Three new species of the segmented spider genus Qiongthela (Mesothelae, Liphistiidae) from Hainan Island, China

Abstract We report three new species of the segmented trapdoor spider genus Qiongthela Xu & Kuntner, 2015 collected from Hainan Island, China based on morphological characters: Q. dongfangsp. nov. (♂♀), Q. nankaisp. nov. (♂♀), Q. yalinsp. nov. (♂♀). We also provide the GenBank accession codes of the DNA barcode gene, cytochrome c oxidase subunit I (COI), of the type specimens of all three new species to aid future identification.

When examining the specimens collected from Hainan Island, we diagnosed three new Qiongthela species. Here, we describe these new species based on genital morphology of both males and females. Furthermore, we provide the genetic distances of intraspecific and interspecific relationships with the closest species based on the DNA barcode gene, cytochrome c oxidase subunit I (COI), to support our descriptions, and also provide the COI sequences of type specimens for future identifications.

Materials and methods
All specimens in this study were collected from Hainan Island, China. We collected them alive, checked for their maturity status, removed the right four legs of adult specimens, preserved the legs in 100% ethanol, and kept them at -80 °C for molecular work. The remains were preserved in 80% ethanol as vouchers for morphological identification and examination. We took juvenile/subadult males back to the laboratory, reared them until they reached sexual maturity, removed the right four legs, and preserved them as described above. All the types and voucher specimens are deposited at the College of Life Sciences, Hunan Normal University, Changsha, Hunan Province, China.
We examined and dissected the specimens using an Olympus SZX16 stereomicroscope. We removed the soft tissues of female genitalia using 10 mg/ml trypsase (Bomei Biotech Company, Hefei, Anhui, China) for at least 3 hours at room temperature. We took photographs of male and female genitals under an Olympus BX53 compound microscope using a CCD digital camera. We conducted all measurements using an MC170HD digital camera mounted on a Leica M205C stereomicroscope and presented the measurements in millimeters. Leg and palp measurements are given in the following order: leg total length (femur + patella + tibia + metatarsus + tarsus), palp total length (femur + patella + tibia + tarsus).
We extracted total genomic DNA from spider legs using the Animal Genomic DNA Isolation Kit (Kangwei Biotech, Beijing, China) following the manufacturer's protocols. We used the primer pair LCO1490/HCO2198 (Folmer et al. 1994) to amplify COI sequences under the following PCR reaction protocol: initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 1 min, annealing at 40 °C for 1 min, and elongation at 72 °C for 30 s; and final extension at 72 °C for 7 min (Xu et al. 2015c). The 25 μl PCR reactions consisted of 12.5 μl of 2×Taq MasterMix (KangWei Biotech, Beijing, China), 1 μl of each forward and reverse 10 μM primer, 1 μl of genomic DNA, and 9.5 μl of double-distilled H 2 O. The PCR products were examined using agarose gel electrophoresis (1% agarose). All PCR products were purified and sequenced at Tsingke Biotechnology Company (Changsha, China). We downloaded all the COI sequences of known Qiongthela species from NCBI and calculated genetic distances based on the standard DNA barcode alignment using MEGA v6.0 (Tamura et al. 2013).
Female genitalia. Two pairs of similar-sized receptacular clusters along the anterior margin of the bursa copulatrix, with short genital stalks ( Fig. 3H-K).
Variation. Males and females vary in body size. Range of measurements in males (N = 2): BL 10. in  Remarks. The maximum and mean intraspecific genetic distances of Q. dongfang sp. nov. are 0.3% and 0.2% based on Kimura 2-parameter (K2P) model, respectively (N = 5). We calculated the interspecific genetic distance between the holotypes of the two closest species. The genetic distances between Q. dongfang sp. nov. and Q. jianfeng (GenBank accession code: KP229838 (paratype); we used the sequence of paratype because the DNA barcodes of the holotype and paratype are identical; Xu et al. 2017), Q. nankai sp. nov., and Q. yalin sp. nov. are 7.7%, 15.7%, and 9.1% based on K2P, respectively. Diagnosis. Males of Q. nankai sp. nov. can be distinguished from those of Q. qiongzhong, Q. yalin sp. nov. and Q. yinggezui by the straight tegular marginal apophysis (Fig. 5A, D), and the cymbial projection short and thick (Fig. 5G); from those of Q. australis by the conductor with a bent apex (Fig. 5C, E, G), and the longer tegular marginal apophysis (Fig. 5A, C, D, F); from those of Q. dongfang sp. nov., Q. jianfeng and Q. sanya by the tegular terminal apophysis with an abruptly narrowed and hooked apex ( Fig. 5A-E); from those of Q. nui by the tegular marginal apophysis with a blunt edge ( Fig. 5A-G); from those of the rest of Qiongthela species by the contrategulum with two distal edges (Fig. 5A, D). Females of Q. nankai sp. nov. differ from those of Q. dongfang sp. nov. and Q. wuzhi by the middle receptacular clusters situated close to each other (Fig. 5H-K); from those of Q. bawang and Q. qiongzhong by the two pairs of receptacular clusters similar in size and shape (Fig. 5H-K); from those of the rest of Qiongthela species by similar-sized receptacular clusters, and all situated along the anterior margin of the bursa copulatrix ( Fig. 5H-K).
Palp. Cymbium with a short projection dorsally (Fig. 5G); paracymbium unpigmented and unsclerotised prolaterally, with numerous setae at the tip (Fig. 5A-C). Contrategulum with an irregular dentate edge proximally and two edges distally: the inner one dentate, and the outer one sharp, semi-translucent (Fig. 5A, D, F). Marginal apophysis of tegulum long, with a blunt apex distally, distal portion of similar width as basal portion (Fig. 5A, D), a proximally directed terminal apophysis of the tegulum with few denticles, abruptly narrowing to a hooked apex (Fig. 5A-E). Conductor situated ventro-proximally on the embolus, the basal portion fused with the embolus, distal portion free, narrowing to a bent apex (Fig. 5A-E). Embolus largely sclerotised, smooth ventrally, with several longitudinal ribs retrolaterally, and with a wide, flat opening of the sperm duct distally (Fig. 5A, D, E).
Female genitalia. Two pairs of receptacular clusters along the anterior margin of the bursa copulatrix, of similar size and shape, and the middle ones close to each other, with very short genital stalks (Fig. 5H-K).
Etymology. The species epithet, a noun in apposition, refers to the type locality. Diagnosis. Males of Q. yalin sp. nov. resemble those of Q. sanya, but can be distinguished from the latter by the narrower conductor base (Fig. 7A, D) and by the cymbium with a longer and more slender projection (Fig. 7G); from those of Q. dongfang sp. nov. by the tegular marginal apophysis slightly longer and with a blunt apex (Fig. 7A, D), and the cymbium with an elongated projection (Fig. 7G); from those of the rest of Qiongthela species by the contrategulum with a smooth edge proximally (Fig. 7F, G). Females of Q. yalin sp. nov. differ from those of Q. sanya by the middle receptacular clusters having short, indistinct genital stalks (Fig. 7I, L); from those of Q. australis, Q. yini and Q. yinggezui by the smaller middle receptacular clusters compared with the lateral ones ( Fig. 7H-M); from those of the rest of Qiongthela species by the middle receptacular clusters situated along the anterior margin of the bursa copulatrix, the laterals located slightly on the dorsal wall of the bursa copulatrix, and the trapezoidal bursa copulatrix (Fig. 7H-M).
Palp. Cymbium with a slender projection dorsally (Fig. 7G); paracymbium unpigmented and unsclerotised prolaterally, with numerous setae at the tip ( Fig. 7A-C). Contrategulum with a smooth edge proximally and two edges distally: the inner one with fine, small denticles, the outer one smooth, sharp, semi-translucent, fused with the inner at the middle of the contrategulum (Fig. 7A, D, F). Tegulum with a long, slightly curved, distally blunt marginal apophysis (Fig. 7A, D), the proximally directed terminal apophysis with a dentate margin, continuously narrowing to a rounded, hooked apex ( Fig. 7A-E). Conductor situated ventro-proximally on the embolus, fused with the embolus at the basal portion, distal portion free, narrowing to a slightly bent apex ( Fig. 7A-E). Embolus largely sclerotised, with a wide, flat opening of the sperm duct distally, ventrally smooth, retrolaterally with several longitudinal ribs (Fig. 7B, C, E).
Female genitalia. The middle receptacular clusters along the anterior margin of the bursa copulatrix, the lateral ones located slightly on the dorsal wall of the bursa copulatrix; the middle ones smaller than the lateral ones, with indistinct genital stalks; bursa copulatrix trapezoidal (Fig. 7H-M).
Etymology. The species epithet, a noun in apposition, refers to the type locality.  Remarks. Both maximum and mean intraspecific genetic distances of Q. yalin sp. nov. are 0% based on K2P (N = 5). The interspecific genetic distance between Q. yalin sp. nov. and Q. sanya (GenBank accession code: MN911990) is 7.2% based on K2P.