A new species of the Southeast Asian genus Opisthotropis (Serpentes: Colubridae: Natricinae) from western Hunan, China

A new species of natricine snake of the Southeast Asian genus Opisthotropis Günther, 1872 is described from western Hunan Province of China based on both mitochondrial DNA and morphological data. The new species is morphologically most similar and genetically most closely related to O. cheni Zhao, 1999 and O. latouchii (Boulenger, 1899), but possesses considerable genetic divergence (p-distance 5.1%-16.7%) and can be differentiated from all other congeners by a combination of the following morphological characters: (1) body size large (total length 514-586 mm) and strongly built; (2) dorsal scale rows 17 throughout, feebly keeled anteriorly and moderately keeled posteriorly; (3) ventral scales 147-152, subcaudal scales 54-62; (4) preocular absent, loreal elongated and touching orbit; (5) supralabials 8-9, fifth and sixth entering obit; (6) anterior temporals short, length 1.74-2.04 times longer than width; (7) maxillary teeth subequal, 28-30; (8) dorsal surface of head with distinct irregular yellow stripes and markings edged with ochre; (9) body with clear black and yellow longitudinal streaks, partly fused to several lighter patches or thicker stripes anteriorly; and (10) venter pale yellow, with asymmetric blackish speckles along outer margin. We present an updated diagnostic key to all members of the genus Opisthotropis, and recommendations on the ecological study for the group are provided.

Despite the remarkable ecological adaptations and rich diversity of Opisthotropis, little attention has been given to its taxonomy until recently (Chuaynkern et al., 2014;David et al., 2011;Iskandar & Kamsi, 2009;Murphy et al., 2008;Wang et al., 2017). This is particularly true for congeners in southern China. Prior to 2010, no modern taxonomic work on Chinese Opisthotropis had been conducted, and our understandings of the genus were based on study that dated back to the nineteenth century (Pope, 1935;Smith, 1943;Taylor, 1965;Zhao & Adler, 1993;Zhao et al., 1998;Zheng, 1992). Although a few recent taxonomic works using genetic methods have revealed surprising endemism and cryptic diversity within the genus, these studies have focused on areas around the Fujian-Guangdong Coast Subregion of southern China, with many areas in southern China remaining under-surveyed in regard to Opisthotropis diversity (Wang et al., 2017;Yang et al., 2011Yang et al., , 2013. As Opisthotropis congeners are ecological specialists with narrow niches and limited dispersal abilities, it is possible that previous records of widespread congeners may represent overlooked distinct and cryptic species (Wang et al., 2017;Zhao et al., 1998;Zhao, 2006).
During field surveys of Hunan Province in 2017, three adult specimens of Opisthotropis were collected in western Hunan, China. Detailed morphological comparisons and phylogenetic analyses showed that the western Hunan population of Opisthotropis represents a distinct evolutionary lineage that can be diagnosed readily from closely related congeners. Therefore, we describe the Hunan population of Opisthotropis as a new species herein.

Sampling
Three adult Opisthotropis specimens (two females and one male) were collected from Guzhang, western Hunan, China on 22, 23, and 24 August 2017 (Figures 1, 2, 3A, 4A, 6). After each collection, the temperature and pH of the water were measured immediately using a liquid-in-glass thermometer and digital pH meter (calibrated), respectively. After euthanization, liver tissues were taken and preserved in 95% ethanol for DNA extraction, with all specimens preserved in 10% formalin in the field and transferred to 75% ethanol for permanent storage after fieldwork. All vouchered specimens and tissue samples were deposited in the Museum of Herpetology, Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (CAS).

DNA extraction, amplification, and sequencing
Genomic DNA was extracted from macerated liver tissue samples using a TIANamp Genomic DNA kit (Tiangen Biotech, China), according to the protocols of the manufacturer. Mitochondrial gene cytochrome b (cyt b) was targeted and amplified using primers L14919 (5'-AACCACCGTTGTTATT CAACT-3') and H16064 (5'-CTTTGGTTTACAAGAACAATGC TTTA-3') (Burbrink et al., 2000;Guo et al., 2012). Polymerase chain reactions (PCR) were performed at 25 μL volume, and amplified DNA was produced after initial denaturing for 7 min at 94 °C, 41 cycles of denaturation for 40 s at 94 °C, annealing for 30 s at 46 °C, extension for 1 min at 72 °C, and final extension for 8 min at 72 °C. The PCR products were purified using   commercial kits and sequenced in both directions by an ABI 3730xL sequencer (Applied Biosystems, Foster City, CA, USA). Sequence editing and management were performed in Geneious Pro 4.8.4 (Kearse et al., 2012). All new sequences were deposited in GenBank (Table 1).
Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted. The DNA substitution model was calculated by PartitionFinder 2 (Lanfear et al., 2017) using Akaike Information Criterion (AIC), which is a GTR+I+G model for the first and second codon positions and a TIM+G model for the third position. The BI analyses were conducted using MrBayes 3.1.2, as described in Ronquist & Huelsenbeck (2003). Two independent runs of four Markov Chains for 10 000 000 generations were summarized by the BI, and sampled every 100 generations. The first 25% were discarded as "burn-in".
Bayesian posterior probability (BPP) was determined to test the confidence of tree topology, where nodes with BPP≥95% were considered strongly supported. Convergence and effective sample size (ESS) of the parameters were investigated in Tracer 1.6 (Rambaut & Drummond, 2013).   GenBank accession No. marked with * represents sequence used in the genetic distance analysis to represent respective species; "-" indicates missing voucher information.
The ML analyses were performed in RAxML 8.2.10 (Stamatakis, 2014) under the most complex substitution model (GTRGAMMA) based on the AIC model assessment results. Partitions were unlinked and bootstrap proportions (BSP) were investigated with 1 000 bootstrap replicates using the fast bootstrapping algorithm, otherwise under default parameters. Nodes of the ML tree with BSP≥70 were significantly supported.
Uncorrected pairwise distance (p-distances) of the sequenced cyt b data among congeners was calculated using MEGA 6 (Tamura et al., 2013).

Morphological analyses
In addition to the three newly collected specimens, a total of 33 vouchered specimens were also examined for morphological data (Appendix). As the genus is understudied and contains cryptic diversity, only topotypic or paratopotypic specimens were examined if available to avoid taxonomic confusion. Additional museum abbreviations include Yibin University, Yibin, China (YBU); Museum of Biology, Sun Yat-Sen University, Guangzhou, China (SYS); Institute of Ecology and Biological Resources, Vietnamese Academy of Science and Technology, Hanoi, Vietnam (IEBR); Zoological Museum, Vietnam National University, Hanoi, Vietnam (VNUH).
Measurements were taken by Jin-Long Ren with a digital slide-caliper to the nearest 0.1 mm, except for total lengths (TL), which were measured using a measuring tape to the nearest 1 mm. Measurement methods and their definitions followed Zhao (2006), and included: total length (TL), snoutvent length (SVL), tail length (TaL), head length (HL), and head width (HW). In addition, the following morphometric characters were examined in this study: rostral length (RL): distance from tip of snout to anterior edges of eyes; rostral width (RW): maximum distance between supralabials at the anterior edges of eyes; interorbital distance (IOD): distance between upper edges of eyes; eye width (EW): maximum horizontal eye width; distance between the lower margins of eye and of lip (SoL); maximum loreal length (LoL); maximum loreal depth (LoD); maximum anterior temporal length (ToL); and maximum anterior temporal depth (ToD).

RESULTS
For the 29 aligned sequences of the congeners, a total of 406 variable and 367 parsimony informative sites were identified. The uncorrected pairwise sequence divergence of 11 sampled Opisthotropis species ranged from 5.0% to 17.8% (Table 2). The three sampled individuals of the Hunan population shared identical haplotypes for cyt b and possessed a genetic divergence of 5.6% and 5.1% from O. cheni and O. latouchii, respectively. Such divergences are higher than the recognized species level in Opisthotropis (5.0% between O. cheni and O. latouchii), with the intraspecific genetic divergences of O. latouchii and O. cheni ranging from 0.0% to 0.5%. Table 2 Uncorrected pairwise sequence divergence among cyt b mtDNA gene sequences of Opisthotropis species For phylogenetic analyses, a majority rule consensus tree inferred from BI (-ln L=-6 531.011 3; average standard deviation of split support=0.002 005; ESS>200) was consistent with the ML tree ( Figure 5). Opisthotropis was recovered as monophyletic with current samplings. Although interspecific relationships among congeners within Opisthotropis were not fully resolved, the final consensus tree yielded high support (BPP>0.95; BSP>70) for key nodes concerning the relationship between the Hunan population with recognized congeners. The Hunan population was recovered as a monophyletic clade (BPP=1.00; BSP=100) within Opisthotropis, which was most closely related to O. cheni (BPP=0.97; BSP=93). Opisthotropis latouchii is the sister taxon to the node, and together all three species constitute the O. latouchii species group ( Figure 5).
Detailed morphological comparisons showed that the Hunan population possessed a suite of morphological characteristics that can be readily distinguished from all recognized congeners, including number of maxillary teeth, head scalation, and ornamentation patterns (Table 3; Figures 1-4). Therefore, according to both molecular and morphological evidence, we describe the Hunan population of Opisthotropis as a new species below. Diagnosis A large-sized species of Opisthotropis diagnosed by the following morphological characters: (1) head barely distinct from neck; (2) body and tail moderately slender; (3) prefrontal single, much broader than long; (4) nostrils directed upwards; (5) eyes small; (6) maxillary teeth subequal; (7) anterior neck dorsal scales smooth, middle body with faint keels, tending to moderately keeled rear body and on tail.

Coloration of holotype in life:
In life, the dorsal surface of the head is brownish yellow, mottled with small brownish black spots and patches. Lighter irregular markings are present on the internasals, prefrontal, and frontal, which do not sharply separate the darker center from the brownish yellow background color. Brownish ocher stripes are observed on the posterior edges of frontal and parietals, forming several irregular broken boundary lines. In front of the stripes on the parietals, six thicker oblique stripes are observed. Color patterns are similar on the lateral surface, supralabials 3-6 (3-7 on right) and infralabials 2-8 brownish yellow with anterior blackish borders. The blackish edge of upper part of supralabials 6-8 (7-9 on right), postoculars, and lower part of anterior temporal forming a thick "eyebrow" streak pattern, connecting with the outer most irregular black lateral stripe extended to tail tip. From supralabial 5 to neck, darker brownish yellow background color gradually transitions into light yellow. Tongue is purple with pink markings, gradually turning white at apex. Dorsal scales from row 3 to 15, each scale is black with a subtriangular or rectangular yellow center, thus constituting thirteen clear longitudinal stripes along the whole trunk just after the posterior edges of the parietals. Before 24th ventral position, dorsal scale rows 1 and 17 pale yellow entirely or marked with minute black spots, black upper edges gradually appearing on these two rows of scales after 24th ventral position, forming an extra yellow stripe along each side. Along the vertebral scales on the back, these yellow stripes fuse to seven conspicuous irregular patches or stripes anteriorly, only eight yellow stripes observed at the base of the tail. Chin and anterior venter pale yellow, venter becoming lighter posteriorly. The ventral scales are cream with bold yellow posterior margins, asymmetric black spots present along the outer margins, somewhat transparent. Subcaudals resemble ventrals but with a black longitudinal joining stripe after the eleventh subcaudal scale.
Coloration in preservation Specimens fixed in formalin and preserved in ethanol resemble the coloration of live animals. However, the yellow longitudinal stripes faded to a dull yellow hue, and the cream venter changed to uniform beige and was no longer transparent (Figures 2A, 2B).
Variation Paratypes generally resemble holotype in morphological characters (Figures 1C, 1D, 2C, 2D; Table 3). However, the male paratype CIB109998 differs from the female specimens (CIB109999 and CIB 110000) by smaller body size (TL 514 mm vs. 529-586 mm in females), shorter anterior temporals (ToL/ToD 1.74 vs. 2.02-2.04 in females), and rather irregular yellow patterns within each dorsal scale (vs. regular) ( Figures 1C, 1D, 2C, 2D). As the sample size is small, it is unknown whether such differences observed between the two sexes represent sexual dimorphism in this species.
Distribution and natural history This species is only known from the type locality presently (Figure 6). Opisthotropis zhaoermii sp. nov. inhabits small, fast-flowing mountain streams in forested areas, with water temperature and pH between 19.9-21.2 °C and 7.85-7.93, respectively. Being nocturnal, individuals were seen swimming at the edge of the backwater of travertine waterfalls from 2100h to 0100h at night (Figure 7). The holotype was collected during a heavy rainstorm that caused the water to become extreme turbid, with water temperature and pH measurements of 19.5 °C and 8.13, respectively.
When handled, individuals never stroked or displayed threating postures. Instead, individuals struggled violently and released a musky, pungent, and enduring defensive odor, much like that of O. balteata and O. kuatunensis (Pope, 1935). The scales of the snakes become dehydrated and crimped quickly after leaving the water for about 10 min, but recovered rapidly when returned to water. Similar to other congeners in China, the new species may prey on earthworms, tadpoles, freshwater isopods, crabs, and small fish (Pope, 1935). Other herpetofauna, including Quasipaa boulengeri, Odorrana schmackeri, Lycodon rufozonatum, L. fasciatus, and Deinagkistrodon acutus, were observed sympatric with the new species.
Etymology The specific epithet, zhaoermii, is derived from the name of an internationally renowned Chinese herpetologist, Prof. Er-Mi Zhao, who unfortunately passed away on 24 December 2016. Designation of this specific epithet honors his great contribution to herpetological research in China, specifically to his contribution to the taxonomy of Opisthotropis, including the first description of the sister species (i.e., O. cheni, see above) of the newly found species. We suggest Zhao's Mountain Stream Snake as its English common name, and "Zhao Shi Hou Leng She" (赵氏后棱蛇) as its Chinese common name.   Table 3).

Key to species of Opisthotropis
With the description of this new species, an updated diagnostic key to all species of the genus Opisthotropis is provided here to facilitate future taxonomic work.

DISCUSSION
Although recent taxonomic work has shed light on the taxonomy of Chinese Opisthotropis, research has been focused on the Pearl River Valley only (Wang et al., 2017;Yang et al., 2013), and diversity of the genus in most parts of southern China remained overlooked. Our discovery of the new species Opisthotropis zhaoermii in the Yangtze River Basin highlights the underestimated diversity of the genus in southern China and calls for further fieldwork and continuous taxonomic studies of the group in the under-surveyed regions of southern China. Despite the recent taxonomic discoveries of Opisthotropis in southern China and northern Indochina, many of the newly described species are known from only a handful of specimens, and in some case, from a single holotype from the type locality (David et al., 2011;Yang et al., 2013;Ziegler et al., 2008). The low numbers of vouchered specimens and geographic samplings have resulted in limited knowledge on the morphological variations, distribution patterns, and conservation of members of the genus. Furthermore, few studies have reported detailed natural history data of congeners, including color in life, behaviors, reproductive biology, and population statuses. As ecological niche differentiation may be a driver of species diversification in Opisthotropis (Yang et al., 2011), it is important to collect ecological data for different congeners. Here we reported on water temperature and pH data of an Opisthotropis species for the first time, and recommend that future studies collect ecological data of Opisthotropis in the field, including the addition of more parameters such as flow speed of water, canopy coverage, humidity, and prey community structures. We recommend future studies to explore the ecology of this unique group of aquatic snakes and investigate the possible ecological speciation mechanisms of the group.