﻿A new species of Achalinus Peters, 1869 (Squamata, Xenodermidae) from Hunan Province, China

﻿Abstract A new species, Achalinushunanensissp. nov., is described from middle and western Hunan Province based on the results of molecular systematics and morphological characters. It diverges from known congeners by a significant genetic divergence (p-distance 3.2%–16.9% based on CO1 mitochondrial gene), and it can be distinguished from all known congeners by the following morphological characters: (1) all dorsal scales strongly keeled, 23 rows throughout the body, the outmost one strongly keeled and enlarged; (2) tail relatively short, TaL/TL 0.221 ~ 0.225; (3) maxillary teeth 23; (4) the suture between internasals 2 × as long as that between prefrontals; (5) loreal one, subrectangular, LorH/LorL 0.62 ~ 0.70; (6) supralabials 6, the 4th and 5th touch the eye; (7) the two anterior temporals in contact with eye; (8) ventrals 163–165, subcaudals 69–72, not paired. This raises the number of known species of Achalinus to 24.

During our field work, two specimens were collected from Hunan Province, China (Fig. 1).They could be assigned to Achalinus by body small, slender, and cylindrical; whole body scales strongly keeled, lanceolate-shaped, and metallic luster; and preocular and postocular absent, loreal and temporals contacting the eyes directly.However, further morphological and molecular analyses revealed that these specimens comprise a separate taxon from other known species.Thus, we described them as a new species herein.

Molecular phylogenetic analyses
Two specimens of the genus Achalinus were collected in Hunan Province, China: CIB 119039 was collected in Huaihua City, and CIB 119040 was collected in Ningxiang County, and they were deposited in Chengdu Institute of Biology (CIB) of Chinese Academy of Sciences (CAS).Genomic DNA were extracted from preserved muscle tissues of them using QIAamp DNA Mini Kit (QIAGEN, Changsheng Biotechnology Co. Ltd).A fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1) was amplified using the primer pairs dglco and dghco (Meyer et al. 2005).The polymerase chain reaction (PCR) was performed in 25 μL of 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.
CO1 sequences (681 bp) were input in MEGA11 (Tamura et al. 2021) and aligned by MUSCLE (Edgar 2004), and then the uncorrected pairwise distances (p-distance) were calculated in MEGA11.IQ-TREE 1.6.12 was used to conduct the maximum likelihood (ML) analysis (Nguyen et al. 2015) under the best-fit model TN+F+I+G4 selected by Modelfinder according to BIC (Kalyaanamoorthy et al. 2017).Ultrafast Bootstrap Approximation (UFB) node support as assessed by using 5000 ultrafast bootstrap replicates and the UFB ≥ 95 was considered significantly supported (Hoang et al. 2018).The single branch tests were made using SH-like approximate likelihood ratio test (SH-aLRT) via 1000 replicates, and the SH ≥ 80 was also considered supported well (Stephane et al. 2010).For Bayesian inference (BI), the best-fitting model HKY+I+G was selected by jModelTest 2.1.10identified via BIC (Darriba et al. 2012) on CIPRES (Miller et al. 2010).The Bayesian inference analysis was conducted using MrBayes v. 3.2.1 (Ronquist et al. 2012) under HKY+I+G this model, and four chains run was calculated for 10 million generations, sampled every 1000 with the first 25% of samples discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of ≤ 0.005.Bayesian posterior probabilities (BI) ≥ 0.95 were considered supported well.
We also estimated divergence time by BEAST v. 2.6.7 using CO1 sequences dataset (Bouckaert et al. 2019).Three calibration constraints were used: the divergence time between Achalinus and other Xenodermatidae: 38.6 Mya; the divergence time between Xenodermus and Fimbrios + Parafimbrios: 29.67 Mya; and the divergence time between Fimbrios and Parafimbrios: 17.66 Mya (Kumar et al. 2022).Two independent searches of 20 million generations were conducted, sampling every 1000 iterations with 25% of the initial samples discarded as burn-in.Tracer v. 1.7.2 was used to evaluate estimate ESS for all parameters (Rambaut et al. 2018).

Molecular systematics
All Achalinus samples cluster in a monophyletic group with high supporting values (SH 100/ UFB 100/ BI 1), and they can be divided into six clades, although the relationships among these clades are still unresolved (Fig. 2   The genetic distances range from 5.0% (A.timi and A. vanhoensis) to 18.1% (A.dehuaensis and A. meiguensis) among the known Achalinus species studied in this work (Table 2), of which, within the Clade F, the genetic distances among the four known species range from 5.8% (A.ningshanensis and A. yangdatongi) to 9.6% (A.juliani and A. ningshanensis), while the lineage composed by those two newly collected specimens range from 3.2% to 8.8% divergent from congeners.
The results above indicate that the Hunan samples are close to the species A. ningshanensis but consist independent evolution lineage.

Species
Combined the results of molecular systematics and morphological characters above, the specimens newly collected in this work represent a new species, and we describe it herein.Etymology.This new species is named after its known distribution range, which is endemic to Hunan Province.The Chinese name is suggested as "湖南脊蛇" (Hú Nán Jǐ Shé) and the English name "Hunan Odd-scale Snake" or "Hunan Burrowing Snake" is suggested.Temporals 2+2+4, 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 two small scales which contact the parietals.Supralabials 6, 4 th and 5 th contact the eye, the last one much elongated.One mental.Two chin shields, similar length.Infralabials 5/6, the first one contact with each other after the mental and before the 1 st chin shields, 1 st -3 rd /1 st -4 th touch the 1 st chin shields.
Dorsal scales lanceolate and strongly keeled; 23 rows throughout the body; those of the outmost rows on both sides significantly enlarged and strongly keeled.Ventrals 163, with two preventrals; anal entire; subcaudals 69, not paired.
Coloration of holotype.In life, dorsum dark, slightly metallic, vent blackbrown, dark brown near the margin, grey in the margin.A yellowish brown patch on the head occipital.The head ventral anterior part dark brown and posterior part yellowish white (Shu et al. 2014).In preservation, dorsum brown, vent anterior part grey and posterior part light brown.Ventral side of tail brown.The head ventral anterior part brown and posterior part grey.
Distribution and habits.Achalinus hunanensis sp.nov. is currently only known from Hunan Province, China: Hecheng District, Huaihua City and Ningxiang County, Changsha City (880-1020 m a.s.l.).The holotype was found at night, near a mountain stream (AT 24 °C, RH 80%) with shrubs under subtropical evergreen broadleaves forest.It was moving from leaf litter to the stream.Earthworms were found at the same place, which we speculated as its prey (Shu et al. 2014).

Discussion
Based on molecular evidence, the newly collected Achalinus specimens in this study are most closely to A. ningshanensis but a genetic differentiation (p-distance 3.2%) already exists between these two groups (Fig. 2, Table 2).The newly collected Achalinus specimens and its sister group A. ningshanensis have separate distribution ranges at south of Yangtze River and north of Yangtze River, respectively, isolated by Three Gorges (Fig. 1).In addition, their estimated divergence time was at 0.48 Mya (Fig. 3), which broadly coincides with the formation of Three Gorges of the Yangtze River (0.30 ~ 0.12 Mya) (Zhang et al. 2018), hindering their communication and driving allopatric speciation.Moreover, combining their distinct morphological differences (Table 5), we describe them as a new species.Currently, 24 Achalinus species are reported.
Achalinus ater was first recorded in Hunan Province only based one specimen (Shen et al. 2014;Shu et al. 2014;Gao et al. 2022).However, this study found that this record was a misidentification and this specimen was designed as the new species holotype.Therefore, A. ater recorded on Hunan reptile check list should be transferred as A. hunanensis.
Due to the secretive life history and morphological similarities, many cryptic species may be "hidden" within known widely distributed species, such as A. spinalis, A. rufescens, and A. ater (Wang et al. 2019;Yang et al. 2022), and the description of A. hunanensis sp.nov.indicated that further study is necessary to conduct by using different Achalinus species and geographic populations to revise the mystery snakes and reveal their evolutionary history.

Figure 2 .
Figure 2. Phylogenetic tree of the genus Achalinus inferred from CO1 gene fragment (681 bp) using Maximum Likelihood.The tree nodes present the supporting values: SH-like approximate likelihood ratio test, Ultrafast Bootstrap Approximation and Bayesian posterior probabilities, respectively (SH, %/UFB, %/BI) (the ones lower than 50 are displayed as "-").Achalinus ningshanensis is noted in blue and A. hunanensis sp.nov. is noted in red.

Figure 3 .
Figure 3. Divergence date estimation (Mya) and 95% HPD (in square bracket) of Clade F. Achalinus ningshanensis is in blue and A. hunanensis sp.nov. is in red.

Figure 4 .
Figure 4.The holotype (CIB 119039, subadult male) of Achalinus hunanensis sp.nov.A dorsolateral view B ventral view C right side of middle body view D dorsal head view E left side of head view F right side of head view G ventral head view.Photographs by SCS.

Figure 5 .
Figure 5.The paratype (CIB 119040, subadult male) of the Achalinus hunanensis sp.nov.A dorsolateral view B ventral view C dorsal head view D right side of head view E ventral head view.Photographs by SCS.

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