﻿Discovery of a new cryptic Achalinus Peters, 1869 (Serpentes, Xenodermidae) species from Hunan Province, China

﻿Abstract A new species, Achalinusshenisp. nov., from central Hunan Province is described, based on the results of molecular systematics and morphological characters according to five specimens. Our molecular phylogeny inferred from the mitochondrial CO1 gene fragment revealed that this new species is most closely related to A.yunkaiensis, but a considerable amount of genetic divergence exists between them (p-distance ranging from 5.8% to 6.4%) and much distinct genetic divergence exists compared with other known Achalinus species (p-distance ranging from 10.4% to 15.8%), supporting its validity. Morphologically, it can be distinguished from its congeners by: (1) dorsal scales strongly keeled, 23 rows throughout the body, the outmost row smooth and significantly enlarged; (2) tail relatively short, TaL/TL 0.183 ~ 0.224; (3) the suture between internasals subequal to the suture between prefrontals; (4) loreal one, subrectangular, LorH/LorL 0.53 ~ 0.57; (5) ventrals 161–170, anal entire, subcaudals 55–61, not paired; (6) the length of supraocular equal to or longer than the length of upper anterior temporal; and (7) vertebral line inconspicuous and subcaudal streak absent. Currently, 27 species of Achalinus are known in the world, amongst which 20 species are distributed in China. Moreover, a key to species of the genus Achalinus is provided in this study.


Introduction
The odd-scaled snakes (burrowing snakes), genus Achalinus Peters, 1869, are a group of small to medium-sized, nocturnal, fossorial, low-aggressive and non-venomous snakes, widely distributed in Vietnam, China and Japan (Zhao et al. 1998;Zhao 2006).Currently, 26 species of this genus have been documented and more than half of them (17 species) were described in the past five years (Wang et al. 2019;Ziegler et al. 2019;Li et al. 2020;Luu et al. 2020;Miller et al. 2020;Hou et

Molecular phylogenetic analyses
Five specimens were collected in the Hunan Province of China: four specimens (ANU20230012-ANU20230015) were collected in Lianyuan City and one specimen (CIB 119043) was collected in the Nanyue District (Fig. 1).Snakes were humanely euthanised with an injection of 0.7% tricaine methanesulphonate (MS222) solution and fresh liver tissue was extracted and immediately preserved in 95% ethanol.The specimens were fixed in 10% formalin for one day, subsequently preserved in 75% ethanol and deposited in the Anhui Normal University Museum (ANU) and Chengdu Institute of Biology (CIB) of Chinese Academy of Sciences (CAS), respectively.Sampling procedures involving live snakes were in accordance with the Wild Animals Protection Law of China.
Genomic DNA was extracted from the preserved liver tissues using QIAamp DNA Mini Kit (QIAGEN, Changsheng Biotechnology Co. Ltd.).A fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene was amplified using the primer pairs: dglco and dghco (Meyer et al. 2005).The polymerase chain reaction (PCR) was performed in 25 μl reactant with the following cycling conditions: 95 °C for 4 min; 35 cycles of denaturing at 95 °C for 30 s, annealing at 48 °C for 30 s and extending at 72 °C for 60 s; and a final extending step of 72 °C for 10 min (Wang et al. 2019).PCR products were sequenced by Beijing Qingke New Industry Biotechnology Co., Ltd.
For our phylogenetic analysis, 38 sequences were used (Table 1), amongst which 33  were obtained from GenBank including 30 sequences of 23 Achalinus species and three sequences of Fimbrios klossi Smith, 1921, Parafimbrios lao Teynié, David, Lottier, Le, Vidal & Nguyen, 2015 and Xenodermus javanicus Reinhardt, 1836, which were used as outgroups (Ma et al. 2023b).CO1 sequences (618 bp) were input in MEGA11 (Tamura et al. 2021) and aligned by MUSCLE (Edgar 2004).Then we calculated the uncorrected pairwise distances (p-distance) in MEGA11.IQ-TREE 1.6.12 was performed to conduct the Maximum Likelihood (ML) analysis (Nguyen et al. 2015) under the best-fit model TN+F+I+G4 computed by ModelFinder according to Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al. 2017).Ultrafast Bootstrap Approximation (UFB) node support was assessed by using 5000 ultrafast bootstrap replicates and the UFB (%) ≥ 95 was considered significantly supported (Hoang et al. 2018).The single branch tests were conducted by SH-like approximate likelihood ratio test (SH-aLRT) by 1000 replicates and the nodal support (SH, %) ≥ 80 was also considered supported well (Stephane et al. 2010).The Bayesian Inference (BI) analysis was conducted via MrBayes (Ronquist et al. 2012) in PhyloSuite 1.2.3 (Zhang et al. 2020) by using a four chains run calculated for 10 million generations under the best model TN+F+I+G4, sampling every 1000 with the first 25% of samples discarded as burn-in and the nodal support Bayesian posterior probabilities (BI, %) ≥ 95 were considered significantly supported.
Morphological descriptions followed Zhao (2006) and Ma et al. (2023b): three measurement characters were measured to the nearest 0.1 mm using a Deli Stainless Ruler (No. 8460): snout-vent length (SVL), tail length (TaL) and total length (TL); other measurement characters were measured to the nearest 0.01 mm using a Deli Digital Vernier Caliper (DL91150): head length (HL), head width (HW), eye horizontal diameter (ED), loreal height (LorH), loreal length (LorL), length of the suture between internasals (LSBI), length of the suture between prefrontals (LSBP), length of supraocular (SPOL: horizontal distance between anterior and posterior tip of supraocular) and length of upper anterior temporal (ATUL: horizontal distance between anterior and posterior tip of upper anterior temporal).We also directly compared the length of the sutures between internasals and prefrontals (LSBI vs. LSBP).Scalation features and their abbreviations are as follows: loreals (Loreal), supralabials (SPL), infralabials (IFL), the number of infralabials touching the first pair of chin shields (IFL-1 st Chin), supraoculars (SPO), temporals (TEM), the number of anterior temporals touching the eye (aTEM-Eye), ventral scales (VEN), subcaudal (SC), entire or divided of the cloacal plate (Anal), dorsal scale rows (DSR) (counted at one-head-length behind the head, at midbody, at one-head-length before the cloacal plate).We also counted the number of maxillary teeth (MT) under the microscope.Bilateral scale counts were given as left/right.
Based on the molecular results above, these specimens are supported to be an unnamed taxon.
Therefore, combining the results of molecular systematics and morphological characters mentioned above, these five specimens, newly collected from Hunan Province, represent a new species and we describe it herein.Type material.Holotype.ANU20230014 (field number HSR23019, Fig. 3), an adult male, collected on 21 March 2023 (27°55′11″N, 111°55′3″E; 408 m a. s. l.), Qixingjie Town, Lianyuan City, Hunan Province, China by the team of Song Huang.
Etymology.The species name "sheni" is named for the memories of the Chinese herpetologist, Prof. You-Hui Shen (沈猷慧), who worked in Hunan Normal University and made great contributions to the herpetological research of China, particularly in Hunan Province where the new species is found.We suggest "Shen's Odd-scale Snake" or "Shen's Burrowing Snake" as its English name and "沈氏脊蛇" (Shěn Shì Jǐ Shé) as its Chinese name.the anterior two contact the eye, the lower anterior temporal much larger, the upper medium temporal much larger, the upper posterior temporal much larger and separated from the other side one by one scale.Supralabials 6, 4 th -5 th contact the eye, the last one much elongated.One mental.Two chin shields, the anterior pairs longer than the posterior pairs.Infralabials 5, the first one contact with each other after the mental and before the 1 st chin shields, 1 st -3 rd touch the 1 st chin shields.
Colouration of holotype in life.Scales tinged weakly iridescent and metallic lustre.Dorsum dark brown and the five innermost dorsal scale rows a little darker, forming an inconspicuous longitudinal vertebral line.Chin shields are tan.Ventrals generally light brown, darker on both sides, free margins of ventral scales greyish-white.Ventral side of tail brownness.
Colouration of holotype in preservation.The dorsal surface of the body uniformly brownish-black, slightly tinged with iridescence and the longitudinal vertebral line a little darker.Chin shields light brown.Ventrals generally creamybrown, darker on both sides, free margins of ventral scales greyish-white.Ventral side of tail light brown.
Variation.Measurements, body proportions and scale counts are listed in Table 3.All paratypes are very similar to the holotype, except in the following: (1) para- The native vegetation in the type locality is subtropical evergreen broad-leaved forests.Areas near the locality where the specimen CIB 119043 was collected is largely covered with artificial coniferous forest dominated by Cryptomeria spp.This new species' population status requires further investigation.The conservation status for the new species is recommended to rate as data deficient (DD).

Discussion
The description of A. sheni sp.nov.brings the total number of Achalinus known species to 27, with 20 species distributed in China, amongst which 16 species are endemic to China.Amongst these, four Achalinus species have been reported in the Hunan Province, A. hunanensis (the north mountain area in western Hunan Province), A. spinalis (the mountain area in north-western Hunan Province, the west mountainous and hilly areas in southern Hunan Province and the north mountainous and hilly areas in eastern Hunan Province), A. jinggangensis (the east mountainous and hilly areas in southern Hunan Province) and A. yunkaiensis (the southern mountain area in western Hunan Province) (Gao et al. 2022;Ma et al. 2023a) and the description of A. sheni sp.nov.(the hilly area in central Hunan Province) raises this number to five.
Achalinus is a group of poorly-known snakes as many species only have a single voucher specimen: A. damingensis, A. panzhihuaensis, A. pingbianensis, A. timi, A. tranganensis, A. vanhoensis and A. zugorum (Ziegler et al. 2019;Li et al. 2020;Luu et al. 2020;Miller et al. 2020;Hou et al. 2021;Ha et al. 2022; Yang et al. 2023) and several species (e.g. A. hainanus and A. werneri) do not have any sequence data accessioned.Consequently, this lack of adequate taxonomic sampling and genome-scale data results in our current situation where the population status, distribution pattern and evolution history of taxa within this genus are unclear (Miller et al. 2020).Therefore, it is paramount to conduct further survey work in these regions to learn more about these snakes.

Figure 1 .
Figure 1.Distribution of Achalinus sheni sp.nov.and A. yunkaiensis.A. sheni sp.nov.: the type locality (red star) and another site (red dot). A. yunkaiensis: the type locality (black star) and other three sites (black dots).

Figure 2 .
Figure 2. Phylogenetic tree of the genus Achalinus inferred from the CO1 gene fragment (618 bp) using Maximum Likelihood.The support values of each node present on the tree: SH / UFB / BI (the ones lower than 50 are displayed as "-").A. yunkaiensis is noted in blue and A. sheni sp.nov. is noted in red.

Figure 3 .
Figure 3. Holotype (ANU20230014, adult male) of Achalinus sheni sp.nov.A dorsolateral view B ventral view C right side of middle body view D light side of head view E right side of head view F dorsal head view G ventral head view.Photos by Yu-Hao Xu.

Table 1 .
Localities, voucher information, GenBank numbers and references for all samples used in this study.

Table 5 .
Comparisons of main morphological characters of Achalinus sheni sp.nov.and A. yunkaiensis.