First description of the male and hemipenial morphology of Opisthotropis haihaensis Ziegler et al., 2019 (Serpentes, Natricidae), with updated diagnosis and distribution

Biodiversity Data Journal 13: e167521 doi: 10.3897/BDJ.13.e167521 Taxonomy & Inventories First description of the male and hemipenial morphology of Opisthotropis haihaensis Ziegler et al., 2019 (Serpentes, Natricidae), with updated diagnosis and distribution Tierui Zhang , Jinlong Ren , Maoliang Li , Yuhao Xu , Xinge Wang , Nikolay A. Poyarkov , Tan Van Nguyen , Song Huang ‡ The Anhui Provincial Key Laboratory of Biodiversity Conservation and Ecological Security in the Yangtze River Basin, College of Life Sciences, Anhui Normal University, Wuhu, China § CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Ecological Restoration and Biodiversity Conservation Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, China | State Key Laboratory of Plateau Ecology and Agriculture, Xining, China ¶ Department of Vertebrate Zoology, Lomonosov Moscow State University, Moscow, Russia # The School of Medicine & Pharmacy, Duy Tan University, Da Nang, Vietnam ¤ Center for Entomology & Parasitology Research, Duy Tan University, Da Nang, Vietnam « The Anhui Provincial Key Laboratory of Biodiversity Conservation and Ecological Security in the Yangtze River Basin, College of Life Sciences, Anhui Normal University, W, China Corresponding author: Tan Van Nguyen ([email protected]), Song Huang ([email protected]) Academic editor: Chelmala Srinivasulu Received: 02 Aug 2025 | Accepted: 09 Oct 2025 | Published: 20 Nov 2025 Citation: Zhang T, Ren J, Li M, Xu Y, Wang X, Poyarkov NA, Nguyen TV, Huang S (2025) First description of the male and hemipenial morphology of Opisthotropis haihaensis Ziegler et al., 2019 (Serpentes, Natricidae), with updated diagnosis and distribution. Biodiversity Data Journal 13: e167521. https://doi.org/10.3897/BDJ.13.e167521 Abstract Background The Haiha Mountain Stream Keelback, Opisthotropis haihaensis Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, 2019, was originally described based on a single female specimen from northern Vietnam. To date, only two female specimens have been reported, rendering O. haihaensis one of the least studied members of its genus. ‡ § § | ‡ ¶ #,¤ « © Zhang T et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. New information During recent herpetological surveys in Guangxi Zhuang Autonomous Region, southern China, one male and one female specimen of Opisthotropis were collected from Qinzhou City. Phylogenetic analyses and detailed morphological comparisons identified these individuals as O. haihaensis. Based on these new specimens, we provide the first description of the male of this species, including hemipenial morphology, along with revised diagnostic features and new data on its natural history and distribution. Keywords Natricidae, Opisthotropis, sexual dimorphism, subtropical moist broadleaf forest Introduction The genus Opisthotropis Günther, 1872 (Natricinae) comprises stream-dwelling snakes distributed from southern China and the Indochina Peninsula to the Ryukyu Archipelago, Sumatra, and the Philippines (Poyarkov et al. 2023, Uetz et al. 2025). Currently, 25 species are recognized in the genus, including nine described within the past two decades (Teynié et al. 2014, Wang et al. 2017, Wang et al. 2017, Ren et al. 2017, Ziegler et al. 2019, Wang et al. 2020). Owing to their aquatic and cryptic lifestyles, many Opisthotropis species remain poorly known and are often represented solely by their type series (Stuart and Chuaynkern 2007, Teynié et al. 2014, Wang et al. 2020, Gao et al. 2024). One such example is Opisthotropis haihaensis Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, a member of the O. maculosa complex together with O. maculosa Stuart & Chuaynkern and O. hungtai Wang, Lyu, Zeng, Lin, Yang, Nguyen, Le, Ziegler & Wang. Species within this complex share the following combination of features: a single prefrontal scale; dorsal scale rows in 15–15–15; smooth dorsal scales; a dark brown to blackish dorsum on the body and tail, with each scale bearing a pale yellow spot; yellow chin shields with dark mottling; and yellow ventral surfaces with dark brown lateral margins and scattered brown flecks (Stuart and Chuaynkern 2007, Yang et al. 2011, Ziegler et al. 2018, Wang et al. 2020). The maculosa complex was first recorded in China by Yang et al. (2011), based on specimens from Guangdong Province and Guangxi Zhuang Autonomous Region (ZAR). Subsequently, Ziegler et al. (2019) described O. haihaensis as a distinct species based on a single female from Tai Chi Village, Quang Son Commune, Hai Ha District, Quang Ninh Province, northern Vietnam. This specimen had previously been misidentified as O. maculosa sensu Stuart & Chuaynkern, which was originally described from Phu Wua Wildlife Sanctuary, Boong Klar District, Nong Khai Province, Thailand (Nguyen et al. 2018). Later, Wang et al. (2020) re-examined the Chinese records assigned to O. maculosa by Yang et al. (2011), describing O. hungtai as a new species from Guangdong 2Zhang T et al Province and Guangxi ZAR. They also confirmed the presence of O. haihaensis in China for the first time, based on an adult female specimen from Shiwandashan National Nature Reserve, Fangchenggang District, Guangxi ZAR, and recommended the removal of O. maculosa from the Chinese herpetofauna. Based on two female specimens, O. haihaensis was initially diagnosed by the following combination of characters: total length 500–509 mm in adult females; relatively long tail (tail length/total length ratio 0.22); internasal not in contact with loreal; prefrontal not contacting supraocular; frontal contacting preocular; one preocular and one or two postoculars; temporals 1+1; eight supralabials, with the 4 and 5 entering the orbit; 22– 24 maxillary teeth; anterior chin shields longer than posterior; 164–169 ventrals (plus two preventrals); 75–79 subcaudals; nasal cleft directed towards first supralabial; dorsal scales in 15–15–15 rows, smooth; tail scales smooth or indistinctly keeled; chin shields yellow with dark mottling; and body and tail dorsum dark, each scale with a pale yellow spot (Ziegler et al. 2019, Wang et al. 2020). Despite its recent description, the lack of male specimens has hindered a comprehensive understanding of the species’ diagnostic characters and distributional limits. During a recent survey in Qinbei District, Qinzhou City, Guangxi ZAR, China, two specimens (one male and one female) referable to Opisthotropis were collected. Morphological comparisons and phylogenetic analyses confirmed their identity as O. haihaensis. In this study, we provide the first detailed morphological description of the male, including hemipenial morphology, update the species’ known distribution, and revise its diagnosis based on new material. Materials and methods Sampling Two specimens of Opisthotropis were collected from Qinbei District, Qinzhou City, Guangxi ZAR, China. These specimens were humanely euthanized with 0.7% tricaine methanesulfonate (MS222) solution. Fresh liver tissue was extracted and immediately preserved in 95% ethanol. Specimens were preserved in 75% ethanol for permanent storage, and deposited in Anhui Normal University Museum (ANU). Sampling procedures involving live snakes were approved by the Animal Ethics Committee of Anhui Normal University and complied with the Wild Animals Protection Law of China. Phylogenetic analyses Total genomic DNA was extracted from preserved liver tissue with OMEGA Tissue DNA Kit D3396 (Omega Bio-Tek, Norcross, GA, USA). A fragment of the mitochondrial cytochrome b (Cyt b) gene was amplified using the primer pair L14910 (5’- GACCTGTGATMTGAAACCAYCGTTGT-3’) and H16064 (5’-CTTTGGTTTACAAGAA CAATGCTTTA-3’) (Burbrink et al. 2000). The double-stranded products were sequenced by Sangon Biotech (Shanghai, China), and raw sequences were assembled using th th First description of the male and hemipenial morphology of Opisthotropis ... 3 SeqMan in the DNASTAR software package (Burland 2000). The sequences obtained from this study were uploaded to DNA Data Bank of Japan (DDBJ). Following Gao et al. (2024), 34 sequences from 18 known Opisthtropis species and three out-group species, Hebius johannis (Boulenger), Natrix natrix (Linnaeus) and Rhabdophis leonardi (Wall) were obtained from GenBank and incorporated into our dataset (Table 1). DNA sequences were aligned by the Clustal W algorithm with default parameters (Thompson et al. 1997) and trimmed with gaps partially deleted in MEGA X (Kumar et al. 2018). Bayesian inferences (BI) was conducted in MRBAYES v. 3.2.7a (Ronquist et al. 2012) under the GTR + I + G model on Phylosuite v1.2.3 (Zhang et al. 2020, Xiang et al. 2023). In the BI analysis, three independent runs were conducted with 1 × 10 generations and sampled every 1000 generations with the first 25% samples were discarded as burn-in. In the ML analysis, the bootstrap consensus tree was inferred from 1000 replicates. Maximum likelihood (ML) was conducted under the best-fit substitution model (GTR + I + G) in RaxmlGUI 1.3 (Silvestro and Michalak 2012). Bootstrap proportions (BSP) were investigated with 1,000 bootstrap replicates using the fast-bootstrapping algorithm. Uncorrected pairwise genetic distances (p-distance) of Cyt b gene among congeners were calculated with MEGA X (Kumar et al. 2018). Species Voucher No. NCBI/DDBJ acc. No. Locality Opisthotropis andersonii SYS r001020 KY594732 China: Mt. Wutong, Shenzhen, Guangdong Opisthotropis andersonii SYS r001423 KY594730 China: Tai Tam, Hong Kong Opisthotropis cheni YBU 071040 GQ281779 China: Guangdong Opisthotropis cheni SYS r001422 KY594741 China: Shimentai NR, Yingde, Guangdong Opisthotropis daovantieni CIB 109024 PQ726905 Vietnam: K’Bang, Gia Lai Opisthotropis daovantieni VNMN 2019.01 PQ726906 Vietnam: K’Bang, Gia Lai Opisthotropis durandi NCSM 80739 MK941137 Laos: Phongsaly Opisthotropis guangxiensis GP 746 GQ281776 China: Guangxi Opisthotropis haihaensis IEBR A. 2016.34 MK941139 Vietnam: Hai Ha, Quang Ninh Opisthotropis haihaensis SYS r000537 MN890017 China: Shiwandashan NR, Guangxi Opisthotropis haihaensis ANU 20240076 LC896244* China: Qinbei, Qinzhou, Guangxi Opisthotropis haihaensis ANU 20240077 LC896245*China: Qinbei, Qinzhou, Guangxi Opisthotropis hungtai SYS r000538 MN890018 China: Mt. Wuhuang, Guangxi Opisthotropis hungtai SYS r000946 KY594748 China: Heishiding NR, Fengkai, Guangdong Opisthotropis jacobi IEBR 4329 MG545601 Vietnam: Tam Dao NP, Vinh Phuc 7 Table 1. Information of samples and sequences used in molecular analysis. Notes: Is = Island; NP = National Park; NR = Nature Reserve; WS = Wildlife Sanctuary; * = DDBJ Accession Number. 4Zhang T et al Species Voucher No. NCBI/DDBJ acc. No. Locality Opisthotropis jacobi ZFMK 100818 MG545602 Vietnam: Tam Dao NP, Vinh Phuc Opisthotropis kuatunensis SYS r000998 KY594745 China: Qixiling NR, Yongxin, Jiangxi Opisthotropis kuatunensis SYS r001008 KY594746 China: Wulong, Shanghang, Fujian Opisthotropis laterialis SYS r001080 KY594744 China: Mt. Wutong, Shenzhen, Guangdong Opisthotropis laterialis ZFMK 100806 MF477899 Vietnam: Tay Yen Tu NR, Bac Giang Opisthotropis latouchii GP 647 GQ281783 China: Fujian Opisthotropis latouchii SYS r000670 KY594742 China: Guadun, Wuyishan, Fujian Opisthotropis laui SYS r001161 KY594738 China: Shangchuan Is, Taishan, Guangdong Opisthotropis laui SYS r001170 KY594739 China: Shangchuan Is, Taishan, Guangdong Opisthotropis maculosa FMNH 265798 MK941138 Thailand: Phu Wua WS, Nong Khai Opisthotropis maxwelli SYS r000841 KY594736 China: Nan’ao Is, Guangdong Opisthotropis maxwelli SYS r001053 KY594737 China: Huboliao NR, Nanjing, Fujian Opisthotropis shenzhenensis SYS r001018 KY594727 China: Mt. Wutong, Shenzhen, Guangdong Opisthotropis shenzhenensis SYS r001021 KY594728 China: Sanzhoutian, Shenzhen, Guangdong Opisthotropis tamdaoensis IEBR A. 2016.33 MF477901 Vietnam: Tam Dao NP, Vinh Phuc Opisthotropis voquyi IEBR 4327 MG451046 Vietnam: Tay Yen Tu NR, Bac Giang Opisthotropis voquyi VNMN 06315 MG451047 Vietnam: Tay Yen Tu NR, Bac Giang Opisthotropis zhaoermii CIB 109999 MG012800 China: Guzhang, Hunan Opisthotropis zhaoermii GZNU 320074 MK161471 China: Leishan, Guizhou Hebius johannis GP 897 KJ685708 China: Yunnan Natrix natrix MTD T 9269 HF680010 Denmark: Sønder Borup, Zealand Rhabdophis leonardi SCUM 090009 KF800933 China: Panzhihua, Sichuan Morphological examination Morphological descriptions followed Ziegler et al. (2019), Wang et al. (2020) and Gao et al. (2024). Body measurements and their abbreviations are as follows: eye horizontal diameter (ED), head length (HL), maximum head width (HW), tail length (TaL), total length (TL), snout length (SnL), snout width (SnW), interorbital distance (IOD), distance between the lower margins of eye and of lip (SoL). All measurements, except for tail length and total length, were obtained with a digital slide-caliper to the nearest 0.01 mm. Scalation features and their abbreviations are as follows: dorsal scale row counts (DSR), supralabial counts (SL), infralabial counts (IL), internasal counts (IN), chin shield counts (CS), frontal counts (F), parietal counts (P), preocular counts (PrO), postocular counts First description of the male and hemipenial morphology of Opisthotropis ... 5 (PtO), ventral counts (VEN), subcaudal counts (SC), prefrontal in contact with supraocular or not (PrF-SpO), preocular in contact with frontal (PrO-F), loreal entering orbit (L-orbit), internasals in contact with loreal (IN-L), supralabials in contact with orbit (SL-orbit), infralabials in contact with anterior chin shields (IL-aCS), temporal counts (TEM), cloacal plate entire or divided (CP), dorsal scale surface of tail (DST). The number of ventral scales was counted according to Dowling (1951). Symmetric characters were given as left/right and averages were used in the analyses. Gender of specimen was determined through dissection. The hemipenes of the male specimen was everted from the left side for the hemipenial description. The preparation and measurement method of hemipenes followed Ren et al. (2022). The everted hemipenes were re-inflated with coloured petroleum jelly and then preserved in 75% ethanol. Photographs of the hemipenes were taken using digital camera attached to a tripod head and multifocal photographs were combined and montaged using the Helicon Focus (7.0.2 Pro) software. Hemipenial morphology and terminology followed Dowling and Savage (1960), Zhang et al. (1984) and Ren et al. (2022). Museum and collection abbreviations are as follows: ANU = Anhui Normal University, Wuhu, China; FMNH = Field Museum of Natural History, Chicago, USA; IEBR = Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, Vietnam; KFBG = Herpetological Collection of Kadoorie Farm and Botanic Garden, Hong Kong, China; SYS = Sun Yat-sen University, Guangzhou, Guangdong, China. Taxon treatment Opisthotropis haihaensis Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, 2019 Materials a. scientificName: Opisthotropis haihaensis; taxonID: urn:lsid:biosci.ohiostate.edu:osuc_names:275502; class: Reptilia; order: Squamata; family: Natricidae; genus: Opisthotropis; specificEpithet: haihaensis; scientificNameAuthorship: Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, 2019; country: China; countryCode: CN; stateProvince: Guangxi; county: Qinbei; municipality: Qinzhou; locality: Tianmushan National Nature Reserve, Mt. Xianrending; verbatimLocality: 570 m; verbatimElevation: 1200 m; verbatimLatitude: 22.006365°N; verbatimLongitude: 108.206833°E; verbatimCoordinateSystem: WGS84; eventDate: 25-Jun-24; eventRemarks: collected by Tierui Zhang; individualCount: 1; sex: male; lifeStage: adult; catalogNumber: ANU 20240076; language: en; collectionCode: Reptilia; basisOfRecord: PreservedSpecimen; occurrenceID: 37B587F0-02CC-5439-B859-E7923E8C247A b. scientificName: Opisthotropis haihaensis; taxonID: urn:lsid:biosci.ohiostate.edu:osuc_names:275502; class: Reptilia; order: Squamata; family: Natricidae; genus: Opisthotropis; specificEpithet: haihaensis; scientificNameAuthorship: Ziegler, Pham, Nguyen, Nguyen, Wang, Wang, Stuart & Le, 2019; country: China; countryCode: CN; stateProvince: Guangxi; county: Qinbei; municipality: Qinzhou; locality: Tianmushan 6Zhang T et al National Nature Reserve, Mt. Xianrending; verbatimLocality: 570 m; verbatimElevation: 1200 m; verbatimLatitude: 22.006365°N; verbatimLongitude: 108.206833°E; verbatimCoordinateSystem: WGS84; eventDate: 25-Jun-24; eventRemarks: collected by Tierui Zhang; individualCount: 1; sex: female; lifeStage: adult; catalogNumber: ANU 20240077; language: en; collectionCode: Reptilia; basisOfRecord: PreservedSpecimen; occurrenceID: AAD9F7D6-CE03-591A-AD77-98734F6D0E95 Description of male specimen ANU 20240076 Measurements and scalation. Body slender and cylindrical (TL 428.0 mm); head short and broad, dorsally depressed, barely distinct from neck (HL/HW 1.87); snout moderate (SnL/SnW 0.74); eyes in medium size (ED/SoL 0.97); pupil round; interorbital distance large (IOD/HW 0.66); minute granular asperities absent on head scales; tail relatively long, tapering posteriorly (TaL 113.9 mm, TaL/TL 0.27); nostril oval-shaped, locating in the upper middle part of nasal, directed upwards. Dorsal scales in 15 rows throughout the body; the outermost rows of dorsal scales slightly enlarged, while the rest ones homogeneous in size, smooth throughout; vertebral scales not enlarged; preventrals 1, ventrals 168; cloacal plate divided; subcaudals 87 paired, with single terminal rigid tip, smooth entirely. Rostral crescent-shaped, wider than high, visible from above; nasals large, wider than high, divided below nostril by a distinct furrow; nasals in contact with the first two supralabials, rostral, internasal, prefrontal and loreal; internasals paired, trapezoid, longer than wide, curved outward posteriorly, in contact with rostral, nasal, and prefrontal; prefrontal single, much broader than long, anteriorly pointed, in contact with internasals, nasals, loreals, preoculars, and frontal; frontal single, pentagonal or near equilateral triangle, almost equal in width and length, tapering posteriorly, equal to its distance from tip of snout; parietals nearly 1.8 times as long as wide, parietal sutures nearly equal to frontal in length; 1/1 supraocular, distinctly longer than wide, not in contact with prefrontal; 1/1 loreal, pentagonal, wider than high, not entering orbit, surrounded by second and third supralabials, nasal, prefrontal and preocular; 1/1 preocular, large, hexagonal, higher than wide, reaching frontal; subocular absent; 2/2 postocular, upper pair is almost twice the length and width of lower pair; 7/7 supralabials, 1 –5 higher than wide, only 5 entering orbit on left lateral, 4 and 5 entering orbit on right lateral (4-1-2/3-2-2), 7 largest; 1+1/1+1 temporals, anterior one very long and narrow, in broad contact with 5 –7 supralabials and parietals, posterior one more strongly developed; infralabials 8/8, first pair in contact behind small mental, 1 –5 / 1 –5 in contact with anterior chin shields, 5 infralabial largest; anterior chin shields longer than posterior ones, posterior chin shields do not contact with each other. Dentition. Maxillary teeth 22, subequal, densely set, without diastema. Coloration in life (Figs 1, 2). Scales on dorsum of head glossy black with scattered yellow flecking; ventral of head yellow with brownish black mottling on the margins of each scale; upper lip yellow with brownish-black anterior and lateral margins on each st th th th th th th th st th st th th First description of the male and hemipenial morphology of Opisthotropis ... 7 supralabial; dorsum of body and tail glossy black with iridescence above, with single bright yellow spot on each scale, yellow spots becoming larger on sides of body; ventrals yellow with brownish black lateral margins; subcaudals yellow with brownish black anterior and lateral margins. Coloration in preservation. Coloration and pigmentation patterns of newly preserved specimens (with 75% ethanol) significantly faded, bright yellow spots on dorsals and scattered yellow flecking on dorsum of head turn milky white, background color of ventrals fades to pale yellow. Gloss of scales remains distinct. Hemipenis (Fig. 3). Fully everted hemipenis is relatively long, measuring 15.71 mm in length and 2.52 mm in width; slender, single, cylindrical, slightly clavate apically. Organ entirely spinose, with spines relatively uniform in size and distribution; spines slightly sparser in the proximal region; no basal nude area present. Basal hook absent. Sulcus spermaticus unforked, centripetal, extending straight to the apex; sulcus lips well developed, broad, and smooth along the entire length. When retracted, the hemipenis reaches the level of the 31 subcaudal scale. Description of additional female specimen ANU 20240077 Measurements and scalation. Body slender and cylindrical (TL 485.0 mm); head short and broad (HL/HW 1.76), dorsally depressed, barely distinct from neck; snout moderate (SnL/SnW 0.60); eye small, (ED/SoL 0.81); pupil round; interorbital distance large (IOD/HW 0.60); minute granular asperities absent on head scales; tail relatively long, tapering posteriorly (TaL 94.9 mm, TaL/TL 0.20); nostril oval-shaped, locating in the upper middle part of nasal, directed upwards. st Figure 1. Live photographs of Opisthotropis haihaensis (specimen ANU 20240076, male) in situ from Qinzhou, Guangxi, China. Photographed by T.R. Zhang.  8Zhang T et al Dorsal scales in 15 rows throughout the body; the outermost rows of dorsal scales slightly enlarged, while the rest ones homogeneous in size, smooth throughout; vertebral scales not enlarged; preventrals 1, ventrals 165; cloacal plate divided; subcaudals 71, paired, with single terminal rigid tip, smooth entirely. Rostral crescent-shaped, wider than high, visible from above; nasals large, wider than high, divided below nostril by a distinct furrow; nasals in contact with the first two supralabials, rostral, internasal, prefrontal and loreal; internasals coalesced into one, pentagonal, wider than long, concaved inward posteriorly, in contact with rostral, nasal, and prefrontal; prefrontal single, much broader than long, anteriorly pointed, in contact with internasals, nasals, loreals, preoculars, and frontal; frontal single, pentagonal or near equilateral triangle, almost equal in width and length, tapering posteriorly, equal to its distance from tip of snout; parietals nearly 1.8 times as long as wide, parietal sutures nearly equal to frontal in length; 1/1 supraocular, right supraocular small and partially coalesced with upper postocular, distinctly longer than wide, not in contact with prefrontal; 1/1 loreal, pentagonal, wider than high, not entering orbit, surrounded by second and third supralabials, nasal, prefrontal and Figure 2. Opisthotropis haihaensis prior to preservation (specimen ANU 20240076, male): A dorsal view of whole body; B ventral view of whole body; C dorsal view of head; D ventral view of head; E right lateral view of head; F left lateral view of head; G close-up of mid-body dorsal scales. Photographs by T.R. Zhang.  First description of the male and hemipenial morphology of Opisthotropis ... 9 7The 6 supralabial is not elongated, approximately equal to or shorter than half the length of the anterior temporal O. haihaensis –The 6 supralabial is distinctly elongated and nearly equal in length to the anterior temporal O. hungtai 8A lateral black stripe separating pale venter from dark dorsum; loreal not entering orbit O. lateralis – No such lateral stripe; loreal entering orbit or not 9 9 Loreal not entering orbit, 1.4–1.7× as long as deep; last supralabial longest O. maxwelli –Loreal entering orbit or not, more than 1.7× as long as deep; last supralabial smaller than preceding one 10 10 Loreal in contact with second supralabial; tail length 15–20% of total length; dorsum without yellow longitudinal stripes or crossbars O. andersonii –Loreal not contacting second supralabial; tail length 20–23% of total length; dorsum with yellow longitudinal stripes or crossbars 11 11 Body size small, total length shorter than 419 mm; maxillary teeth ≤ 25 O. latouchii –Body size moderate or large, total length longer than 419 mm; maxillary teeth ≥ 25 12 12 Maxillary teeth 25–28; anterior temporals elongated, maximum anterior temporal length / depth ratio 2.63–3.63; dorsum dark olive with yellow crossbars O. cheni – Maxillary teeth 28–30; anterior temporals short, maximum anterior temporal length / depth ratio 1.74–2.04; dorsum with longitudinal yellow stripes O. zhaoermii Analysis In this study, 1 060 base pairs (bp) of Cyt b sequences were successfully obtained for alignment, including two newly generated sequences of O. haihaensis. Both ML and BI analyses yielded highly congruent topologies (Fig. 6), consistent with findings from previous studies (Ren et al. 2017, Wang et al. 2017, Ziegler et al. 2019, Wang et al. 2020). The genus Opisthotropis was recovered as a monophyletic group within the scope of the current sampling (BSP = 83, BPP = 0.99). All O. haihaensis individuals clustered within a distinct and well-supported clade (BSP = 100, BPP = 1.00), nested within the Opisthotropis clade. Infraspecific uncorrected p-distances among the O. haihaensis th th 16 Zhang T et al specimens were 0.2% –4.5% (Suppl. material 1). The p-distances between O. haihaensis and all analyzed Opisthotropis species varied from 13.3%–13.4% (for O. hungtai) to 18.0%–18.3% (for O. daovantieni Orlov, Darevsky & Murphy). The following is a detailed description of the additional specimens of O. haihaensis. Discussion Wang et al. (2020) considered the number of supralabials to be a key diagnostic character distinguishing Opisthotropis haihaensis from O. hungtai, with the former bearing eight supralabials and the latter seven. However, our findings indicate that supralabial counts may vary depending on the position of the oral fissure relative to the posterior margin of the seventh supralabial, potentially leading to misinterpretation. We also re-checked the supralabial counts of O. haihaensis specimen SYS r000537 and found it actually has 7 supralabials (8 in Yang et al. (2011), Wang et al. (2020)). To minimize diagnostic ambiguity caused by variable supralabial counts, we propose an alternative diagnostic character: in O. haihaensis, the 6 supralabial is not elongated and is approximately equal to or shorter than half the length of the anterior temporal, whereas in O. hungtai, the 6 supralabial is distinctly elongated and nearly equal in length to the anterior temporal. th th Figure 6. ML phylogenetic tree estimated from Cyt bsequences depicting phylogenetic relationships of Opisthotropis (numbers above branches are BSP/BPP).  First description of the male and hemipenial morphology of Opisthotropis ... 17 Among the 25 currently recognized species of Opisthotropis, hemipenial morphology has been described in only 13 species (see Table 5) and several of these descriptions remain brief or lack sufficient detail (Pope 1935, Zhang et al. 1984, Stuart and Chuaynkern 2007, Yang et al. 2011, Ziegler et al. 2018, Ren 2019, Gao et al. 2024). Based on the most recent summaries by Gao et al. (2024), the general hemipenial morphology of Opisthotropis is characterized by the following traits: relatively short and stout hemipenis, shallowly bilobed in a “Y” shape, noncalyculate and noncapitate (except in O. alcalai and O. maculosa); uniform coverage with small spines or spinules; a single enlarged basal hook on the proximal region of the truncus (except in O. maculosa); and a single, undivided, centripetal sulcus spermaticus extending to the base or distal portion of the inner right lobe (except in O. daovantieni). Within this framework, Opisthotropis haihaensis and O. maculosa exhibit unique hemipenial morphology in possessing a single, cylindrical hemipenis entirely spinose and lacking a basal hook distinguishing them from other congeners. Presence or absence of the basal hook and morphology of sulcus serve as significant taxonomic diagnostic criteria, as they could directly influence reproductive behavior and reflect adaptive evolution in reproductive strategies. The hemipenis of Opisthotropis hungtai, the species most closely related to O. haihaensis, remains undescribed. Yang et al. (2011) briefly noted that the hemipenis of specimen SYS r0538 is cylindrical and extends to the level of the 12 subcaudal and provided an image of KFBG 2002.01. However, the structure shown does not appear to be fully everted, rendering any morphological inference inconclusive. Detailed documentation of hemipenes in Opisthotropis hungtai and other poorly characterized or undocumented species is essential for evaluating patterns of hemipenial variation and their potential phylogenetic significance. Species Shape Ornamentation Enlarged basal hook Sulcus spermaticus Relative length of retracted hemipenis Source O. alcalai Single Uniformly spinose throughout 1 Undivided, extends to apical tip 11 Brown and Leviton (1961) O. andersonii ? Beset with spines proximally and papillalike processes distally 1 ? ? Pope (1935) O. daovantieni Bilobed, lobes welldeveloped Spinose throughout, spines approximately homogeneous in size, proximal fourth sparser, without basal naked area 1 Bifurcate, centripetal, reaching apex of each lobe 17 Gao et al. (2024) th th th Table 5. Documented hemipenial morphology of Opisthotropis species. Notes: Relative length of retracted hemipenis was defined by the subcaudal it can reach at the distalmost; “?” indicates missing data. 18 Zhang T et al Species Shape Ornamentation Enlarged basal hook Sulcus spermaticus Relative length of retracted hemipenis Source O. guangxiensis Bilobed, short and thin, slightly stouter at the proximal half Spinose throughout, proximal third sparser. 1 Undivided, reaching apex of the right lobe ?Ren (2019), Gao et al. (2024) O. haihaensis Single, cylindrical, slightly clavate apically Spinose throughout, spines approximately homogeneous in size, proximal sparser slightly, without basal naked area. Absent Undivided, centripetal, reaching apex of hemipenis 31 This study O. hungtai Cylindrical ? ? ? 12 Yang et al. (2011) O. jacobi Bilobed, lobes short, inwards curved Covered with small spines, curved backwards. The terminal area of the hemipenis between the lobes without spines. Upper truncus of hemipenes with ring of enlarged spines 1 Undivided, stretching to the slightly smaller lobe only. ?Ziegler et al. (2018) O. kuatunensis Slightly divided at the tip Spinous, spines are set in longitudinal rows and connected by fleshy ridges, recurved proximally and gradually decrease in distal. 1 Undivided 10 Pope (1935) O. lateralis ? Beset with spines proximally and papillalike processes distally 2 ? ? Pope (1935) O. latouchii Bilobed, slightly forked at the tip Beset with hookshaped spines proximally and papillalike processes distally 1 Undivided, reaching apex of the right lobe 6 –8 Pope (1935), Zhang et al. (1984), Ren (2019) O. laui Bilobed, slightly elongated Beset with hookshaped spines proximally and papillalike processes distally 1 ? ? Ren (2019) O. maculosa Single, cylindrical Basal area naked, remainder of hemipenis uniformly ornamented with spines Absent Undivided, centripetal, reaching apex of hemipenis 12 Stuart and Chuaynkern (2007) st th th th th th First description of the male and hemipenial morphology of Opisthotropis ... 19 Species Shape Ornamentation Enlarged basal hook Sulcus spermaticus Relative length of retracted hemipenis Source O. voquyi Bilobed, lobes short, inwards curved Covered with small spines, curved backwards. The terminal area of the hemipenes between the lobes without spines. Upper truncus of hemipenes with ring of enlarged spines. 1 Undivided, stretching to the slightly smaller lobe only. ?Ziegler et al. (2018) O. zhaoermii Bilobed, right lobe shorter than the left lobe Covered with small spines. The terminal area of the hemipenis between the lobes and basal area naked. Spines around basal hook moderately enlarged, spines on the remainder of hemipenis approximately homogeneous in size 1 Undivided, reaching apex of the right lobe ?Gao et al. (2024) Opisthotropis haihaensis is currently known from only four specimens collected at three localities in northern Vietnam and southern China. It inhabits clear, slow-flowing mountain streams within evergreen broad-leaved forests habitats that are increasingly threatened by deforestation and other anthropogenic disturbances. Given its narrow known distribution and extremely limited number of recorded specimens, we recommend a provisional IUCN Red List status of Near Threatened (NT). This classification is precautionary, pending further field surveys and population assessments, and reflects the urgent need for more comprehensive ecological and conservation data. Acknowledgements We thank Ying-Yong Wang and Shuo Qi from Sun Yat-sen University for their assistance in examining the Opisthotropis haihaensis specimen SYS r000537. We thank Jun-Jie Huang from Chengdu Institute of Biology for photographing the hemipenis. We also thank Ngoc Quynh Nguyen and Duc Trong Nguyen (SIFASV, Vietnam) for their assistance in preparing the figures and map. We are sincerely grateful to the anonymous reviewer for their valuable comments and suggestions, which helped improve the manuscript. This research was supported by Science and Technology Projects of Xizang Autonomous Region, China (XZ202301ZY0036G), the National Natural Science Foundation of China (NSFC 31471968), the Rufford Foundation (Grant No. 45888-2-P; data analysis), the Russian Science Foundation (RSF Grant No. 22-14-00037-P: data analysis) and National Animal Collection Resource Center, Sun Yat-sen University (The Museum of Biology), Guangzhou, China. 20 Zhang T et al References • Brown WC, Leviton AE (1961) Discovery of the snake genus Opisthotropis in the Philippine Islands: with description of a new species. Occasional Papers of the Natural History Museum of Stanford University. • Burbrink FT, Lawson R, Slowinski JB (2000) Mitochondrial DNA phylogeography of the polytypic North American rat snake (Elaphe obsoleta): a critique of the subspecies concept. 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