﻿A new species of the genus Hebius (Squamata, Natricidae) from Yunnan, China

﻿Abstract A new species of the genus Hebius Thompson, 1913 is described from Yingjiang County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China, based on molecular and morphological evidence. It can be distinguished from its congeners by the following set of characters: (1) dorsal scale rows 19–17–17, feebly keeled; (2) ventrals 146–151; (3) nasal complete, nostril in the middle of the nasal; (4) supralabials 9, the fourth to sixth in contact with the eye; (5) infralabials 10–11, the first 5 touching the first pair of chin shields; (6) preoculars 2; (7) postoculars 3; (8) temporals 3, arranged in two rows (1+2); (9) maxillary teeth 31, the last 4 slightly enlarged, without diastema; (10) tail comparatively long, TAL/TL ratio 0.334 in the male; (11) dorsolateral series of irregular orange or ochre yellow blotches, extending from the neck to the posterior part of the tail; and (12) venter pale orange, tips of ventrals with subrectangular black blotches. All Hebius specimens were strongly recovered as monophyletic, in which Hebiustaronensis (Smith, 1940) and Hebiusvenningi (Wall, 1910) were monophyletic as sister to the Yingjiang County specimens. According to the p‐distance of cytochrome b, the new species differs from its congeners by 9.7–15.4%.

Currently, there are 51 valid species in the genus Hebius, of which 26 are known to occur in China (Hauser et al. 2022;Li et al. 2022;Ma et al. 2023;Xu et al. 2023;Uetz et al. 2024).Furthermore, recent morphological and molecular phylogenetic analyses of Hebius have shown that the weak species delimitation within this genus may be due to underestimated diversity (Guo et al. 2014;Kizirian et al. 2018;Liu et al. 2018;Zhou et al. 2019;Hou et al. 2021).
Our morphological and molecular results support the presence of a new snake species, based on two specimens collected from Yingjiang County, Yunnan Province, China (Fig. 1) during field surveys in July 2023 and February 2024.The specimens could be identified as members of Hebius by having the following combination of morphological characters: (1) 2 supralabials in contact with nasal; (2) maxillary teeth in a continuous series, gradually larger posteriorly in the series or the last two teeth abruptly enlarged, the diastema before the distinctly enlarged posterior maxillary teeth absent; (3) internasals broad anteriorly, nostrils lateral; and (4) color pattern usually comprising a dorsolateral series of dark dots, forming two longitudinal stripes.However, these specimens could not be assigned to any known species (Guo et al. 2014;Ren et al. 2018).Furthermore, molecular analyses also revealed that the Yingjiang County specimens differed from those of other congeners.

Sampling
One drowned adult female and one road-killed subadult male specimens were collected from Yingjiang County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China.Sex was determined by tail dissection.Liver tissues were extracted and preserved in 95% ethanol.The specimens were preserved in 75% ethanol, and deposited in the Anhui Normal University Museum (ANU) and Qinghai University Museum (QHU).All sampling and procedures involving snake specimens were performed in accordance with the Wild Animals Protection Law of the People's Republic of China and approved by the Institutional Ethics Committee of Anhui Normal University (protocol code AHNU-ET2021025) and Qinghai University (protocol code SL-2023028).
For phylogenetic analysis, 56 sequences were selected (Table 1), among which 54  were obtained from the National Center for Biotechnology Information (NCBI), including 51 sequences from 31 Hebius species and three outgroups: Trachischium monticola (Cantor, 1839), Herpetoreas platyceps (Blyth, 1854), and Herpetoreas burbrinki Guo, Zhu, Liu, Zhang, Li, Huang & Pyron, 2014, and aligned using MEGA X software (Kumar et al. 2018).Tree inference was performed in IQ-TREE v. 1.6.12(Nguyen et al. 2015) under the maximum likelihood (ML) model, and 5000 replicates of ultrafast bootstrap were used to estimate the Ultrafast Bootstrap Approximation (UFB) node support.The SH-like approximate likelihood ratio test (SH-aLRT) was conducted with 1000 replicates.In addition, we calculated the uncorrected pairwise distances (p-distances) using the MEGA X software (Kumar et al. 2018).

Morphological examination
Morphological characters were described for the newly collected specimen and compared with other key references (Günther 1875;Wall 1910;Wall 1925;Bourret 1934;Taylor 1934;Gressitt 1937;Smith 1940;Dowling 1951aDowling , 1951b;;David and Das 2003;Zhao 2006;David and Vogel 2010;Guo et al. 2014;Liu et al. 2018;Ren et al. 2018;Purkayastha and David 2019;Zhou et al. 2019;Ziegler et al. 2019;David et al. 2021;Huang 2021;Hou et al. 2021;Hauser et al. 2022;Li et al. 2022;Ma et al. 2023;Xu et al. 2023).The measurements and scale counts followed those of Dowling (1951aDowling ( , 1951b)), Zhao (2006), andHuang (2021).A ruler with 1 mm accuracy was used to measure the snout-vent length (SVL), measured from the tip of the snout to the anterior edge of the vent; tail length (TAL), measured from the anterior edge of the vent to the tip of the tail; and total length (TL), defined as the sum of the SVL and TAL.All other measurement characteristics were measured to the nearest 0.01 mm using digital calipers: head length (HL), measured from the tip of the snout to the posterior margin of the mandible; head width (HW), measured from the widest part of the head in dorsal view; and eye diameter (ED), measured from the most anterior corner of the eye to the most posterior corner.Scalation features and their abbreviations are as follows: supralabials (SL); infralabials (IL); loreals (LOR); preoculars (PRO); postoculars (PO); Chin; infralabials touching the first pair of chin shields (IFL-1 st Chin); temporals (TEMP); supraoculars (SPO); and three dorsal scale row (DSR) counts: 1) counting from one head length behind the head, 2) at midbody (namely at SVL/2), and 3) at one head length before the vent; ventral scales (VS); cloacal plate (CP); and subcaudal (SC).In addition, we also examined the number of maxillary teeth (MT).

Phylogenetic relationships
The ML tree was reconstructed from a fragment of the mitochondrial Cyt b gene (Fig. 2).Due to the poor quality at both ends of the newly generated sequences, we cut the sequences to obtain a final length of 725 base pairs (bp).

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9.8-10.9Etymology.The specific name citrinoventer comprises the Latin words "citrinus" (yellowish-orange or orange) and "venter" (the belly or underside), based on the pale orange venter of the new species.According to its type locality Yingjiang County, Yunnan Province, China, the name we suggest is Yíng Jiāng Fù Liàn Shé (盈江腹链蛇) in Chinese and Yingjiang Keelback Snake in English.Comparisons.In many characters, Hebius citrinoventer sp.nov. is similar to H. venningi (Wall, 1910) and H. taronensis (Smith, 1940).However, the new species can be distinguished from H. venningi by (1) 19 DSR on the anterior part of the body (vs.17  6) venter pale orange, tips of ventrals with subrectangular black blotches (vs.pale areas of the venter are yellowish-ochre or yellowish-brown).For more detailed information and visual comparisons, please refer to Table 3 and Fig. 6.
Head scalation: Rostral pentagonal, wider than high, visible from above; nasal entire, subpentagonal, about twice as wide as high; internasals 2, trapezoidal, in broad contact with each other, narrowing anteriorly; prefrontals 2, pentagonal, wider than long, in contact with loreal; frontal narrow, pentagonal, longer than wide, shield-like, slightly concave in the middle on both sides; SPO 1 on each side, hexagonal, much longer than wide; LOR 1/1, subrectangular, wider than long; PRO 2/2, upper one larger than lower one; PO 3/3; SL 9/9, the first 2 in contact with nasal, the 2 nd to 4 th in contact with the loreal, 4 th to 6 th entering orbit, the 7 th and 8 th largest; TEMP 3/3, arranged in two rows (1+2), the anterior temporal long and trapezoidal; chin shields in 2 pairs, the posterior pair longer than anterior one and separated by several small scales; IL 11/11, first pair in contact behind the mental, 1 st to 5 th touching the first pair of chin shields, the 5 th and 7 th largest.
Coloration of the fresh specimen: Dorsal surface of the head is olive-brown and scattered with pale-brown vermiculate stripes or irregular blotches.A pale, irregular yellow-ochre oblique streak is directed upward on both sides of the head, extending from the temporal region to the nape.The upper half of the 1 st to 8 th SL is olive-brown, the lower half is pale brown, and the 9 th is completely olive-brown.Ventral surface of head creamy yellow, the edges of partial scales have irregular black-gray patches.
Body olive-brown, darker on the top than on the sides.A faint, yellow-ochre or rusted dorsolateral stripe extends from the neck to the end of the tail, on the upper part of the 5 th to the lower part of the 7 th scale rows in the anterior part of the body, and the upper part of the 4 th to 6 th in the middle and posterior parts of the body, accompanied by a series of conspicuous, pale orange or ochre yellow irregular blotches, about two scales in diameter.The orange or ochre yellow blotches are not symmetrically distributed on both sides of the body but are arranged in a staggered manner, that is, the blotch on the left side of the body corresponds to the area between the two blotches on the right side of the body, and vice versa.Above and below the orange blotch, there is a slightly smaller, dull blackish-brown irregular blotch.The blackish-brown blotches above are arranged in a staggered pattern in the middle of the body, forming a checkered pattern with the background color.
Ventral anterior pale orange, darker toward the rear, and scattered with a few small black spots.The outermost edge of the ventral is black.Outer one-sixth of the ventrals with subrectangular black blotches on each side, producing an irregular, continuous ventrolateral stripe, which merges with the dark ventral edge in the posterior part of the body.The ventral surface of the tail is uniform pale orange with black-brown edge; a thin, brown-black stripe extends on the ventral part of the tail, formed by the inner margins of the SC, extending from the 1 st SC to the end of the tail.
Coloration in preservation: In preservation, the background color of the dorsal body changed to brownish-black, and the checkered pattern on dorsal surface has disappeared or faded.An indistinct pale-brown dorsolateral stripe extends from the neck to the end of the tail and is accompanied by a series of ochre yellow, irregular blotches.Head brownish-black, upper half of the 1 st to 8 th supralabials brownish-black, lower half gray white, the 9 th completely brownish-black.The infralabials mainly black-gray, the left half of the 5 th to 11 th pale gray, and the right half very dark gray.Ventral surface of the head cream, the edges of the partial scales had irregular very dark gray patches.The ventral surface of the body cream anteriorly, darker toward the rear, and the posterior part is light creamy yellow.In addition, the rest of the color pattern is similar to that seen in life.
Variation.The paratype has a similar coloration in preservation as the holotype, but the subcaudals are almost completely black, with only the inner margins being creamy yellow.In scalation features, there is the following variation: the paratype has fewer infralabials (10 vs. 11) and more ventrals (151 vs. 146).The measurements and scalation features of the series (N = 2) are listed in Table 4.
Distribution and habitat.Hebius citrinoventer sp.nov. is currently only known from Yingjiang County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China: Tongbiguan Town (1300 m a.s.l.) (Fig. 7).The holotype (ANU20230016) was found drowned in a fish-catching cage placed by local  residents in a wide stream at approximately 7:00 am, and we presume that it may have fallen into the trap the preceding night while attempting to catch fish in the cage.The paratype (QHU2024005) was found road-killed on the side of the road next to a stream after a light rain between 21:00 and 22:00.Both specimens were found in a well-preserved monsoon forest habitat.

Discussion
In this study, we combined morphological and molecular analyses of specimens in the genus Hebius to provide robust evidence for the identification of new species.due to the lack of morphological and molecular data on the topotype, the classification status of these two specimens requires further study.The new species, Hebius citrinoventer sp.nov., has some morphological features common to H. venningi and H. taronensis, but can still be distinguished from them in the following characters: dorsal scale rows, number of temporal and ventral scales, and venter color pattern.Molecular phylogenetic analysis also separated the new species H. citrinoventer sp.nov.and provided strong support for its placement as a sister taxon.Moreover, the new species also possessed a considerable level of genetic divergence from 9.7-10.0%for H. venningi and 10.9-11.2% for H. taronensis in Cyt b.In addition, the new species is geographically isolated by the Hengduan Mountains, which plays an important role as a geographical barrier in speciation.
The genus Hebius is a highly diverse group distributed throughout eastern and southern Asia.Owing to the semi-aquatic habitats of this genus, specimen collection is relatively difficult, and the population and distribution data for many species is insufficient, which poses obstacles to conservation.Yingjiang County, where the new species was found, lies in the southwest Yunnan Province and is one of the most biodiverse regions in China.Although the discovery site of this new species is legally protected, the holotype derived from the fish trap and the road-killed paratype clearly indicates that this species is still influenced by human activities.Further surveys and evaluation of the population of the new species should be performed, and further consideration should be given to incorporating it in the local protected animal lists for protection.
In China, most species of Hebius are known from Yunnan Province, and the identification of Hebius citrinoventer sp.nov.brings the total number of Hebius species in China to 27, of which 20 are reported in Yunnan Province.This result further illustrates that reptile diversity in Yunnan is still underestimated.Therefore, more specific surveys may help to better understand the biodiversity in southwest China.

Conclusion
Here, we describe a new species of the genus Hebius, Hebius citrinoventer sp.nov., based on two specimens collected from Yingjiang County, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China.The discovery of this new species has brought the total number of known species in the genus Hebius to 52.Currently, Hebius citrinoventer sp.nov. is only known to be distributed in southwest Yunnan Province, China.Since Yingjiang County is close to the borders of Myanmar, this species also probably occurs in the adjacent area of this country.However, the detailed distribution range, population size, and feeding habits of the new species have not yet been elucidated, and further research and evaluation of the population of the new species should be conducted.
Uncorrected p-distances (%) among the Hebius species based on partial mitochondrial Cyt b gene.

Figure 2 .
Figure 2. Maximum likelihood tree of the genus Hebius inferred from Cyt b.The nodes supporting values on branches are presented as SH-like approximate likelihood ratio test (SH) / Ultrafast Bootstrap Approximation (UFB); ones under 50% are omitted.Tips for the new species in the present study are shown in red.

Table 1 .
GenBank accession numbers, localities, and voucher information for all specimens used in this study.

Table 3 .
Comparisons of main morphological characters between H. citrinoventer sp.nov., H. taronensis and H. venningi.Abbreviations as per Material and methods.

Table 4 .
Main morphological characters of Hebius citrinoventer sp.nov.Abbreviations as per Material and methods.
David et al. (2021)phylogenetic analysis, we found that the sequence KJ685679 (voucher number: GP 1618) from Myanmar clustered together with H. taronensis and had a very low p-distance of approximately 0.3% in Cyt b.However, it was referred to as Hebius sp.inGuo et al. (2014)but tentatively as H. venningi inDavid et al. (2021).Because the specimen was not examined, we conservatively assigned it to H. taronensis.Additionally, the uncorrected p-distance between specimen AMNH 147155 from Vietnam and H. optatus was 9.8%, which clearly reached the inter-species level; therefore, we proposed the specimen as H. cf.optatus.Likewise, the phylogenetic structure showed that the specimens(GP 1940 and AUP-00062), which were once considered Hebius bitaeniatus, clustered in different clades: H. craspedogaster, H. johannis, H. metusia and H. octolineatus were clustered together with low support and formed a sister group with specimen GP 1940; the specimen AUP-00062 was clustered with H. septemlineatus with high support (SH 93 / UFB 99), and the uncorrected p-distance was 4.2%.However, ZooKeys 1206: 255-274 (2024), DOI: 10.3897/zookeys.1206.123841Yu-Hao Xu et al.: A new Hebius species