally, and 5 spines retrolaterally, I spine strong, conspicuous, tip bifurcated. Cymbium constricted medially, attaching to an earlobe-shaped process. Embolus triangular, bearing a basal tooth. Median apophysis sclerotized, divided into 4 pine needle like structures. Conductor membranous, reduced ( Figure 4B).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with shorter legs. Total length 2.27 ( Figure 5A-B Etymology. The specific name refers to the type locality; noun. Description. Male (holotype). Total length 2.28 ( Figure 6A Figure 6C-D): tibia with 5 slender spines prolaterally and 5 slender spines retrolaterally, with I spine longest. Cymbium not wrinkled, earlobe-shaped process small. Embolus triangular, prolateral lobe small, oval. Median apophysis sclerotized, divided into 2 pine needle like structures.

Diagnosis. This new species is similar to
Conductor broad, C shaped in ventral view ( Figure 6B).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with longer legs. Total length 2.76 ( Figure 7A-B  Figure 7C): spermathecae coiled, atrium triangular, anterior margin of atrium with short hairs. Etymology. The specific name refers to the type locality; noun.    Description. Male (holotype). Total length 2.25 ( Figure 8A Figure 8C-D): tibia with 5 long spines prolaterally and 5 spines retrolaterally, with tibia I spine longest. Cymbium not wrinkled, earlobe-shaped process small, cymbium double the length of bulb. Embolus triangular, prolateral lobe reduced. Median apophysis sclerotized, divided into 15 pine needlelike structures. Conductor reduced ( Figure 8B).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with shorter legs. Total length 2.50 ( Figure 9A-B  Figure 10C-D): tibia with 3 long spines prolaterally, and 5 spines retrolaterally, with I spine longest, tip bifurcated. Cymbium not wrinkled, earlobe-shaped process absent, cymbium double the length of bulb. Embolus spoon-shaped; prolateral lobe reduced. Median apophysis sclerotized, divided into 2 sharp pine needlelike structures. Conductor reduced ( Figure 10B).
Female (one of the paratypes). Similar to male in color and general features, but smaller and with shorter legs. Total length 2.10 ( Figure 11A-B Etymology. The specific name refers to the type locality; noun.    Description. Male (holotype). Total length 2.50 ( Figure 12A).       Figure 18B) (conductor C shaped in L. danxia, bamboo leaf-shaped in L. yaoi), from L. yaoi by the tibia I spine slender, asymmetrically bifurcated ( Figure 18C) (tibia I spine strong in L. yaoi); from L. danxia by the unwrinkled cymbium ( Figure 18C-D) (cymbium constricted and wrinkled at 1/3 in L. danxia).
Female. Similar to male in color and general features, but larger and with longer legs. Total length 2.50 ( Figure 19A-B  Figure 20D), conductor bamboo leaf-shaped in ventral view ( Figure 20B) Figure 20C-D): tibia with 3 long setae prolaterally, 5 spines retrolaterally, with I spine strong, longest, tip curved. Cymbium with no wrinkle medially, earlobe-shaped process small. Bulb with spoon-shaped embolus, prolateral lobe small. Median apophysis absent. Conductor bamboo leaf-shaped in ventral view ( Figure 20B).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.25 ( Figure 23A-B  Figure 24D), conductor C shaped ( Figure 24B) Figure 24C-D): femur with 4 spines ventrally, tibia with 2 long setae prolaterally, 2 long setae and 5 slender spines retrolaterally, the spines equally strong, spine I longest. Cymbium not wrinkled, earlobe-shaped process small. Embolus triangular, prolateral lobe reduced. Median apophysis absent. Conductor C shaped in ventral view ( Figure 24B).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 1.93 ( Figure 25A-B) Etymology. The specific name refers to the type locality; noun.
Diagnosis. This new species is similar to L. jiulong Lin & Li, 2010, but can be distinguished by the male pedipalpal tibia with 6 spines retrolaterally, tibia II spine thickest, I, II spines equally length, tibia II spine asymmetrically bifurcated ( Figure 26D), median apophysis absent, conductor reduced ( Figure 26B) (I, II spines equally strong, tibia II spine longest and bifurcate, tibia I spine half the length of II, median apophysis broad and smooth, conductor rugose, triangular in L. jiulong).
Female (one of the paratypes). Similar to male in color and general features, but smaller and with shorter legs. Total length 2.38 ( Figure 27A-B) Figure  27C): spermathecae coiled, atrium fusiform.
Female (one of the paratypes). Similar to male in color and general features, but smaller and with shorter legs. Total length 2.81 ( Figure 29A-B Figure 29C): spermathecae coiled, atrium fusiform, anterior margin of atrium with one large pointed process medially, and covered with short hairs.
Distribution. China (Guizhou). Etymology. The specific name refers to the type locality; noun.
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.15 ( Figure 31A-B Figure 31C):      spermathecae coiled, atrium semicircular, anterior margin of atrium with one pointed process medially, and covered with short hairs.  Figure  32D), conductor bamboo leaf-shaped in ventral view ( Figure  32B) (tibia with 1 long seta, 6 spines retrolaterally in L.  Figure 32C-D): tibia with 6 spines retrolaterally, arranged equidistantly. Embolus triangular, prolateral lobe absent. Median apophysis absent. Conductor bamboo leaf-shaped in ventral view ( Figure 32B).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with shorter legs. Total length 2.88 ( Figure 33A-B  Figure 34D), conductor C shaped ( Figure 34B) (tibia I spine strong, conductor triangular in L. chiosensis; tibia spines slender, cymbium with 1 strong spine on the earlobeshaped process, conductor reduced, embolus with 1 tooth basally in L. panbao Wang & Li sp. nov.).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with shorter legs. Total length 1.95 ( Figure 35A-B Figure 36D), conductor reduced, embolus with 1 tooth basally ( Figure 36B) (tibia I spine stronger than other spines, conductor triangular in L. chiosensis; tibia I, II and III spines equally strong, stronger than other spines, conductor C shaped in L. panbao Wang & Li sp. nov.).

Female (one of the paratypes).
Similar to male in color and general features, but larger and with shorter legs. Total length 2.50 ( Figure 37A-B Figure 37C): spermathecae coiled, atrium triangular, anterior margin of atrium covered with short hairs. Etymology. The specific name refers to the type locality; noun.

Female (one of the paratypes).
Similar to male in color and general features, but smaller and with shorter legs. Total length 2.13 ( Figure 39A-B Etymology. The specific name refers to the type locality; noun.   Figure 40C-D): tibia with 5 slender spines retrolaterally, spines I, II and III equally strong, stronger than other spines, spine I longest. Cymbium constricted medially, retrolaterally attaching to 1 curved spine and an earlobe-shaped process, with 1 short spine. Embolus triangular, and prolateral lobe absent. Median apophysis shaped like pointed process, with 3 sclerotized spots distally. Conductor C shaped ( Figure 40B).

Female (one of the paratypes).
Similar to male in color and general features, but smaller and with shorter legs. Total length 2.30 ( Figure 41A-B). Carapace 0.93 long, 0.80 wide.      Figure 42D) (tibia with 5 spines retrolaterally, spine I strong and longest in L. liping, 6 slender spines in L. parlonga); median apophysis triangular ( Figure 42B) (median apophysis like pointed process in L. liping; ligulate in L. parlonga); from L. parlonga by the cymbium retrolaterally with 1 horn-shaped spine on the earlobe-shaped process in L. parlonga.

Female (one of the paratypes).
Similar to male in color and general features but larger and with shorter legs. Total length 2.38 ( Figure 43A-B Etymology. The specific name refers to the type locality; noun. Diagnosis. This new species is similar to L. rudicula , but can be distinguished by the male pedipalpal tibia with 6 spines retrolaterally ( Figure 44D), prolateral lobe indistinct ( Figure 44C), conductor broad and long, distal edge undulate ( Figure 44B) (5 spines retrolaterally, prolateral lobe oval, conductor short, C shaped in L. rudicula).
Female (one of the paratypes). Similar to male in color and general features, but smaller and with shorter legs. Total length 1.93 ( Figure 45A-B Etymology. The specific name refers to the type locality; noun.      Diagnosis. This new species can be distinguished from all other species of the genus by the male pedipalpal cymbium with one curved, short spine medially in retrolateral view, median apophysis triangular, spermathecae not tightly twisted, just spiraled in the female. Description. Male (holotype). Total length 2.13 ( Figure 46A) Figure 46C-D): tibia with 3 long setae prolaterally, and 5 slender spines retrolaterally, spine I longest. Cymbium not wrinkled, earlobe-shaped process indistinct, and with 1 curved, short spine retrolaterally. Bulb with spoon-shaped embolus, prolateral lobe indistinct. Median apophysis triangular in ventral view. Conductor thin, triangular in ventral view ( Figure 46B).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 1.70 ( Figure 51A-B Figure 52D), (tibia with 4 long setae prolaterally, 5 slender spines retrolaterally, with spine I longest, spines II III equal length, conductor thin, triangular in L. lujia Wang & Li sp. nov.; tibia with 3 long setae prolaterally, 1 long seta and 5 spines retrolaterally, with spine I strongest, tip asymmetrically bifurcated, conductor broad, distal edge undulate in L. jinsha; tibia with 3 long setae prolaterally, 6 slender spines retrolaterally, with spine I longest, conductor semicircular in L. quinquespinata; tibia with 4 slender spines prolaterally, 5 slender spines retrolaterally, with spines I, II equal length, cymbium with 2 long, curved spines on earlobe-shaped process retrolaterally, conductor semicircular in L. gubin Wang & Li sp. nov.); from L. gubin and L. quinquespinata by the base of median apophysis slightly swollen ( Figure 52B) (base of median apophysis distinctly swollen, 4 times the width of tip in L. gubin Wang & Li sp. nov.; 3 times in L. quinquespinata).
Female (one of the paratypes): similar to male in color and general features, but with a larger body size and shorter legs. Total length 1.95 ( Figure 53A-B Etymology. The specific name refers to the type locality; noun. Diagnosis. This new species is similar to L. shibingensis and L. wuming Wang & Li sp. nov., but can be distinguished by the median apophysis index finger-like in prolaterally view, tip bifurcated ( Fig. 54D ) (median apophysis small, triangular in    Description. Male (holotype). Total length 2.07 ( Figure 54A) Figure  54C-D): tibia with 4 long setae prolaterally, 5 large spines retrolaterally, with I spine strong, located medially. Cymbium constricted medially, attaching an earlobe-shaped process retrolaterally. Embolus triangular, bearing a basal tooth, prolateral lobe oval. Median apophysis index finger-like in prolateral view, tip bifurcated. Conductor bamboo leaf-shaped in ventral view ( Figure 54B).

Female (one of the paratypes).
Similar to male in color and general features, but smaller and with shorter legs. Total length 2.02 ( Figure 55A-B Figure 55C): spermathecae coiled, atrium fusiform. Etymology. The specific name refers to the type locality; noun.

Diagnosis.
This new species is similar to L. kangsa Wang & Li sp. nov., and L. shibingensis but can be distinguished by the male pedipalpal bulb embolus without basal tooth ( Figure 56B), (embolus with basal tooth in the two species mentioned above); from L. kangsa Wang & Li sp. nov. and L. shibingensis by the tibia spine I located at the base of the tibia ( Figure 56D) (tibia spine I located medially in L. kangsa Wang & Li sp. nov. and L. shibingensis).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 3.02 ( Figure 57A-B Figure 57C): spermathecae coiled, atrium triangular.          Etymology. The specific name refers to the type locality; noun.

Leptonetela shanji
Diagnosis. This new species is similar to L. penevi Wang & Li, 2016 and L. changtu Wang & Li sp. nov. but can be distinguished by on the male pedipalpal bulb median apophysis long, and half the length of bulb ( Figure 64B) (median apophysis short, 1/5 the length of bulb in L. palmate, and L. changtu Wang & Li sp. nov.); male pedipalpal tibia spines slender, equally strong ( Figure 64D) (tibia spines I II equally strong, stronger than other spines in L. penevi, tibia spines I II III equally strong, stronger than other spines in L. changtu Wang & Li sp. nov.).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.08 ( Figure 65A-B Etymology. The specific name refers to the type locality; noun. Diagnosis. This new species is similar to L. tianxingensis, but can be distinguished by on the male pedipalpal bulb conductor longer than median apophysis ( Figure 66B) (conductor shorter than median apophysis in L. tianxingensis); male pedipalpal tibia III spine strong ( Figure 66D) (tibia III spine slender in L. tianxingensis).
Female (one of the paratypes): similar to male in color and general features, but with a larger body size and longer legs. Total length 1.98 ( Figure 67A-B  Figure 67C): spermathecae coiled, atrium triangular.      Figure 68C-D): tibia with 5 large spines retrolaterally, tibia spine I longest, spines I and II equally strong, stronger than others. Cymbium not constricted. Embolus triangular, prolateral lobe oval. Median apophysis palm-shaped, teeth of median apophysis reduced to sclerotized spots. Conductor semicircular ( Figure 68B).

Leptonetela changtu
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.70 ( Figure 69A-B Etymology. The specific name refers to the type locality; noun.

Diagnosis.
This new species is similar to L. niubizi Wang & Li sp. nov. but can be distinguished by the male pedipalpal tibia with 5 spines retrolaterally, with I spine strongest, tip bifurcated, the other 4 spines slender, 2 of them longer than I spine ( Figure  70D); tip of median apophysis with 5 small teeth, and 1 ox hornshaped large teeth ( Figure 70B) Figure 70C-D): tibia with 3 long setae prolaterally, and 5 spines retrolaterally, with spine I strongest, tip bifurcated, and the other 4 spines slender, 2 of them longer than spine I. Cymbium constricted medially, attached to an earlobe-shaped process retrolaterally. Embolus triangular, prolateral lobe absent. Tip of median apophysis with 5 small teeth, and 1 horn-shaped large teeth. Conductor broad C tile-shaped in ventral view ( Figure  70B).
Female (one of the paratypes). Similar to male in color and general features, but with a larger body size and shorter legs. Total length 2.60 ( Figure 73A-B Figure 73C): spermathecae coiled, atrium triangular.        Figure 74C-D): tibia with 2 spines prolaterally and 5 spines retrolaterally, with spine I strongest, tip asymmetrically bifurcated. Cymbium constricted medially, attaching a small earlobe-shaped process retrolaterally. Embolus triangular, prolateral lobe oval. Median apophysis short, palmshaped, distal edge with 5 small teeth. Conductor C tile-shaped in ventral view, tip of conductor undulate ( Figure 74B).

Distribution. China (Guizhou).
Female (one of the paratypes). Similar to male in color and general features, but larger and with longer legs. Total length 2.05 ( Figure 75A-B Figure 76C-D): tibia with 2 long setae prolaterally, and 5 spines retrolaterally, with spine I strongest, tip asymmetrically bifurcated. Cymbium constricted medially, attaching a small earlobe-shaped process retrolaterally. Embolus triangular, prolateral lobe indistinct. Median apophysis harrow-like, with 10 small teeth distally. Conductor smooth, C tile-shape in ventral view ( Figure 76B).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.60 ( Figure 77A-B Figure 78C-D): tibia with 3 long setae prolaterally, 5 large spines retrolaterally, with spine I strongest, tip asymmetrically bifurcated. Cymbium not constricted, earlobe-shaped process absent. Embolus triangular, prolateral lobe absent. Median apophysis with 6 needle-shaped teeth distally. Conductor C tileshape in ventral view ( Figure 78B).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 1.70 ( Figure 79A-B Figure 79C): spermathecae coiled, atrium fusiform, anterior margin of atrium with one mastoid process medially. sharp teeth distally ( Figure 80B), tibia II spine strongest ( Figure  80C) (tibia spine I strongest, tip asymmetrically bifurcated, median apophysis with 6 small teeth distally in L. sexdentata and L.  Figure 80B).

Leptonetela meiwang
Female. Similar to male in color and general features, but larger and with shorter legs. Total length 1.75 ( Figure 81A-B Figure 82B) (median apophysis with 6 teeth distally in L. arvanitidisi, 3 teeth in L. paragamiani and 5 teeth in L. erlong Wang & Li sp. nov.); tibia spine I strongest, tip asymmetrically bifurcated, spines II, III equally strong, stronger than other 2 ( Figure 82C) (tibia II-V spines slender, curved, and equally strong in L. arvanitidisi and L. erlong Wang & Li sp. nov., tibia III-V spines equally strong, slender than II spine in L. paragamiani); from L. arvanitidisi by    the conductor C tile-shaped ( Figure 82B) (conductor triangular in L. arvanitidisi).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.00 ( Figure 83A-B Etymology. The specific name refers to the type locality; noun. Diagnosis. This new species is similar to L. arvanitidisi Wang & Li, 2016, L. paragamiani Wang & Li, 2016 and L. tawo Wang & Li sp. nov. but can be distinguished by on the male pedipalpal bulb median apophysis with 5 teeth distally ( Figure 84B) (median apophysis with 6 teeth in L. arvanitidisi, 4 teeth in L. tawo Wang & Li sp. nov. and 3 teeth L. paragamiani); from L. paragamiani and L. tawo Wang & Li sp. nov. by the tibia spines II-V slender, curved, and equally strong ( Figure 84D) (tibia spines II, III equally strong, stronger than other 2 in L. tawo Wang & Li sp. nov., spines III-V equally strong, more slender than spine II in L. paragamiani); from L. arvanitidisi by the conductor C tile-shaped ( Figure 84B) (conductor triangular in L. arvanitidisi).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.30 ( Figure 85A-B Etymology. The specific name refers to the type locality; noun.
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.40 ( Figure 87A-B Etymology. The specific name refers to the type locality; noun. Diagnosis. This new species is similar to L. thracia  and L. dabian Wang & Li sp. nov., but can be distinguished by the male pedipalpal tibia with 7 long setae prolaterally, 5 slender spines retrolaterally, with spines I, II, III equally strong, stronger than others ( Figure 88D) (tibia with 4 long setae prolaterally, 5 slender spines retrolaterally, with spines I, II equally strong, stronger than others in L. thracia; 3 slender spines prolaterally, 5 slender retrolaterally spines equally strong in L. dabian Wang & Li sp. nov.); tip of median apophysis bent downwards, with 5 larger teeth distally ( Figure  88B) (tip of median apophysis not bent, with 4 teeth distally in L. thracia; tip of median apophysis bent upwards, with 3 larger teeth distally in L.  Figure 88C-D): tibia with 7 long setae prolaterally, 5 large spines retrolaterally, with spines I, II, III equally strong, stronger than others. Cymbium constricted medially, attached to a small earlobe-shaped process retrolaterally. Embolus triangular, prolateral lobe oval. Median apophysis bent downwards, with 5 larger teeth distally. Conductor triangular in ventral view ( Figure 88B).
Female: Similar to male in color and general features, but smaller and with shorter legs. Total length 2.08 ( Figure 89A-B Figure 90D) (tibia spine I trifurcate in L. notabilis); from L. shuang Wang & Li sp. nov. by the conductor C tile-shaped, distal edge of median apophysis with 6 teeth ( Figure 90B) (conductor triangular, distal edge of median apophysis with 7 teeth in L. shuang Wang & Li sp. nov.); from L. sexdigiti by the strongly twisted spermathecae (spermathecae loosely twisted in L. sexdigiti).
Female (one of the paratypes). Similar to male in color and general features, but larger and with shorter legs. Total length 2.50 ( Figure 91A-B Figure 91C): spermathecae coiled, atrium fusiform.

Remarks.
Our research showed that this species should be transferred to the genus Leptonetela, based on the result of DNA barcoding and morphological characters such as the pedipalpal femur lacking spines and the tibia with one strong spine retrolaterally.