First female Achalinus sheni identified through DNA and scale analysis

Researchers have identified and described the first known female of the Chinese odd-scaled snake Achalinus sheni, providing the first female-based morphological reference for a species previously represented only by males. The adult female was collected with three males in Xinshao County, Shaoyang City, Hunan Province, China, during a 2022 herpetofaunal survey. Examination of its scales, proportions, coloration and mitochondrial DNA placed it within A. sheni. The finding also revealed a markedly short tail and a distribution of ventral and subcaudal scales that differed from the available male specimens, although the researchers cautioned that the apparent sex-related pattern is based on a single female and cannot yet be generalized.

Achalinus sheni was originally described in 2023 from five male specimens: four from Lianyuan City and one from Nanyue District, both in Hunan. The species was placed within Achalinus, the most diverse genus in the family Xenodermidae, whose members are commonly called odd-scaled snakes. The genus contains 28 recognized species and is distributed across eastern and southeastern Asia, including northern Vietnam, southwestern China and parts of Japan. The original description used mitochondrial cytochrome c oxidase subunit I, or COⅠ, sequence data to show that A. sheni was most closely related to A. yunkaiensis. It also distinguished the two species using the relative lengths of the supraocular and upper anterior temporal scales and the numbers of ventral and subcaudal scales in males.

Because the known series of A. sheni consisted entirely of males, the original work could not compare the sexes or determine whether the characters used to separate A. sheni from A. yunkaiensis remained diagnostic in females. The new material addresses that gap through a combined morphological and molecular analysis. The four Xinshao County snakes were fixed in 75% ethanol and deposited in the Vertebrate Zoology Laboratory at the College of Life Science, Hunan Normal University. DNA was extracted from preserved liver tissue, and a mitochondrial COⅠ fragment was amplified with the primer pairs Chfm4 and Chrm4. The resulting products were sequenced, compared with homologous sequences obtained from GenBank and analyzed using maximum-likelihood methods.

The phylogenetic evidence strongly associated the new female with known members of A. sheni. In the maximum-likelihood tree, the specimen HNNU2022001 clustered with the holotype and other previously identified specimens with a support value of 97. The methods section describes the assembled COⅠ sequences as 639 base pairs, while the phylogenetic figure caption reports a tree inferred from 629 base pairs. Uncorrected sequence distances between the new female and the known A. sheni specimens were 0.7–0.8%. By comparison, the distance between A. sheni and the sampled A. yunkaiensis was 6.2–6.5%. The tree was estimated in IQ-TREE 2 under the best-fit TIM3+F+I+G4 model selected with ModelFinder, and nodal support was assessed through 1,000 ultrafast bootstrap replicates.

The adult female was collected on April 11, 2022, at 27°23′58″N, 111°33′44″E and 420 metres above sea level. It had a total length of 345.1 millimetres, consisting of a 294.8-millimetre snout-to-vent length and a 50.3-millimetre tail. Its body was slender and cylindrical, its head was distinct from its neck and its tail was relatively short, accounting for 14.9% of total length. Previously described males had tail proportions of 18.3–22.4%, while the three new males measured 21.5–22.0%. The female’s tail also tapered abruptly, in contrast to the relatively elongated tails of the males. Because only one female was available, the study could not establish whether these differences consistently characterize the sexes.

Scale counts provided further detail about the female and the three accompanying males. The female had 173 ventral scales and 45 unpaired subcaudals, for a combined total of 218. The males had 156–158 ventral scales, 61–62 subcaudals and combined totals of 217–220. Thus, although the female and males had similar overall counts of ventral plus subcaudal scales, the female had more ventrals and substantially fewer subcaudals, consistent with her shorter tail. All four specimens had 23 dorsal scale rows at the neck, midbody and near the cloaca, an entire cloacal plate, one loreal scale, six supralabials and a 2+2+3 arrangement of temporal scales. The fourth and fifth supralabials contacted the eye in every specimen.

The female’s head measurements were also recorded in detail. Head length was 9.16 millimetres and head width was 3.95 millimetres, producing a head-length-to-width ratio of 2.32. Its horizontal eye diameter was 1.10 millimetres. The suture between the internasal scales measured 1.40 millimetres, nearly equal to the 1.31-millimetre suture between the prefrontals. The frontal was pentagonal and pointed backward, the parietals were bordered by elongated nuchal scales, and no preocular or postocular scales were present. Two anterior temporals touched the eye; the upper was much smaller than the lower, and the lower also contacted the parietal. The supraocular measured 1.49 millimetres compared with 1.63 millimetres for the upper anterior temporal, giving a ratio of 0.91.

Coloration in life provided another observable character. The female’s scales had a subtle iridescent quality that gave its dorsum a reflective brownish-black appearance. The five innermost dorsal scale rows were slightly darker, producing an inconspicuous longitudinal line over the vertebral region. Its chin shields were tan, and the ventral surface was predominantly off-white, with the edges of the ventral scales transitioning from gray-white to black. The underside of the tail was brownish. After preservation, the dorsal surface was uniformly brownish-black with slight iridescence, the chin shields were light brown, and the ventrals were generally pale brown with darker sides and grayish-white free margins. The researchers documented this coloration and provided photographs of the adult female from dorsal, dorsolateral and ventral perspectives.

The comparison with A. yunkaiensis showed that the new female retained several features used in the species diagnosis, but also revealed differences between the known females of the two species. Female A. sheni had five infralabials, whereas the compared female A. yunkaiensis had six. The supraocular-to-upper-anterior-temporal ratio was 0.91 in A. sheni and 0.55–0.65 in A. yunkaiensis. The A. sheni female also had 173 ventral scales and 45 subcaudals, compared with 144–156 ventrals and 51–55 subcaudals in female A. yunkaiensis. The new males from Xinshao County differed somewhat from the original males: they had 156–158 ventral scales rather than 161–170, while their 61–62 subcaudals were similar to or slightly higher than the original range of 55–61.

The study documents a single female specimen and therefore cannot establish the full extent of sexual variation within A. sheni, variation among populations or the causes of the observed differences in tail proportion and scale distribution. Its principal result is the first morphological and molecular identification of a female A. sheni, together with a direct comparison between the sexes represented in the available material. The new female expands the recorded character range for the species and provides a reference for recognizing the sex-specific anatomy seen in this individual. Establishing whether its short tail and scale arrangement are typical of female A. sheni will require additional female specimens, while the new locality also provides another verified occurrence in Hunan based on both morphology and mitochondrial sequence data.

Subject of Research: Morphological and mitochondrial identification of the first known female Achalinus sheni from Hunan, China

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

Article References: Ma, Y.-H., Liu, J.-Y., Li, H., Zhou, L., Xu, Y.-H., Peng, D.-Y., Zhang, Z.-Q., & Mo, X.-Y. (2024). First description of the female of Achalinus sheni (Serpentes, Xenodermidae), with expanded description of this species. Herpetozoa, 37, 189-195. https://doi.org/10.3897/herpetozoa.37.e124331

Image Credits: AI Generated

DOI: 10.3897/herpetozoa.37.e124331

Keywords: Achalinus sheni, odd-scaled snakes, Xenodermidae, female specimen, snake morphology, mitochondrial COⅠ, molecular systematics, Hunan, China, taxonomy, First, description