Asian Herpetological Research 2015, 6(4): 245–256 ORIGINAL ARTICLE DOI: 10.16373/j.cnki.ahr.140097
A New Species of the Genus Tylototriton (Amphibia: Urodela: Salamandridae) from Eastern Himalaya
Janak Raj KHATIWADA1, 2**, Bin WANG1**, Subarna GHIMIRE3, Karthikeyan VASUDEVAN4, Shanta PAUDEL5 and Jianping JIANG1*
1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China 2 University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China 3 Tribhuvan University, Central Department of Zoology, Kirtipur, Kathmandu, Nepal 4 CSIR-Centre for Cellular and Molecular Biology (CCMB), Laboratory for the Conservation of Endangered Species, Uppal Road, Hyderabad 500048, India 5 Himalayan Research and Conservation Nepal, GPO Box 8975 EPC 5997, Kathmandu, Nepal
Abstract A new species of the genus Tylototriton is described from eastern Himalaya based on molecular and morphological comparisons. The new species is diagnosable from the closely-related species by having light brown colouration in dorsal region in life, flat and blunt snout, greatly separated dorsolateral bony ridges on head and straight- thick tailfin. In addition to head morphology, the new species is also morphologically distinguishable from its closely- related species Tylototriton shanorum by having 16 dorsal warts and average smaller Snout Vent Length (SVL).
Keywords Tylototriton himalayanus sp. nov., eastern Himalaya, morphology, molecular phylogeny, taxonomy
1. Introduction some cryptic species are still need to be confirmed (Nishikawa et al., 2013a; Nishikawa et al., 2014; Zhang The salamandrid genus Tylototriton Anderson, 1871 et al., 2013). Some of the newly-described species are ranges across eastern Himalaya, Indochina and South found on the edge of the distribution of Tylototriton, China, and presently includes 21 species (Fei et al., for example, T. notialis, T. uyenoi and T. shanorum are 2012; Frost, 2015; Le et al., 2015). The genus is divided from the southern distributional margin of this genus into two subgenera, subgenus Tylototriton regarded as (Nishikawa et al., 2013a; Nishikawa et al., 2014; T. verrucosus species group and subgenus Yaotriton Stuart et al., 2010). Eastern Himalaya is reported as the referred as T. asperrimus species group (Dubois and westernmost distributional edge of the genus Tylototriton. Raffaëlli, 2009; Fei et al., 2005). Noticeably, 15 species Tylototriton salamanders here have been named as T. have been newly described in recent six years (Chen et verrucosus but found genetically differentiated from al., 2010; Fei et al., 2012; Hou et al., 2012; Le et al., other populations of Tylototriton (Weisrock et al., 2006; 2015; Nishikawa et al., 2013a, b; Nishikawa et al., 2014; Nishikawa et al., 2014). These reports indicated that Phimmachak et al., 2015; Shen et al., 2012; Stuart et the Himalayan population of this genus was probably a al., 2010; Yang et al., 2014; Zhao et al., 2012), which new species. However, this conclusion was supported by greatly promote species diversity of Tylototriton. But, only one sample from Nepal without any morphological * Corresponding author: Prof. Jianping JIANG, from Chengdu Institute description. of Biology, Chinese Academy of Sciences, Chengdu, China, with his research focusing on taxonomy, systematics and evolution of amphibians This study, therefore, was carried out in the Illam and reptiles. District, eastern Nepal and Darjeeling, northeastern E-mail: [email protected] ** These authors contributed equally to this work. India in 2011–2012. Three populations of Tylototriton Received: 25 December 2014 Accepted: 27 July 2015 from eastern Nepal and one from northeastern India 246 Asian Herpetological Research Vol. 6 were surveyed. In this study, mitochondrial DNA haplotypes of the ND2 gene sequence dataset. MP and morphological characters were used to evaluate analysis, which was implemented in PAUP version the taxonomic status of these Tylototriton populations 4.0b10 (Swofford, 2003), involved heuristic searches of (Figure 1). 1 000 random-addition replicates using tree bisection- reconnection (TBR) branch swapping. Bootstrap supports 2. Materials and Methods (bs) for nodes of the resulting MP tree were assessed by analyses of 1 000 bootstrap replicates. For Bayesian 2.1 Molecular analysis We obtained tissue of 39 analyses, the best-fit substitution model was selected Tylototriton salamanders from three sites in Illam District, under the Bayesian Information Criterion by the program Mechi of Nepal, one sample from Darjeeling, India and jModeltest 2.1.4 (Darriba et al., 2012). The best-fit one sample of T. verrucosus from the type locality Husa, substitution model for the ND2 dataset was GTR + I + G. Longchuan County, Yunnan Province of China (Table 1; BI analyses were conducted in the program Mrbayes 3.1.2 Figure 1). (Ronquist and Huelsenbeck, 2003). Two dependent runs Total genomic DNA was extracted from muscle were initiated each with four simultaneous Markov Chain or liver preserved in 95% ethanol using the DNeasy Monte Carlo (MCMC) chains for 30 million generations Tissue Kit (QIAGEN). Fragments of the complete and sampled every 1 000 generations. The convergence NADH dehydrogenase subunit 2 (ND2) gene and part of of chains and burn-in period of all runs were examined cytochrome b (cytb) gene were amplified and sequenced. by plots of log-likelihood scores and low standard The primers and PCR conditions for amplifying cytb deviation of split frequencies, then we discarded the first gene fragment were chosen according to Moritz et al. 25% generations as burn-in, and then used the remaining (1992), and those for amplifying ND2 gene according to trees to create a 50% majority-rule consensus tree and Shen et al. (2012). PCR products were sequenced with to estimate Bayesian posterior probabilities (BPP). To bi-directional sequencing using primers for PCR. New further visualize the degree of genetic splits among the sequences were deposited in GenBank under accession Himalayan population and the closely-related species, we numbers KT765132–KT765212. reconstructed phylogenetic network using Splittree 4.11.3 For phylogenetic analysis, we downloaded ND2 gene (Huson and Bryant, 2006) based on cytb gene sequence sequences of 33 samples of 19 known Tylototriton species dataset, and the supports of lineages were evaluated by from GenBank (Table 1). We also downloaded ND2 gene 1 000 bootstrap replicates. sequences of each species of Echinotriton andersoni and 2.2 Morphological analyses Following the work by Pleurodeles waltl from Genebank as outgroups according Nishikawa et al. (2014), eastern Himalayan Tylototriton to Zhang et al. (2008). GenBank accession numbers of was regarded as the sister lineage to T. shanorum from all ND2 sequences used here are presented in Table 1. Myanmar. So we firstly compared the morphological For further investigation of the genetic split between the characters of Himalayan specimens with the holotype Himalayan population and its congeners, we downloaded T. shanorum as described by Nishikawa et al. (2014). cytb gene sequences of 146 individuals of 19 T. shanjing Furthermore, Himalayan salamander was previously populations (Yu et al., 2013), 3 samples of T. shanorum, regarded as T. verrucosus (Frost, 2015). So we also and 1 for each of T. kweichowensis and T. taliangensis compared the morphology between the Himalayan (GenBank accession numbers of them are JX854457– population and topotypic T. verrucosus from the type JX854515, JX854517, JX444703, EF627452 and locality (Husa, Longchuan County, Yunnan Province of EF627455). China). Similarly, considering the taxonomic debate and All DNA sequences were aligned using Clustal X confusion between T. verrucosus and T. shanjing, we also 1.83 (Thompson et al., 1997) with default settings and included in our morphological comparison T. shanjing manually adjusted. Pairwise divergences (uncorrected samples from the type locality (Jingdong County, Yunnan p-distance) between species on ND2 dataset were Province of China) and Baoshan population from Baoshan determined in MEGA 6.0.5 (Tamura et al., 2013). City, Yunnan Province of China. Haplotypes were identified using DAMBE 5.3.109 (Xia, A total of 52 adult males and 39 adult females were 2013). included for morphological comparisons. Among Maximum parsimony (MP) and Bayesian inference them, 13 females and 32 males were from Himalayan (BI) analyses were conducted to reconstruct the population. Out of total 45 samples, only 39 samples phylogenetic relationships among the taxa based on were used for molecular analysis. The information for No. 4 Janak Raj KHATIWADA et al. A New Species of Tylototriton 247
Figure 1 Localities for samples used in this study of Tylototriton. Sample numbers were given in Table 1. The samples without locality information were not shown on the map. Asterisk denotes the type locality of T. himalayanus sp. nov. specimens used in morphological comparison is given knee); LF (distance from knee to tip of longest toe). The in Table 3. Specimens were deposited in Natural History sex and maturity status were identified by checking the Museum, Tribhuvan University, Soyambhu, Kathmandu, state of cloaca (Anders et al., 1998; Schleich and Kästle, Nepal (NHMTU) and Chengdu Institute of Biology, 2002; Sparreboom, 2013). Morphological measurements Chinese Academy of Sciences, Chengdu, China (CIB, were made using a digital dial caliper to the nearest 0.01 CAS; Appendix 1). The following 21 measurements mm and weights of individual species were measured by were taken for morphometric analyses: total length (TL, digital weight machine to the nearest 0.01 gm. from tip of snout to tip of tail); snout-vent length (SVL, Morphological differences between populations were from tip of snout to posterior edge of vent); head length examined by Student’s t-test (for parametric data) and (HL, from jugular fold to snout tip); head width (HW, Mann-Whitney U Statistic (for non-parametric data), maximum head width); snout length (SL, from tip of based on SVL and 20 ratio values to SVL (R, %). Based snout to the anterior corner of eye); lower jaw length on 20 log10-transformed values of the metric characters, (LJL, from jaw angle to tip of lower jaw); eyelid-nostril Principal Component Analysis (PCA) was used to see the overall morphological variation by BiodiversityR package length (ENL, minimum distance between eyelid and (Kindt and Coe, 2011) in R 2.13.1 (R Development nostril; internarial distance (IND, minimum distance Core Team, 2009). In the Nepal Himalaya population, between the external nares); upper eyelid width (UEW, female group were significantly larger than male group greatest width of upper eyelid); upper eyelid length (UEL, in total length (Mann-Whitney U Statistic = 60.000, P < greatest length of upper eyelid); axilla-groin distance 0.001) and SVL (Mann-Whitney U Statistic = 125.000, (AGD distance between axilla and groin); trunk length P = 0.039). Therefore, we separately analyzed the (TRL, from gular fold of throat to anterior tip of vent); male and female groups. Indian specimens collected tail length (TAL: from anterior tip of cloaca to tip of tail): from Darjeeling District were not included for further vent length (VL: maximum length of cloacal opening); morphological comparisons due to lack of information basal tail width (BTAW: tail width measured at the base about male and female populations. of tail); medial tail width (MTAW: tail width measured at the medial section of tail); basal tail height (BTAH: tail 3. Results height measured at the base of tail); medial tail height (MTAH: tail height measured at the medial section of 3.1 Molecular analyses Aligned dataset of ND2 tail); length of arm (LA: distance from axilla to tip of contained 1 035 bps including 447 variable sites and elbow); LH (length of hand: distance from elbow to 273 parsimony informative sites, and the dataset of longest finger); LT (length of tibia: distance from groin to cytb contained 753 bps including 157 variable sites 248 Asian Herpetological Research Vol. 6
Table 1 Voucher specimens, localities and GenBank accession numbers for samples used in the molecular phylogenetic analyses based on ND2 dataset.
Sample ID Species Voucher Locality GenBank accession No. Ingroup 1 T. asperrimus CIB-200807055 Jinxiu County, Guangxi Province, China KC147815 2 T. notialis FMNH 271120 Boualapha, Khammouan, Laos HM462061 3 T. hainanensis MVZ230352 Jianfengling, Hainan Province, China DQ517850 4 T. vietnamensis ROM 35364 Quang Thanh, Cao Bang Province, Vietnam HM462056 5 T. wenxianensis CIB20090527 Wenxian County, Gansu Province, China KC147813 6 T. broadoridgus CIB-200085 Sangzhi County, Zhangjiajie, Hunan Province, China KC147814 7 T. dabienicus HNNU1004II024 Dabie Mountains, Anhui Province, China KC147812 8 T. liuyangensis CSUFT 20100108 Liuyang County, Hunan Province, China KJ205598 9 T. lizhenchangi KUHE 42316 Yizhang, Hunan Province, China AB769532 10 T. panhai / Phu Hin Rong Kla NP, Phitsanulok, Thailand AB830735 11 T. taliangensis CIB-GG200110183 Shimian County, Ya'an, Sichuan Province, China KC147819 12 T. kweichowensis MVZ230371 Daquan County, Yunnan Province, China DQ517851 13 T. daweishanensi 201303001 Pingbian County, Yunnan, China Zhao et al. (2012) 14 T. yangi NMNS 3114 Pingbian County, Yunnan, China AB830739 15 T. yangi KUHE 42282 China (Pet trade) AB769546 16 T. uyenoi KUHE 19147 Doi Suthep, Chiang Mai, Thailand AB830733 17 T. uyenoi / Doi Lahnga, Chiang Rai, Thailand AB830728 18 T. pulcherrima KUHE 46406 Yunnan, China (Pet trade) AB830738 19 T. shanjing KUHE 43489 Myanmar (Pet trade) AB830723 20 T. shanjing KUHE 43578 Myanmar (Pet trade) AB830724 21 T. shanjing CAS 234479 Pianma, Lushui County, Yunnan Province, China HM462055 22 T. shanjing KUHE 43579 Myanmar (Pet trade) AB830725 23 T. shanjing MVZ 219763 Jingdong County, Yunnan Province, China DQ517852 24 T. shanjing CAS 215071 Pianma County, Yunnan Province, China HM462054 25 T. shanjing NMNS 3682 Jingdong County, Yunnan Province, China AB830721 26 T. cf. shanjing KUHE 42342 Myanmar (Pet trade) AB830722 27 T. cf. shanjing KUHE 42348 Myanmar (Pet trade) AB769544 28 T. shanorum CAS 230899 Taunggyi, Shan, Myanmar HM770087 29 T. shanorum CAS 230933 Taunggyi, Shan, Myanmar AB922822 30 T. shanorum CAS 230940 Taunggyi, Shan, Myanmar AB922823 31 T. verrucosus KIZ 201306055 Husa, Longchuan County, Yunnan Province, China AB922818 32 T. verrucosus KIZ 201306056 Husa, Longchuan County, Yunnan Province, China AB922819 33 T. verrucosus CIB YN2009060638 Husa, Longchuan County, Yunnan Province, China KT765172 34 T. verrucosus MVZ (no number) Nepal DQ517854 35 T. himalayanus sp. nov. CIB 201406246 Mai Pokhari 1, Illam, Mechi, Nepal KT765173 36 T. himalayanus sp. nov. CIB 201406247 Mai Pokhari 1, Illam, Mechi, Nepal KT765174 37 T. himalayanus sp. nov. CIB 201406252 Mai Pokhari 2, Illam, Mechi, Nepal KT765175 38 T. himalayanus sp. nov. CIB 201406253 Mai Pokhari 2, Illam, Mechi, Nepal KT765176 39 T. himalayanus sp. nov. CIB 201406254 Mai Pokhari 2, Illam, Mechi, Nepal KT765177 40 T. himalayanus sp. nov. CIB 201406255 Mai Pokhari 2, Illam, Mechi, Nepal KT765178 41 T. himalayanus sp. nov. CIB 201406256 Mai Pokhari 2, Illam, Mechi, Nepal KT765179 42 T. himalayanus sp. nov. CIB 201406257 Mai Pokhari 2, Illam, Mechi, Nepal KT765180 43 T. himalayanus sp. nov. CIB 201406258 Mai Pokhari 2, Illam, Mechi, Nepal KT765181 44 T. himalayanus sp. nov. CIB 201406259 Mai Pokhari 2, Illam, Mechi, Nepal KT765182 45 T. himalayanus sp. nov. CIB 201406260 Mai Pokhari 2, Illam, Mechi, Nepal KT765183 46 T. himalayanus sp. nov. CIB 201406261 Mai Pokhari 2, Illam, Mechi, Nepal KT765184 47 T. himalayanus sp. nov. CIB 201406262 Mai Pokhari 2, Illam, Mechi, Nepal KT765185 48 T. himalayanus sp. nov. CIB 201406263 Mai Pokhari 2, Illam, Mechi, Nepal KT765186 49 T. himalayanus sp. nov. CIB 201406264 Mai Pokhari 2, Illam, Mechi, Nepal KT765187 50 T. himalayanus sp. nov. CIB 201406265 Mai Pokhari 2, Illam, Mechi, Nepal KT765188 51 T. himalayanus sp. nov. CIB 201406266 Mai Pokhari 2, Illam, Mechi, Nepal KT765189 52 T. himalayanus sp. nov. CIB 201406267 Mai Pokhari 2, Illam, Mechi, Nepal KT765190 53 T. himalayanus sp. nov. CIB 201406268 Mai Pokhari 2, Illam, Mechi, Nepal KT765191 54 T. himalayanus sp. nov. CIB 201406269 Mai Pokhari 2, Illam, Mechi, Nepal KT765192 55 T. himalayanus sp. nov. CIB 201406270 Mai Pokhari 2, Illam, Mechi, Nepal KT765193 56 T. himalayanus sp. nov. CIB 201406271 Mai Pokhari 2, Illam, Mechi, Nepal KT765194 No. 4 Janak Raj KHATIWADA et al. A New Species of Tylototriton 249
(Continued Table 1)
Sample ID Species Voucher Locality GenBank accession No. 57 T. himalayanus sp. nov. CIB 201406272 Mai Pokhari 2, Illam, Mechi, Nepal KT765195 58 T. himalayanus sp. nov. CIB 201406273 Mai Pokhari 2, Illam, Mechi, Nepal KT765196 59 T. himalayanus sp. nov. CIB 201406274 Mai Pokhari 2, Illam, Mechi, Nepal KT765197 60 T. himalayanus sp. nov. CIB 201406275 Mai Pokhari 2, Illam, Mechi, Nepal KT765198 61 T. himalayanus sp. nov. CIB 201406276 Mai Pokhari 2, Illam, Mechi, Nepal KT765199 62 T. himalayanus sp. nov. CIB 201406277 Mai Pokhari 2, Illam, Mechi, Nepal KT765200 63 T. himalayanus sp. nov. CIB 201406278 Mai Pokhari 2, Illam, Mechi, Nepal KT765201 64 T. himalayanus sp. nov. CIB 201406279 Mai Pokhari 2, Illam, Mechi, Nepal KT765202 65 T. himalayanus sp. nov. CIB 201406280 Mai Pokhari 2, Illam, Mechi, Nepal KT765203 66 T. himalayanus sp. nov. CIB 201406281 Mai Pokhari 2, Illam, Mechi, Nepal KT765204 67 T. himalayanus sp. nov. CIB 201406282 Mai Pokhari 2, Illam, Mechi, Nepal KT765205 68 T. himalayanus sp. nov. CIB 201406283 Bagh Khor, Illam, Mechi, Nepal KT765206 69 T. himalayanus sp. nov. CIB 201406284 Bagh Khor, Illam, Mechi, Nepal KT765207 70 T. himalayanus sp. nov. CIB 201406285 Bagh Khor, Illam, Mechi, Nepal KT765208 71 T. himalayanus sp. nov. CIB 201406286 Bagh Khor, Illam, Mechi, Nepal KT765209 72 T. himalayanus sp. nov. CIB 201406287 Bagh Khor, Illam, Mechi, Nepal KT765210 73 T. himalayanus sp. nov. CIB 201406288 Bagh Khor, Illam, Mechi, Nepal KT765211 74 T. himalayanus sp. nov. 3150 WIIM 38.6.1.3 Namthing Pokhri, Darjeeling,West Bengal, India KT765212 outgroup 75 Echinotriton andersoni MVZ232187 DQ517774 76 Pleurodeles waltl MVZ186112 DQ517814 and 103 parsimony informative sites. Five haplotypes 3.2 Morphological comparisons Morphometric were recognized from 41 samples from the Himalayan comparisons between the Himalayan population and populations. The number of variable sites among these the closely-related species are shown in Table 3, haplotypes was only four. MP and BI analyses yielded and morphological differences on several diagnostic nearly identical topologies (Figure 2 A). The genus characters among them are presented in Table 4. Tylototriton was weakly supported as a monophyly by MP Himalayan population versus T. shanorum Himalayan analysis (bs = 70), but not supported by BI analysis (bpp Tylototriton showed distinct morphological differences = 0.85). Tylototriton was divided into two major clades, from T. shanorum (Table 4). Himalayan population has Clade A (bs = 91 and bpp = 1.00) and Clade B (bs = 93 16 dorsal warts but T. shanorum has 14 dorsal warts and bpp = 1.00) which were corresponding to subgenus (Nishikawa et al., 2014). Moreover, the average SVL Tylototriton and subgenus Yaotriton, respectively. Five of Himalayan male population (71.92 ± 6.10 mm, range haplotypes recognized from all eastern Himalayan 87.70–63.10) were smaller than holotype T. shanorum samples were mixed together and formed a monophyletic (76.0 mm). Furthermore, the head of the Himalayan clade (bs = 100 and bpp = 1.00) which were clustered population had longer length than width but that of T. with the clade including T. shanorum samples (bs = 85 shanorum has greater width than length. In addition, and bpp = 0.95) in Clade A. But our result revealed that Himalayan population showed great differences with T. the Himalayan population was paraphyletic with the clade shanorum in coloration from head to tail (Table 4). containing T. verrucosus, T. shanjing, T. anguliceps, T. Himalayan population versus the topotypic T. uyenoi, T. daweishanensi and T. yangi (bs = 90 and bpp verrucosus In male, the SVL of Himalayan population = 0.99). As note, the topotypic T. verrucosus were deeply was smaller than topotypic T. verrucosus (Mann-Whitney nested with the T. shanjing populations. The phylogenetic U-test = 6.00, P = 0.014). Himalayan population was network analyses based on cytb dataset similarly strongly significantly larger on RSL, RIND, RTL and RBTAH supported the splits between the Himalayan population ratio % value of SVL but significantly smaller on and its closely-related species (bs = 100; Figure 2 B). RENL, RBTAW and RMTAW than T. verrucosus (P < Interspecific genetic uncorrected p-distance of ND2 in 0.05). Himalayan female population was significantly the genus Tylototriton ranged from 0.7%–14.2% (Table smaller than T. verrucosus only on RBTAW (P < 0.01). 2). The p-distance between the Himalayan population Himalayan population could also be separated from the and the other species of Tylototriton ranged from 5.2% to topotypic T. verrucosus on grooves on the tail, state of 12%. warts and coloration (Table 4). 250 Asian Herpetological Research Vol. 6
Himalayan population versus the topotypic T. shanjing 20
0.061 of Jingdong population In male, the SVL of Himalayan population and topotypic T. shanjing were similar (Mann-
19 Whitney U-test = 14, P = 0.097). Himalayan population 0.062 0.052 was significantly larger than T. shanjing of Jingdong
18 population on RTL, RHL, RHW, RSL, RENL, RIND, 0.065 0.019 0.064 RTRL, RTAL, RVL, RBTAH, RLA, RLH, RLT and
17 RLF of percentage SVL ratio value (P < 0.05). In female 0.028 0.064 0.021 0.062 population, Himalayan Tylototriton was significantly larger than T. shanjing on RTL, RHL, RHW, RSL, RENL, 16 0.043 0.049 0.070 0.046 0.067 RIND, RUED, RTRL, RTAL, RVL, RBTAH, RMTAH, RLA, RLH, and RPPLF (P < 0.05). They were also 15 0.072 0.068 0.075 0.092 0.072 0.086 different in the shape of head, development of grooves on the tail, state of warts and coloration (Table 4). 14 0.076 0.047 0.039 0.043 0.067 0.040 0.066 Himalayan population versus T. shanjing of Baoshan population In male, the SVL of Himalayan population . 13 0.007 0.074 0.050 0.037 0.046 0.070 0.044 0.066 and T. shanjing of Baoshan population were similar (Mann-Whitney U-test = 26.00, P = 0.20). Himalayan
12 population was significantly larger only on RENL (P < Tylototriton 0.059 0.061 0.081 0.063 0.052 0.057 0.061 0.054 0.053 0.05) and significantly smaller on RUEL and RBTAW
11 (P < 0.05) than T. shanjing of Baoshan population; but 0.063 0.075 0.079 0.100 0.088 0.072 0.075 0.082 0.074 0.071 in female, the percentage SVL ratio value showed no significant difference in any character between these two 10 0.105 0.107 0.120 0.119 0.126 0.142 0.131 0.126 0.123 0.127 0.120 populations. The results of the PCAs exhibited the overall 9 0.111 0.096 0.095 0.106 0.107 0.113 0.118 0.129 0.114 0.125 0.116 0.108 morphological difference among the Himalayan population and its closely-related species. In male, 8 0.071 0.093 0.091 0.094 0.097 0.095 0.127 0.103 0.098 0.104 0.103 0.102 0.100 the first two principal components axis explained for 52.8% of the total variation; and in female, the first two 7 0.074 0.088 0.094 0.111 0.092 0.100 0.104 0.109 0.131 0.122 0.104 0.106 0.108 0.109 principal components axis explained for 57.5% of the total variation. We constructed PCA plots of the first 6 principal component (PC1) versus the second (PC2) for 0.034 0.069 0.076 0.091 0.088 0.090 0.093 0.096 0.122 0.108 0.094 0.098 0.103 0.099 0.102 the male and female groups, respectively (Figure 3). Both
5 PCA graphs of male and female groups showed that the 0.037 0.049 0.071 0.080 0.100 0.111 0.087 0.110 0.097 0.095 0.099 0.126 0.092 0.098 0.097 0.105 Himalayan population was clearly separated from the
4 closely-related species. In addition, on PCA graph of male 0.095 0.080 0.095 0.082 0.099 0.097 0.100 0.110 0.095 0.100 0.101 0.135 0.112 0.113 0.113 0.105 0.109 (Figure 3 A), the topotypic T. verrucosus was clustered
3 with T. shanjing populations especially with the Baoshan 0.042 0.089 0.076 0.090 0.081 0.092 0.096 0.087 0.085 0.092 0.095 0.128 0.105 0.094 0.100 0.102 0.102 0.101 population, but on the PCA graph of female, T. verrucosus was separated from T. shanjing populations (Figure 3 B). 2 0.047 0.049 0.090 0.081 0.093 0.088 0.100 0.101 0.095 0.097 0.098 0.103 0.130 0.109 0.102 0.108 0.106 0.109 0.106 The Himalayan specimens are different from its closely-related species on colorations and the shape of 1 -distance of the mtDNA ND2 sequences used in this study between members of the genus p -distance of the mtDNA 0.048 0.048 0.044 0.087 0.077 0.092 0.089 0.103 0.108 0.092 0.099 0.113 0.096 0.100 0.129 0.113 0.113 0.108 0.116 0.109 snout, dorsolateral bony ridges on head and tailfin (Figure 4). In life, the dorsal region of the Himalayan individuals is dark to light brown, and in preservation, body colour faded to a lighter blackish brown (Figure 4 A and G). But the dorsal region of the topotypic T. verrucosus specimens are blackish brown with dark to light brown warts (Figure 4 H) both in life and preservation, and T. shanjing T. asperrimus 1. T. notialis 2. T. hainanensis 3. T. T. vietnamensis 4. T. T. wenxianensis 5. T. T. broadoridgus 6. T. T. dabienicus 7. T. T. liuyangensis 8. T. 9. T. lizhenchangi 9. T. T. panhai 10. T. Table 2 Genetic uncorrected Table T. taliangensis T. 11. T. kweichowensis 12. T. T. daweishanensi 13. T. T. yangi 14. T. T. uyenoi 15. T. T. anguliceps 16. T. T. pulcherrima 17. T. T. shanjing 18. T. T. shanorum 19. T. T. verrucosus 20. T. T. himalayanus sp. nov. 21. T. have blackish-brown background and yellow warts on No. 4 Janak Raj KHATIWADA et al. A New Species of Tylototriton 251
Figure 2 Bayesian inference (BI) tree based on mtDNA ND2 sequences (A) and the phylogenetic network constructed by the program Splittree based on mtDNA cytb sequences (B). Values on branches of the tree are bootstrap support (bs) values from the Maximum parsimony analysis and Bayesian posterior probabilities (bpp). Values on branches of the network were bootstrap supports (bs). Sample ID on the phylogenetic tree (A) was shown in Table 1.
Figure 3 PCA plots of the first principal component (PC 1) versus the second (PC 2) for the male (A) and female (B) groups of the eastern Himalayan Tylototriton population and its closely-related species. dorsal region (Figure 4 I and J). Snout of the Himalayan T. shanorum (Nishikawa et al., 2014). specimens is more flat, broad and blunt than other species Based on the distinct morphological and molecular (Figure 4 K, L, M and N; Table 4). The dorsolateral bony characteristics of the eastern Himalayan specimens from ridges on head of the Himalayan specimens were found to other species of the genus Tylototriton, we described the be wider separated than those of other populations (Figure eastern Himalayan Tylototriton as a new species. 4 K, L, M and N; Nishikawa et al., 2014). The tailfin of the Himalayan specimens is straighter and thicker than 3.3 Description others (Figure 4 C, D, E and F). The base of tail has Tylototriton himalayanus sp. nov. distinct grooves and showed similar character with T. Holotype NHMTU17A-0098, adult male (Figure 4 verrucosus but this character is absent in T. shanjing and A and B), SVL 73.95 mm, was collected by Janak Raj 252 Asian Herpetological Research Vol. 6
Table 3 The morphometric differences among the Himalayan Tylototriton population, topotypes of T. verrucosus, topotypes of T. shanjing and T. shanjing from Baoshan city of China. All measurements are presented as Mean ± SD and range in parentheses (in mm).
Himalayan population topotypic T. verrucosus topotypic T. shanjing T. shanjing from Baoshan 32 (Male) 13 (Female) 3 (Male) 2 (Female) 14 (Male) 22 (Female) 3 (Male) 2 (Female) 141.05 ± 6.42 152.55 ± 11.70 156.89 ± 1.06 185.93 ± 8.25 138.47 ± 11.01 146.09 ± 9.82 154.00 ± 0.91 162.52 ± 0.42 TL (153.53–130.92) (186–136.20) (158.12–156.23) (191.77–180.09) (158.34–114.55) (162.34–130.33) (161.07–142.67) (162.82–162.22) 71.92 ± 6.10 76.06 ± 7.53 85.75 ± 6.97 94.22 ± 0.14 74.78 ± 6.95 80.62 ± 7.56 71.38 ± 2.34 82.74 ± 0.90 SVL (87.70–63.10) (97.00–66.00) (92.88–78.94) (94.33–94.12) (89.13–61.64) (87.28–51.67) (74.21–69.53) (83.38–82.1) 17.63 ± 2.08 19.73 ± 2.42 21.31 ± 2.95 25.075 ± 0.26 16.79 ± 1.55 17.333 ± 1.40 19.59 ± 0.63 19.96 ± 1.83 HL ( 21.30–13.91) (24.00–15.95) (23.54–17.96) (25.26–24.89) (20.13–14.17) (20.15–14.86) (20.08–18.88) (21.26–18.66) 16.55 ± 0.98 17.97 ± 2.03 19.37 ± 1.40 25.96 ± 5.19 15.01 ± 1.29 16.01 ± 0.98 17.04 ± 1.21 16.89 ± 0.65 HW (19.25–14.65) (23.00–15.70) (20.72–17.91) (29.64–22.29) (16.44–11.21) (17.63–13.95) (18.17–15.75) (17.35–16.43) 14.90 ± 0.97 15.13 ± 2.61 14.56 ± 0.87 15.80 ± 0.64 12.91 ± 1.29 13.37 ± 1.03 14.76 ± 1.97 15.49 ± 0.04 SL (18.00–13.49) (21.5–10.50) (15.17–13.57) (16.26–15.35) (15.43–10.17) (14.94–11.00) (16.52–12.63) (15.52–15.46) 4.77 ± 0.64 4.82 ± 0.82 17.13 ± 2.46 20.66 ± 0.77 4.13 ± 0.71 4.07 ± 0.53 18.61 ± 2.17 18.84 ± 0.28 ENL (5.77–3.42) (6.20–3.76) (18.93–14.32) (21.21–20.11) (5.77–2.94) (5.25–3.25) (20.21–16.14) (19.04–18.64) 6.07 ± 0.98 6.24 ± 1.05 5.13 ± 0.71 5.60 ± 0.08 4.51 ± 0.73 4.83 ± 0.56 4.74 ± 0.46 4.14 ± 0.78 IND (9.32–4.50) (9.32–4.93) (5.89–4.48) (5.66–5.54) (6.16–3.55) (5.76–3.31) (5.03–4.21) (4.70–3.59) 2.37 ± 0.72 2.86 ± 0.74 2.26 ± 0.20 3.01 ± 0.10 2.13 ± 0.31 2.37 ± 0.39 2.62 ± 0.46 2.35 ± 0.03 UED (3.99–1.41) (4.30–1.76) (2.49–2.11) (3.09–2.94) (2.70–1.64) (3.02–1.63) (3.02–2.12) (2.38–2.33) 3.59 ± 0.70 4.09 ± 0.72 4.78 ± 1.11 4.88 ± 0.39 4.39 ± 0.53 4.59 ± 0.49 5.28 ± 0.29 5.19 ± 0.13 UEL (5.00–1.95) (5.33–3.30) (5.52–3.5) (5.16–4.6) (5.54–3.65) (5.43–3.67) (5.57–4.98) (5.29–5.10) 18.18 ± 4.68 19.35 ± 2.47 20.63 ± 2.37 26.17 ± 3.89 18.66 ± 1.91 19.98 ± 1.42 21.68 ± 2.14 22.13 ± 0.79 OL (38.34–11.20) (24.00–15.00) (22.98–18.24) (28.93–23.42) (21.90–15.12) (22.19–17.17) (23.92–19.65) (22.69–21.57) 38.53 ± 5.03 39.26 ± 7.14 43.22 ± 3.97 52.30 ± 8.64 37.78 ± 6.81 40.68 ± 3.28 37.21 ± 2.86 42.99 ± 3.62 AGD (52.26–31.63) (53.00–30.84) (46.01–38.68) (58.42–46.19) (52.26–30.74) (49.79–34.74) (40.52–35.51) (45.56–40.43) 57.47 ± 7.02 58.63 ± 8.67 58.54 ± 8.00 71.17 ± 3.26 54.28 ± 8.39 58.73 ± 5.28 56.90 ± 5.86 58.41 ± 1.93 TRL (76.66 – 45.00) (72.30–48.04) (67.74–53.14) (73.48–68.86) (76.66–47.12) (70.5–50.23) (60.34–50.14) (59.78–57.05) 70.45 ± 4.16 76.48 ± 5.19 70.87 ± 6.55 86.97 ± 3.48 61.83 ± 5.90 63.29 ± 6.85 71.79 ± 4.23 78.71 ± 4.77 TAL ( 77.88–60.28) (89.00–70.20) (76.05–63.51) (89.44–84.51) (70.39–49.06) (77.54–47.62) (75.32–67.1) (82.09–75.34) 8.48 ± 1.92 9.52 ± 2.27 8.42 ± 0.59 8.78 ± 1.44 6.88 ± 0.99 6.22 ± 0.82 7.15 ± 0.32 6.82 ± 1.03 VL (12.00 – 5.00) (12.70–6.00) (9.06–7.87) (9.80–7.76) (8.20–5.28) (8.00–4.54) (7.49–6.85) (7.56–6.09) 4.28 ± 0.95 4.67 ± 0.63 7.87 ± 0.74 8.13 ± 1.18 5.24 ± 0.87 5.52 ± 0.67 5.87 ± 0.90 6.55 ± 0.03 BTAW (5.89–1.88) (5.91–3.82) (8.37–7.02) (8.97–7.30) (7.07–3.82) (7.15–4.49) (6.92–5.34) (6.60–6.51) 3.77 ± 1.35 3.83 ± 1.04 5.65 ± 0.73 6.11 ± 0.01 3.90 ± 0.63 4.05 ± 0.54 4.10 ± 1.17 4.55 ± 0.06 MTAW (10.21–2.70) (5.45–2.41) (6.43–4.96) (6.12–6.10) (5.11–3.07) (5.09–2.9) (5.45–3.36) (4.60–4.51) 10.10 ± 1.33 9.83 ± 0.85 9.78 ± 0.36 11.23 ± 1.53 6.713 ± 0.88 6.88 ± 0.68 8.54 ± 0.69 9.03 ± 0.87 BTAH (12.30–7.08) (11.00–8.50) (10.16–9.43) (12.32–10.15) (8.49–5.41) (8.27–5.13) (9.34–8.08) (9.65–8.42) 9.89 ± 1.42 10.22 ± 1.28 11.05 ± 1.84 11.65 ± 1.06 7.04 ± 0.98 7.50 ± 1.01 10.00 ± 0.58 9.84 ± 1.06 MTAH (11.83–6.71) (12.21–7.20) (12.81–9.14) (12.41–10.90) (8.74–5.17) (9.29–5.74) (10.43–9.34) (10.60–9.09) 9.97 ± 1.22 9.83 ± 0.86 10.51 ± 1.00 10.40 ± 1.26 8.37 ± 1.63 8.35 ± 0.80 10.15 ± 0.38 10.56 ± 0.99 LA (12.71–7.40) (11.00–8.40) (11.65–9.76) (11.30–9.51) (12.71–6.49) (9.85–6.82) (10.55–9.79) (11.27–9.86) 16.06 ± 2.15 17.66 ± 1.68 18.87 ± 2.10 19.94 ± 1.40 16.12 ± 1.54 16.92 ± 1.16 18.53 ± 1.59 18.78 ± 0.36 LH (20.00 – 12.00) (20.00–15.49) (21.26–17.27) (20.94–18.95) (19.04–13.47) (18.84–14.09) (20.03–16.85) (19.04–18.53) 8.96 ± 1.17 9.45 ± 1.55 9.01 ± 0.87 9.19 ± 0.58 9.29 ± 1.28 9.67 ± 1.01 11.57 ± 1.46 10.94 ± 0.87 LT (11.3–6.66) (20.00–15.49) (9.85–8.11) (9.60–8.78) (11.76–7.68) (11.43–7.32) (12.68–9.91) (11.56–10.32) 17.99 ± 1.33 19.51 ± 1.94 21.80 ± 2.26 23.73 ± 4.00 17.60 ± 1.84 18.21 ± 1.52 19.57 ± 1.46 19.48 ± 0.66 LF (20.64–14.18) (24.00–16.14) (23.23–19.19) (26.57–20.9) (20.84–14.35) (21.13–15.24) (21.11–18.2) (19.95–19.01)
Khatiwada, Shanta Paudel and Subarna Ghimire from Mai Tribhuvan University was collected by Kalu Ram Khubu Pokhari, Illam District, Mechi, eastern Nepal (27°0'25" N Rai from the same place on June 6, 2003. 87°55'48" E at an elevation of 2110 m) on 09 July, 2014. The holotype and paratypes were deposited in Paratypes NHMTU17A-0098-1, adult male, collected the Natural History Museum, Tribhuvan University, by Janak Raj Khatiwada, Shanta Paudel and Subarna Soyambhu, Kathmandu, Nepal (NHMTU). Ghimire from the same place of the holotype on 09 July, Etymology The specific epithet is derived from the 2014. A 2-C002, female adult, Mechi Multiple Campus, current distribution range of the species in the eastern No. 4 Janak Raj KHATIWADA et al. A New Species of Tylototriton 253
Table 4 Morphological comparison of males between the Himalayan population, T. shanorum, T. verrucosus and T. shanjing. Data for T. shanorum is derived from Nishikawa et al. (2014).
Morphological T. himalayanus sp. nov. 32♂ T. shanorum 1♂ T. verrucosus 3♂ T. shanjing 14♂ characters SVL 71.92 ± 6.10 76 85.75 ± 6.97 74.78 ± 6.95 Broad, flat and blunt snout, Snout is truncate, head wider Bluntly oval, longer than wider, Longer than wider, poorly longer than wider, greatly than longer, poorly greatly Head poorly separated dorsolateral greatly separated dorsolateral separated dorsolateral bony separated dorsolateral bony bony ridges bony ridges ridges ridges Distinct grooves on either side Tail No information Absent of grooves Grooves were poorly developed at the base tail Uniform blackish dark brown Dorsal surface with dark brown, colouration in dorsal region Body is dark brown to black; Trunk is dark brown than head head with bright yellow or but light brown colour in venter slightly lighter than and tail region but ventral Colour orange. The warts are yellowish. dorsolateral region and ventral dorsum; head, warts and side of surface has creamy colouration, Tail and limbs are orange- surface has creamy colouration, tail dull reddish brown warts are ligher brown yellow. Warts are bright yellow. warts are darker brown Lighter brown droso-lateral Dark brown droso-lateral rows Light yellow droso-lateral rows Yellowish droso-lateral rows of Warts rows of warts on either side of of warts on either side of trunk of warts on either side of trunk warts on either side of trunk trunk Elevation 900–2 317 1 457 1 000–2 000 1 500 ranges (masl)
Himalaya. are extended behind eyes. Eye is moderate size with round Diagnosis The new species is a member of sub-genus pupils and black irises with large, granular upper eyelid. Tylototriton. It is diagnosed from other members by Skin of body is granular. Tail compressed laterally, with having the following morphological characters: medium well-developed fin fold, a little shorter than snout-vent size salamander male 87.70–63.10 mm SVL and 97.00– length. Uniform dark brown colouration in dorsal region 66.00 mm in females, dorsolateral bony ridges on head but light brown colour in dorsolateral region, ventral present; quadrate spine absent, broad and flat head, blunt surface has creamy colouration. Tail is comparatively snout, moderate-sized eyes with granular upper eyelids. lighter brown than body colour. The ventral side of digits The two lines of dorsolateral bony ridges on head are is rusty cream but palms and soles are dull cream colour. distinct and greatly separated. The body shows pairs of The cloaca has a small longitudinal slit. Fingers and longitudinal lines of knob-like 16 dorsal warts, with finely toes not webbed. Front and hindlegs muscular, with four granular skin and tail. Tail compressed laterally, with a fingers on the front legs and five toes on the hindlegs; well-developed fin fold. The new species has light brown relative lengths of fingers 3>2>4>1; relative lengths of colour with darker warts in life. toes 3>4>2>5>1. Colour In life, dark to light brown colouration in dorsal Variation A detailed analysis of the morphology and region, light brown colour in dorsolateral region, ventral life history of Tylototriton salamanders from eastern surface has creamy colouration. Tail, hand and feet are Nepal can also be found in Anders et al. (1998). Our comparatively lighter brown than body colour. The ventral study revealed greater sexual dimorphism in salamanders side of digits is rusty cream but palms and soles are dull from the eastern Himalaya. Females tend to be larger cream colour. In preservation, the body color faded to a than males in different morphological traits. Summary lighter blackish brown, and brown color to lighter brown. statistics is provided in Table 3. The SVL in males ranged Description of the holotype Adult male shows the from 87.70–63.10 mm (71.92 ± 6.1 mm) and 97.00–66.00 following measurements with percentage ratio proportions mm (76.06 ± 7.53 mm) in females. Similarly, TL of of SVL 73.95 mm (R: /SVL*100): TL 191%; HL 27%; males was 141.33 ± 6.35 mm and 152.55 ± 11.70 mm in HW 23%; SL 21%; LJL 5%; ENL 7%; IND 12.7%; UEW females and body weight of males were 14.50 ± 1.39 gm 5.3%; UEL 3%; OL 2.7%; AGD 52%; TRL 73% ; TAL in males and 17.69 ± 5.66 gm in females, respectively 91% ;VL 16%; BTAW 2%; MTAW 13%; BTAH 13%; (Table 3). The body temperature was 19.39 ± 0.92 °C in MTAH 12%; LA 13%; LH 21%; LT 14%; LF 25%. Head male and 19.88 ± 0.54 °C in female. There was no distinct is bluntly oval shaped; HL is slightly longer than HW. colour variation between male and female. The ground Snout is blunt, clearly extending beyond the lower lip. colouration varied from dark brown to light brown. Head The bony ridges are present on either side of the head and is blunt and oval, internarial distance smaller than eye to 254 Asian Herpetological Research Vol. 6 nostril length, tail length is almost equal to SVL, medial tail height is similar to basal tail height in males but larger in females, forelimb is shorter than hindlimb, vent length is longer in male compared to female. The dorsal body surface has two series of rounded knoblike 16 black warts that originated nearby the forelimb and extended backward at the end of vent. The clutch sizes vary from 26 to 60 and egg size varies 6 to 10 mm in diameter (Nag and Vasudevan, 2014; Schleich and Kästle, 2002).The fully grown larva has four limbs and three pairs of large gills and longer or little shorter tail than SVL. They have olive-brownish colour thickly speckled with darker markings (Schleich and Kästle, 2002; Sparreboom, 2013). Distribution and natural history Most specimens were collected from permanent and temporary ponds in the Illam District of Nepal (Figure 5). The habitats are characterized as the subtropical hill forest and area is dominated by scattered vegetation, for example, Schima wallichii, Castonopsis indica, Casttonopsis tribuloides, Albizzi sp., Sauraria nepalensis, Rubus ellipticus and Figure 4 The male holotype (NHMTU17A-0098) of Tylototriton himalayanus sp. nov. (A, B, C, G, and K), the male topotype of T. Eupatorium adenophorum. Anders et al. (1998) reported verrucosus (D, H and L), the male topotype of T. shanjing (E, I and the distribution of the new species in five places in the M) and one female of T. shanjing of Baoshan population (F, J and Illam District of Nepal with elevation ranging from N). Dorsal (A, G, H, I and J) and ventral view (B) of the body, left lateral view of the tail (C, D, E and F), and dorsal view of the head 1 100 m to 2 120 m. They also checked the previous (K, L, M and N). Scale bar=20 mm in body and tail, 10 mm in head. record by Shrestha (1988) in Dhankuta District but did not find the species. The distribution of new species is currently known only from the Illam District of Nepal and in Darjeeling District of India (details provided in Nag and Vasudevan 2014). The salamanders are more terrestrial in non-breeding seasons (from October to February) and found hiding under the logs, bushes and stones and come to the breeding ponds in early March or April soon after heavy monsoonal showers (also see Schleich and Kästle, 2002) .
4. Discussions
Salamanders are poor dispersers and highly associated Figure 5 Habitat at the type locality of Tylototriton himalayanus with specific habitat requirements for breeding and sp. nov. in Mai Pokhari, Illam district, Mechi, Nepal (27°0'25" N 87°55'48" E at an elevation of 2 110 m). larval development (Zamudio and Wieczorek, 2007). Long isolation and local climatic adaptation might cause (Tables 3, 4; Figure 2). Furthermore, molecular analyses great differentiation in the morphological and genetic showed that the Nepalese and Indian Tylototriton are characteristics of species resulting in the formation of new species (Eckert et al., 2008). Based on molecular genetically identical (Figure 2). The sampling locations and morphological characters of individuals from eastern from Nepal and current study sites in the Darjeeling Himalaya, we described them as a new species. Our result District, India are in close proximity roughly around 40 revealed that this new species, both morphologically and km. genetically, is greatly distinct from the closely-related The new species is a single representative of the species T. shanorum, T. verrucosus and T. shanjing order Caudata and has a restricted distribution in eastern No. 4 Janak Raj KHATIWADA et al. A New Species of Tylototriton 255
Himalaya (Shrestha, 1989; 1984). Eastern Himalaya References is also regarded as a biodiversity hotspot due to high biological diversity and endemism (Olson et al., 2001). Anders C., Schleich H., Shah K. 1998. Contributions to the Recent study by Nag and Vasudevan (2014) also reported Biology of Tylototriton verrucosus Anderson 1871 from East a new species from 37 locations in Darjeeling Districts, Nepal (Amphibia: Caudata, Salamandridae). In: H Schleich and W Kastle (eds.) Contributions to the Herpetology of South Asia India. This species inhabits in the mountain and hilly (Nepal, India) No. 4. Fuhlrott Museum, Wuppertal, Germany, regions, at elevation range of 900–2 317 m above sea 1–26 level and found living around aquatic habitats, permanent Chen X. H., Wang X. W., Tao J. 2010. A new subspecies of and temporary pools, puddles, lakes and also in the paddy genus Tylototriton from China (Caudata, Salamandridae). 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Species Sample ID Sex Country Province District Locality Unit T. himalayanus NHMTU17A-0098 Male Nepal Mechi Illam Mai Pokhari 1 Illam, Nepal T. himalayanus NHMTU17A-0098-1 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus A 2-C002 Female Nepal Mechi Illam Mai Pokhari 3 Illam, Nepal T. himalayanus 201406246 Male Nepal Mechi Illam Mai Pokhari 1 Illam, Nepal T. himalayanus 201406247 Male Nepal Mechi Illam Mai Pokhari 1 Illam, Nepal T. himalayanus 201406252 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406253 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406254 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406255 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406256 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406257 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406258 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406259 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406260 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406261 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406262 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406263 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406264 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406265 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406266 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406267 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406268 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406269 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406270 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406271 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406272 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406273 Female Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406274 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406275 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406276 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406277 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406278 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406279 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406280 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406281 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406282 Male Nepal Mechi Illam Mai Pokhari 2 Illam, Nepal T. himalayanus 201406283 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406284 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406285 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406286 Female Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406287 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406288 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406289 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406290 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406291 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406292 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406293 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. himalayanus 201406294 Male Nepal Mechi Illam Bagh Khor Illam, Nepal T. shanjing 21590 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21592 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21594 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21599 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21602 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21604 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21607 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21610 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21612 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21614 Female China Yunnan Jingdong CIB, Chengdu, China (Continued Appendix 1)
Species Sample ID Sex Country Province District Locality Unit T. shanjing 21623 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21624 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21625 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21626 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21631 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21632 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21638 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21642 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21644 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21645 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21646 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21651 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21652 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21653 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21658 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21662 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21662 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21664 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21673 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21679 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21688 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21689 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21692 Female China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21695 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21699 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 21702 Male China Yunnan Jingdong CIB, Chengdu, China T. shanjing 98004 Female China Yunnan Baoshan CIB, Chengdu, China T. shanjing 98005 Female China Yunnan Baoshan CIB, Chengdu, China T. shanjing 98006 Male China Yunnan Baoshan CIB, Chengdu, China T. shanjing 98007 Male China Yunnan Baoshan CIB, Chengdu, China T. shanjing 98008 Male China Yunnan Baoshan CIB, Chengdu, China T. verrucosus YN2009060606 Female China Yunnan Husa CIB, Chengdu, China T. verrucosus YN2009060607 Female China Yunnan Husa CIB, Chengdu, China T. verrucosus YN2009060637 Male China Yunnan Husa CIB, Chengdu, China T. verrucosus YN2009060638 Male China Yunnan Husa CIB, Chengdu, China T. verrucosus YN2009060639 Female China Yunnan Husa CIB, Chengdu, China T. verrucosus 2014111701 Female China Yunnan Husa CIB, Chengdu, China T. verrucosus 2014111702 Male China Yunnan Husa CIB, Chengdu, China