First description of the female of Achalinus sheni (Serpentes, Xenodermidae), with expanded description of this species

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Introduction
Achalinus Peters, 1869, commonly known as the oddscaled snakes, is the most diverse genus of the family Xenodermidae (Uetz et al. 2022).It contains 28 recognized species, and is widely distributed in eastern and southeastern Asia, ranging from northern Vietnam to southwestern China, and partly into Japan, from which 21 species occur in China.In recent years, with extensive sampling and molecular phylogenetic methods, more than 20 members of the genus Achalinus have been discovered and described (Wang et al. 2019;Ziegler et al. 2019;Li et al. 2020;Luu et al. 2020;Miller et al. 2020;Hou et al. 2021;Huang et al. 2021;Li et al. 2021;Ha et al. 2022;Yang et al. 2022;Ma et al. 2023aMa et al. , 2023b;;Yang et al. 2023;Zhang et al. 2023;Li et al. 2024).
During a herpetofaunal survey of Hunan, China in 2022, one female and three male Achalinus were collected from Xinshao County, Shaoyang City (Fig. 1).Morphological and molecular phylogenetic analyses revealed that the specimens are A. sheni.Here we provide morphological data for the newly collected female specimen of A. sheni , and extend the description of this species.

Sampling
Four odd-scaled snake specimens were collected from Xinshao County, Shaoyang City, Hunan Province, China (HNNU2022001-HNNU2022004). The four specimens were fixed in 75% ethanol and deposited in the Vertebrate Zoology Laboratory, College of Life Science, Hunan Normal University.

Morphological examination
Morphological descriptions followed Zhao (2006) and Ma et al. (2023b).Abbreviations in this study are as follows: snout-vent length (SVL): snout-vent length from tip of snout to anterior margin of the cloaca; tail length (TaL): tail length from posterior margin of cloaca to tip of tail; total length (TL): from snout tip to tail end; head length (HL): from the tip of snout to the posterior margin of mandible; head width (HW): from the widest part of the head in dorsal view; eye horizontal diameter (ED): from the most anterior edge of the eye to the most posterior edge; loreal height (LorH): measured from the highest point to the lowest point of the loreal in lateral view; loreal length (LorL): from the most anterior edge of the loreal to the most posterior edge of the loreal in lateral view; length of the suture separating the internasals (LSBI); length of the suture separating the prefrontals (LSBP); length of the supraocular (SPOL): horizontal distance between the anterior and posterior tips of the supraocular, and length of the upper anterior temporal (ATUL): horizontal distance between the anterior and posterior tips of the upper anterior temporal.We also directly compared the length of the sutures between the internasals and prefrontals (LSBI vs. LSBP).

Phylogenetic analyses
Genomic DNA was extracted from preserved liver tissue using the TIANamp Genomic DNA Kit.The fragment of the mitochondrial DNA gene encoding cytochrome c oxidase subunit Ⅰ (COⅠ) was amplified using the primer pairs Chfm4 and Chrm4 (Che et al. 2012).The PCR products were sequenced at Shanghai Map Biotech Co., Ltd.The homologous sequences of the Achalinus species and the outgroups species were downloaded from GenBank (Table 1).
The COⅠ sequences (639 bp) were assembled using SeqMan in the DNASTAR software package (Burland, 2000), and compared and aligned using MEGA 7 software (Kumar et al. 2018).The uncorrected pairwise distances (p-distance) were calculated in MEGA 7. Maximum likelihood analysis (Nguyen et al. 2015) was executed using IQ-TREE 2 under the best-fit model TIM3 + F + I + G4 selected by Modelfinder according to AIC.Nodal support was estimated by 1,000 bootstrap replicates using the ultrafast bootstrap feature.

Results
Maximum likelihood trees showed consistent topology.The newly collected female specimen clustered with the specimens (including the holotype) of Achalinus sheni and demonstrated strong support (ML = 97, Fig. 2).The genetic distance (uncorrected p-distance) between the newly collected female specimen and the specimens (including the holotype) of A. sheni was only 0.7-0.8%(Table 2).Description of the female specimen.Adult female with a total length of 345.1 mm (SVL 294.8 mm and TaL 50.3 mm), tail relatively short, TL/ToL 0.149, body slender and cylindrical.Head distinct from neck, rostral small, triangular, only the upper tip is visible from above.Head length 9.16 mm, head width 3.95 mm, HL/HW 2.32.Eyes small, eye width 1.10 mm.Length of the suture between the internasals (LSBI 1.40 mm) subequal to the length of the suture between the prefrontals (LSBP 1.31 mm).Frontal pentagonal pointed backwards, much shorter than the parietals; each parietal bordered with an elongated nuchal, with no preoculars or postoculars.Nostril in anterior portion of nasal scale, posterior margin of nostril with a distinct nostril cleft.A single loreal scale present, extending from the nasal to the eye, distinctly wider than high.Temporals 2+2+3.aTMPs elongated, upper aTMP much smaller than the lower aTMP; upper aTMP and lower aTMP in contact with eye, lower aTMP also in contact with parietal scale.Supralabials 6, 4 th -5 th contact the eye, the last much elongated.A single mental scale present.Two chin shields, the anterior pair longer than the posterior pair.Infralabials 5, the first contacting each other posterior to the mental and anterior to the 1 st chin shields, 1 st -3 rd contact the 1 st chin shields.Dorsal scales 23-23-23, strongly keeled, dorsum with an inconspicuous longitudinal vertebral stripe.Ventrals (VEN) 173.Subcaudals (SC) 45, not paired.Cloacal plate (CP) entire (Table 3).

Achalinus sheni
Coloration of the female specimen in life.Scales possess a subtle iridescent quality which gives the dorsum a distinctive reflective brownish-black appearance.Dorsum dark brown and the five innermost dorsal scale rows a little darker, forming an inconspicuous longitudinal vertebral line.Chin shields are tan.On the ventral surface, an off-white shade prevails, with the edges of the ventral scales gradually transitioning from gray-white to black.Ventral side of tail brownish.
Coloration in preservative.(Fig. 3) The dorsal surface of the body uniformly brownish-black, slightly tinged with iridescence and the longitudinal vertebral line appears a little darker.Chin shields light brown.Ventrals generally pale brown, darker on both sides, free margins of ventral scales grayish-white.Ventral surface of tail light brown.
Extended diagnosis.Upon examining the additional adult males of Achalinus sheni from Xinshao County, it was observed that they possessed fewer ventral scales compared to specimens from the type locality.However, the number of subcaudal scales remained approximately the same for individuals from both locations.Upon inspection of adult female A. sheni collected in Xinshao County, it was noted that it exhibited an extremely short tail and consequently fewer subcaudals and more ventral compared to adult male individuals.
Comparison.Compared to male individuals of A. sheni and A. yunkaiensis, female individuals of both species exhibit similar differences in the number of ventral scales and subcaudals.Compared to males, both female A. yunkaiensis   and female A. sheni have fewer sublabial scales, which is consistent with the common characteristics of this genus.However, the number of sublabial scales of A. sheni is even less than that of female A. yunkaiensis (Table 4).

Discussion
Only five male specimens of the Achalinus sheni have been described in previous studies.This study reports the first discovery of a female of the same species and provides a detailed description and photograph of the female.In terms of morphological characteristics, the male specimens collected in Xinshao County are close to the holotype specimen from Lianyuan City.Some differences were noted in the female specimen: Tail relatively short, TL/ToL 0.149, which was different from that of the male specimens (0.183-0.224); the tail of the female specimen is suddenly tapered, distinct from the elongated tail of the male specimens.Xinshao County and Lianyuan City are geographically adjacent, and the climate is not obviously different.Achalinus sheni is fossorial and difficult to find.Only one female specimen was collected in this study.Whether the apparent differences between the sexes of this species are consistent, whether there are further differences, and the reasons for these differences have not yet been fully investigated and analyzed.

Figure 1 .
Figure 1.Sampling sites of Achalinus sheni: solid red star, the locality of the holotype; solid red circle, the locality of the paratype; solid green triangle, the locality of the newly collected female specimen.

Figure 2 .
Figure 2. Phylogenetic tree of the genus Achalinus inferred from COⅠ gene fragments (629 bp) using Maximum Likelihood.The numbers above the branches represent the supporting values.

Figure 3 .
Figure 3. Adult female (HNNU2022001) of Achalinus sheni.A. Dorsolateral view; B. Ventral view; C. The tail of the male and female; D. Dorsal head view; E. Ventral head view; F. Light side of head view; G. Ventral head view.Photos by Le-Qiang Zhu.

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