Tropical Natural History 20(2): 144–161, August 2020 2020 by Chulalongkorn University A New Species of Tylototriton (Urodela: Salamandridae) from Nan Province, Northern Thailand PORRAWEE POMCHOTE1*, WICHASE KHONSUE1, PANUPONG THAMMACHOTI1, AXEL HERNANDEZ2, PARADA PEERACHIDACHO1, CHATMONGKON SUWANNAPOOM3, YASUHO ONISHI4 AND KANTO NISHIKAWA4, 5 1Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, THAILAND 2Department of Environmental Sciences, Faculty of Sciences and Technics, University Pasquale Paoli of Corsica, Corte 20250, FRANCE 3Division of Fishery, School of Agriculture and Natural Resources, University of Phayao, Phayao 56000, THAILAND 4Graduate School of Global Environmental Studies, Kyoto University, Kyoto 606–8501, JAPAN 5Graduate School of Human and Environmental Studies, Kyoto University, Kyoto 606–8501, JAPAN * Corresponding author. Porrawee Pomchote ([email protected]) Received: 27 February 2020; Accepted: 6 June 2020 ABSTRACT.– A new species of the genus Tylototriton, obtained from Doi Phu Kha National Park, Nan Province, northern Thailand, is described based on molecular and morphological evidence and named herein as Tylototriton phukhaensis sp. nov. The new species is morphologically distinct from the four known Thai Tylototriton species (T. panhai, T. uyenoi, T. anguliceps and T. verrucosus), in having a prominent, narrow, and straight sagittal ridge on the head that distinguishes it from the other Thai species. The molecular analysis also indicated that the new species is a distinct lineage and sister to T. anguliceps and T. uyenoi. The knowledge obtained in this study will greatly contribute to conducting the future conservation of Thai Tylototriton. KEY WORDS: molecular phylogeny, morphology, new species, Thailand, Tylototriton been subdivided into the two subgenera of INTRODUCTION Tylototriton and Yaotriton (e.g., Nishikawa et al., 2013a, 2013b; Phimmachak et al., The salamandrid genus Tylototriton was 2015; Wang et al., 2018). From the above established in 1871 following the discovery records, Thailand is located at the lowest of the first species of the crocodile newt, latitude for this genus. Tylototriton verrucosus, in a flooded rice In Thailand, Pomchote et al. (2008) paddy near Nantin high up between the recognized two different phenotypes of T. Momien and Husa valleys, Yunnan verrucosus that had an allopatric distribution Province, southwestern China (Anderson, and showed morphological differences. 1871). In the following almost 150 years Nishikawa et al. (2013a) analyzed the this species and genus has been found to phylogenetic and morphological characteristics have a wide distribution range in the hills of these populations and described them as new and mountains of the Himalayan region species (T. uyenoi and T. panhai) and also from northeastern India, Bhutan, eastern recognized another cryptic lineage, later Nepal, southern China through northern described as T. anguliceps (Le et al., 2015). Myanmar, northern Thailand to northern At present, four Tylototriton species (T. Vietnam (Wang et al., 2018). The genus has POMCHOTE ET AL. — A NEW SPECIES OF TYLOTOTRITON FROM NAN, THAILAND 145 panhai, T. uyenoi, T. anguliceps, and T. MATERIALS AND METHODS verrucosus) have been confirmed in Thailand (Pomchote et al., 2020), and includes the Sampling southernmost population of the whole genus Field surveys were conducted on 12 June in Asia, T. panhai (Hernandez et al., 2019). 2019 at Doi Dong Ya Wai Mountain, The distribution of urodeles is restricted to DPKNP, Nan Province, northern Thailand mainly the northern hemisphere because, in (Fig. 1) using the visual encounter survey general, they favor cool and moist habitats method. Five specimens were found in an (Webb et al., 1981). In their southernmost isolated temporary swamp located on the distribution, such as in Southeast Asia, mountain at an elevation of 1,795 m amsl. Tylototriton tends to be restricted to middle They were caught by hand and kept in to high elevations in moist and forested plastic boxes for examination. mountains (Hernandez et al., 2018). In All specimens were checked for sex and Thailand, Tylototriton species are distributed maturity using the cloacal characters allopatrically in high mountainous areas at (Pomchote et al., 2008), and were ascribed an altitude of more than 1,000 m above mean as three adult males and two adult females. sea level (amsl) throughout the northern and All five samples were used for molecular northeastern parts (Pomchote et al., 2020). studies (Table 1), while one of the males However, the phylogeographic survey on and both females were used for the the Thai species is not complete yet, which morphological analysis. has been basically been reliant on Pomchote Following previous studies (Pomchote et et al. (2008) without surveying various high al., 2020), live specimens were euthanized and non-continuous mountain populations in by immersion in a solution of tricaine Thailand. From the aforementioned data, it methane sulfonate (MS-222; 5 g/L) for is probable that more unknown newts are about 5 min, measured for morphometrics existent in Thailand. and body weight (BW), as detailed below, From our field survey in Doi Phu Kha and tissue samples of each individual were National Park (DPKNP), Nan Province, obtained by removing the tail tip, and then which is located in the northern part of preserved in 95% (v/v) ethanol for Thailand, we discovered the easternmost molecular analysis. An antiseptic liquid was population of Thai Tylototriton sp. The used to clean the wound for the samples color pattern of the newts found in DPKNP released back in the habitat, whilst those for was rather similar to T. anguliceps, T. uyenoi, voucher specimens and morphological and T. verrucosus but with some differences analysis were preserved in 70% (v/v) in external morphology. Therefore, we used ethanol and temporarily deposited at The mitochondrial (mt) DNA sequence data and Chulalongkorn University Museum of morphological characters to clarify the Natural History (CUMZ). taxonomic status of the Tylototriton sp. Molecular Study from DPKNP. Based on the molecular and After removal, the tail-tip tissue samples morphological analyses, the specimens from were used for molecular analysis. Genomic DPKNP belong to a lineage distinct from DNA extraction, PCR amplification and known species of Tylototriton, and so are sequencing of the mtNADH dehydrogenase named and described as Tylototriton subunit 2 region (ND2) gene were performed phukhaensis sp. nov. herein. as previously reported (Pomchote et al., 2020). 146 TROPICAL NATURAL HISTORY 20(2), AUGUST 2020 FIGURE 1. Current known distribution of Tylototriton phukhaensis sp. nov. from DPKNP, Nan Province (red circle = type locality). The map was modified by Praphatsorn Punsompong We combined the new sequences of the The first three million generations were DPKNP samples with those of the other discarded as burn-in, and we considered species available from GenBank (Table 1). topologies with posterior probabilities (bpp) The optimum substitution models were of 0.95 or greater as significant support selected by Kakusan 4 (Tanabe, 2011). We (Leaché and Reeder, 2002). Pairwise applied a non-partition model to our dataset, comparisons of uncorrected sequence then constructed phylogenetic trees by divergences (p-distance by 988 bps) were maximum likelihood (ML) and Bayesian calculated using MEGA7 (Kumar et al., inference (BI) methods. The ML tree was 2016). searched using RAxML (Stamatakis, 2014) Morphological Study using the gamma model of rate We compared the morphometric heterogeneity option, and the BI tree was characters of the DPKNP newts with those searched with MrBayes v3.2.6 (Ronquist et of three of the four known Tylototriton al., 2012). species found in Thailand (T. anguliceps, T. The robustness of the ML tree was tested uyenoi, and T. verrucosus) using data from using bootstrap analysis (Felsenstein, 1985) Pomchote et al. (2020) because their color with 2,000 replicates, and we regarded tree pattern is rather similar and judged as the T. topologies with bootstrap values (bs) of verrucosus group (Nishikawa et al., 2013a). 70% or more to be significantly supported Furthermore, the DPKNP population has (Huelsenbeck and Hillis, 1993). For BI, two previously been identified as T. verrucosus independent runs of four Markov chains (Nabhitabhata and Chan-ard, 2005), T. were conducted for 10 million generations. uyenoi (Hernandez, 2016), or T. anguliceps POMCHOTE ET AL. — A NEW SPECIES OF TYLOTOTRITON FROM NAN, THAILAND 147 TABLE 1. Samples of Tylototriton species and related species used for molecular analyses. CAS = California Academy of Sciences; CIB = Chengdu Institute of Biology; CUMZ (A) = Natural History Museum of Chulalongkorn University Section Amphibians; KIZ = Kunming Institute of Zoology; KUHE = Graduate School of Human and Environmental Studies, Kyoto University; MVZ = Museum of Vertebrate Zoology, University of California, Berkeley; NMNS = National Museum of Natural Science, Taiwan; VNMN = Vietnam National Museum of Nature; ZMMU = Zoological Museum of Moscow State University. *Topotype. No. Species Voucher number Locality GenBank no. Source 1 T. phukhaensis sp. nov. CUMZ-A-7717 DPKNP, Nan, Thailand MN912573 This study 2 T. phukhaensis sp. nov. CUMZ-A-7718 DPKNP, Nan, Thailand MN912574 This study 3 T. phukhaensis sp. nov. CUMZ-A-7719 DPKNP, Nan, Thailand MN912575 This study 4 T. phukhaensis sp. nov. CUMZ-A-7720 DPKNP, Nan, Thailand MN912576