Molecular Phylogenetics Of The Genus Thermophis And The Description Of A New Species

Hot-spring snakes, Thermophis, a relict genus endemic to the Tibetan Plateau, achieve the world's highest altitude distribution among all snakes, commonly restricted to the proximity of geothermal sites. There are 31 currently known distribution sites of hot-spring, among them 6 sites were discovered by mou expeditions. We found there are some characters differ from T. zhaoermii and T. baileyi.Where are the 3 snakes' systematic position?We sequcned 3 mt DNA genes and 1 n DNA gene from T. zhaoermii, T. baileyi and 3 specimans from Shangri-La. In order to find the molecular systematic position of the three snakes of this study, we retrieved cytb, ND4 and c-mos sequences from Gen Bank for 85 genera(Table 1) of Alethinophidia(primitive + advanced snakes). In order to maximize our dataset of combined genes, three different sequences from two congeneric species were combined to form supraspecific terminals at the generic level. Bayesian Inference(BI) and Maximum likelihood(ML) methods were used to construct phylogenetic trees. The three snakes clustered in the genus Thermophis with clear internal structure and high supports. The genus Thermophis clustered in Xenodontinae.Up to now, taxonomists still have been trying to reach a consensus on the concept of species and methods of species delimitation to determine the validity of a species, relaxed criteria may result in confusion, whereas overly strict criteria may not facilitate consistent communications and actions in the practice of taxonomy and conservation biology. Here, we propose an eclectic and feasible rule-viz., the “four-differences” rule-to determines the validity of a species, also apply to identify a new species. First and foremost, the morphological difference(s) compared with the closest species should be perceptible. To further confirm that the morphological difference(s) possess taxonomic significance rather than representing intraspecific polymorphism, we also need evidence of mitochondrial DNA(matrilineal divergence) and nuclear DNA differences(patrilineal divergence). Finally, the presence of geographical or ecological difference represents the potential for natural reproductive isolation. In this paper, we illustrate the utility of this rule in relation to the taxonomy of hot-spring snakes.For morphological study, we examined 23 characters from the 3 specimens, 12 T. zhaoermii and 11 T. baileyi. These three individuals all have 15 maxillary teeth, are distinguished from T. baileyi(21-24) and T. zhaoermii(16-17). T. shangrila sp. nov. is morphologically distinguished from similar species T. zhaoermii by several characters: a closer distance between the two eyes(distance between the two eyes/head width is 0.56 in females, 0.58 in male, vs. 0.59–0.61 in females, 0.64–0.73 in males), a wider rostral(rostral width/height is 1.70–2.40 in females, 2.10 in male, vs. 1.00–1.50 in females, 1.72–1.86 in males), and a wider mental(mental width/height is 1.85–2.05 in females, 1.45 in male vs. 1.45–1.77 in females, 1.15–1.30 in males). In females, a slightly longer head(head width/length is 0.62–0.65 vs. 0.68–0.71). In males, a more anterior occurrence of the reduction from 10(8, 6) to 8(6, 4) scales in each dorsal row on the tail(30 vs. 34.5–35, 49 vs. 52–61.5, 75 vs. 76.5–77).In order to detect mt DNA differences and n DNA differences, uncorrected pair-wise distances and unrooted trees of all sequences of each of those four genes of Thermophis(from this study and Genbank) were calculated. We found they have differences between 3 specimen and the two known species.The discovery reported in this study expands the distribution region of hot-spring snakes in a southeasterly direction. The new site is the southeastern-most corner of the Tibetan Plateau and the southern-most tip of the Hengduan Mts. The name “Hengduan” means “to transect” and “cut downward” in Chinese. Topographically, in this area, parallel mountain ranges are separated by deep, narrowly incised river valleys. With strongly geographic heterogeneity, Hengduan Mts. had naturally resulted in vicariance restricting gene flow. Such geographical difference could represent the potential for natural reproductive isolation.There are differences in morphology, mt DNA, n DNA, and geography between the new species and T. zhaoermii. This new species meets our proposed eclectic and feasible “four-differences” rule.We found that the quality of habitats around the hot springs has declined because of the increasingly serious exploitationof geothermal energy. The habitat faces an extremely high risk of extinction in the wild. During these fieldworks, only one extent of occurrence of hot-spring snakes had been found, where we had got three samples. The number of individuals considered to be facing a continuing decline. According to IUCN Red List Categories and Criteria(Version 3.1, Second edition, 2001), the new species should be listed as “Critically Endangered”(A2cd+3cd+4cd; B1ab(i, iii, v)).