A New Species of Pareas (Squamata, Pareidae) from Guangxi Province, China

Simple Summary A new species of snail-eating snake in the genus Pareas is described, from the Youjiang District, Baise City, Guangxi Zhuang Autonomous Region, China, based on one male and three juvenile specimens. The maximum likelihood analyses based on Cytochrome b (Cyt b) and NADH dehydrogenase subunit 4 (ND4) indicated the new taxon is different from its congeners. Morphologically, the new species can be diagnosed from the other species by a combination of seven characters. The recognition of the new species brings the number of described Pareas species to 30. Abstract We described a new species of genus Pareas from Baise City, Guangxi Zhuang Autonomous Region, China, based on morphological and molecular evidence. Pareas baiseensis sp. nov. is distinguished from its congeners by the combination of (1) Yellowish-brown body colouration; (2) Frontal subhexagonal to diamond-shaped with its lateral sides converging posteriorly; (3) The anterior pair of chin shields is longer than it is broad; (4) Loreal not in contact with the eye, prefrontal in contact with the eye, two or three suboculars; (5) Rows of 15–15–15 dorsal scales, five rows of mid-dorsal scales keeled at the middle of the body, one vertebral scale row enlarged; (6) 187–191 ventrals, 89–97 subcaudals, all divided, cloacal plate single; (7) Two postocular stripes, the nuchal area forming a dark black four-pointed fork collar with the middle tines shorter than the outside tines. The genetic divergence (uncorrected p-distance) between the new species and other representatives of Pareas ranged from 13.9% to 24.4% for Cytochrome b (Cyt b) and 12.1% to 25.5% for NADH dehydrogenase subunit 4 (ND4). Phylogenetic analyses of mitochondrial DNA gene data recovered the new species from being the sister taxon to (P. boulengeri + P. chinensis) from China.


Sampling
The four individuals of snail-eating snakes (one male, three juveniles) were collected from Daleng Township, Youjiang District, Baise City, Guangxi Zhuang Autonomous Region, China, in November 2022. The collected specimens were fixed in approximately 95% ethanol and subsequently transferred to 75% ethanol for permanent storage. Liver tissue samples were preserved separately in 95% ethanol. The specimens examined in the present study were preserved and deposited at Anhui Normal University Museum (ANU).

Morphological Examination
Referring to the following literature, Dowling 1951, Vogel 2015, and Poyarkov et al., 2022, a total of 21 morphological characters in four specimens of the new species were examined (Table 1). Morphological measurements (all in mm) included: snout-vent length (SVL); tail length (TaL); total length (TL); relative tail length (TaL/TL); head length from snout tip to jaw angles (HL); maximal head width (HW); and eye diameter (ED). Meristic characteristics evaluated were the number of dorsal scale rows counted at one head length behind head (ASR), at mid-body (MSR), namely at SVL/2, and at one head length before vent (PSR); number of enlarged vertebral scale rows (VSE); number of keeled dorsal scale rows at midbody (KMD); number of ventral scales (VEN); number of subcaudal scales (SC); number of cloacal plates (CP); number of supralabials (SL); number of infralabials (IL); number of anterior temporals (At); number of posterior temporals (Pt); number of loreals (LOR); number of preoculars (Preoc); number of suboculars (SoO); and number of postoculars (PoO). We recorded the values for paired head characteristics on both sides of the head (in a left/right order). We measured body and tail lengths with a measuring tape (to the nearest of 1 mm); all other measurements were taken using an electronic slide caliper (to the nearest 0.1 mm).
A maximum likelihood (ML) tree was reconstructed using RaxML v7.2.6 using the GTRGAMMA model with 1000 ultrafast bootstrap (BS) replicates [26,27]. We also calculated the pairwise distances (p-distances) among ingroup taxa using the neighbour-joining method in MEGA X [25,28]. The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/ (accessed on 8 June 2023). The LSID for this publication is urn:lsid:zoobank.org:pub:73634CF1-1ACA-4C07-B8A2-3929E2306558.

Phylogenetic Relationship
The newly generated sequences of Cyt b and ND4 genes of four specimens shared one haplotype for each gene. The sequences were submitted to GenBank (accession numbers, OQ054328 for Cyt b, OQ054329 for ND4). The p-distances based on fragments of Cyt b between the new species and other species of the genus Pareas varied from 13.9% (Pareas boulengeri) to 24.4% (Pareas carinatus) ( Table 2), and those of ND4 varied from 12.1% (Pareas boulengeri) to 25.5% (Pareas berdmorei) ( Table 3). Phylogenetic analyses of mitochondrial DNA data recovered the new species to be the sister taxon to (P. boulengeri + P. chinensis) from China ( Figure 1). Combined with morphological data, the specimens from Baise, Guangxi, China, are considered to be a new species.
The electronic version of this article in Portable Document Format (PDF) will sent a published work according to the International Commission on Zoological No clature (ICZN), and hence the new names contained in the electronic version are tively published under that Code from the electronic edition alone. This published and the nomenclatural acts it contains have been registered in ZooBank, the online tration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be res and the associated information can be viewed through any standard web browser b pending the LSID to the prefix http://zoobank.org/ (accessed on 8 June 2023). The LS this publication is urn:lsid:zoobank.org:pub:73634CF1-1ACA-4C07-B8A2-3929E230

Phylogenetic Relationship
The newly generated sequences of Cyt b and ND4 genes of four specimens s one haplotype for each gene. The sequences were submitted to GenBank (accession bers, OQ054328 for Cyt b, OQ054329 for ND4). The p-distances based on fragments b between the new species and other species of the genus Pareas varied from 13.9% ( boulengeri) to 24.4% (Pareas carinatus) ( Table 2), and those of ND4 varied from 12.1% ( boulengeri) to 25.5% (Pareas berdmorei) ( Table 3). Phylogenetic analyses of mitocho DNA data recovered the new species to be the sister taxon to (P. boulengeri + P. chin from China ( Figure 1). Combined with morphological data, the specimens from Guangxi, China, are considered to be a new species.    Holotype. ANU20220011 (collection number HSR22185), an adult male (Figure 2A), was found in the Daleng Township, Youjiang District, Baise City, Guangxi Zhuang Autonomous Region, China (23.73521N, 106.39544E (DD); ca 781 m a.s.l.). The specimen was collected by Jiaxiang Wu and Yongjin Liu on 25 November 2022 and deposited at Anhui Normal University Museum.

Diagnosis
Pareas baiseensis sp. nov. is distinguished from all other Pareas by a combination of the following characteristics: (1) Yellow-brown body colouration; (2) Frontal subhexagonal to diamond-shaped with its lateral sides converging posteriorly; (3) The anterior pair of chin shields is longer than it is broad; (4) The loreal is not in contact with the eye, prefrontal in contact with the eye, two or three suboculars; (5) Rows of 15-15-15 dorsal scales, five rows of mid-dorsal scales keeled at the middle of the body, one vertebral scale row enlarged; (6) 187-191 ventrals, 89-97 subcaudals, all divided, cloacal plates single; (7) Two postocular stripes, the nuchal area forming a dark black four-pointed fork collar with the middle tines shorter than the outside tines.
Pareas baiseensis sp. nov. differs from P. vindumi, P. victorianus, and P. monticola by the loreal not contacting the eye (vs. the loreal contacting the eye); two or three suboculars (vs. one or suboculars fused with postoculars); five slightly keeled dorsal scale rows at midbody (vs. smooth or 7-11 keeled dorsal scale rows at midbody); and the nuchal area forming a dark black four-pointed fork collar with the middle tines shorter than the outside tines. (vs. nuchal area no collar) [6,7,10,29].
Pareas baiseensis sp. nov. differs from Pareas dulongjiangensis by the nuchal area forming a dark black four-pointed fork collar with the middle tines shorter than the outside tines (vs. two brownish-black longitudinal stripes running on each side of the neck leaving a pale central portion); the loreal not contacting the eye (vs. loreal contacting the eye); absence of preoculars (vs. preoculars being present); two or three suboculars (vs. suboculars fused with postoculars); a higher number of ventrals (187-191 vs. 182); and a higher number of subcaudals (89-97 vs. 76) [13].
Pareas baiseensis sp. nov. differs from Pareas yunnanensis by the dorsal surface of the head, which is light brown with dark brown spots (vs. dorsal surface of head is black); sides of the head with two lateral postorbital stripes (vs. no or one or two indistinct large black spots on each side of the head, no stripe on each side of the head); a higher number of infralabials (9 vs. 6-8); five slightly keeled dorsal scale rows at midbody (vs. 5-7 rows of middorsal scales keeled on the middle part of the body); a higher number of ventrals (187-191 vs. 169-175); and a higher number of subcaudals (89-97 vs. 59-65) [13].

Description of Holotype
An adult male, SVL 428 mm, TaL 151 mm, TL 579 mm, TaL/TL ratio 0.26; body slender, compressed; head elongate, clearly distinct from neck; snout round in dorsal view; eye slightly enlarged, pupil vertical and slightly elliptical; rostral slightly visible in dorsal view; frontal subhexagonal to diamond-shaped with its lateral sides converging posteriorly; nasal scale single; two prefrontals large, in contact with the eye; single loreal not in contact with the eye; temporals 2 + 3/3 + 3; 1/1 supraocular; 1/1 preocular; 4/4 suboculars; 1/1 postoculars; 8/8 supralabial scales; 9/9 infralabials; 191 (+1 preventral) ventrals; 15-15-15 dorsal scale rows, five rows of mid-dorsal scales keeled at the middle of the body; 97 subcaudals; cloacal plate single (Table 4). Colouration: In life, the dorsal surface of the head is light brown with dark brown spots. The dorsum is brown with dark-brown speckling, and there are 34 irregular black cross-bands on the lateral sides of the body from neck to vent. The ventral is creamishyellow with a few small black spots, the background colour gradually darkens to the rear, and the subcaudal scales are light brown. The sides of the head have two lateral postorbital stripes: the upper stripe extends from the temporal area backward extension to the dorsal scales of the neck, where it joins a large black collar around the nape, forming a dark black Ψ-shaped chevron pattern overall; the lower stripe extends backwards past the 9th supralabial, and at the throat contacting the four-pointed fork collar with the middle tines shorter than the outside tines. There are two black lines on the back of the parietal that extend back to the neck, with two lateral postorbital stripes; together, they form a dark black four-pointed fork collar.
In the preserved state, the colouration still resembles the specimen in life, but the dorsum colour fades to yellowish-brown (Figures 3 and 4).

Distribution
This species is currently only known from onelocality, Daleng Township, Youjiang District, Baise City, Guangxi Zhuang Autonomous Region, China. We found the snakes between 10:00 pm to 1:00 am after light rain in November 2022. The habitat environment was a well-preserved subtropical evergreen broad-leaved forest at elevations of 750-790 m.

Discussion
The phylogenetic results of Poyarkov et al. (2022) support the genus Pareas sensu lato being divided into two subgenera (Pareas sensu stricto and Eberhardtia) and six species groups; the subgenus Pareas sensu stricto includes two species groups (P. carinatus and P. nuchalis groups), the subgenus Eberhardtia includes four species groups (P. chinensis, P. hamptoni, P. monticola and P. margaritophorus groups). The members of the subgenus Eberhardtia differ from the members of the subgenus Pareas by the following combination of morphological characters: frontal subhexagonal to diamond-shaped with its lateral sides converging posteriorly; the anterior pair of chin shields is longer than it is broad; a single thin elongated subocular; and the ultrastructure of dorsal scales not ravine-like, having pore and arc structures, with arcs connecting to each other forming characteristic lines [4,8,[37][38][39]. Our phylogenetic results support Pareas baiseensis sp. nov. belongs to the P. chinensis species groups in the subgenus Eberhardtia, but Pareas baiseensis sp. nov. have two or three suboculars, which is inconsistent with the diagnostic characteristic of subgenus Eberhardtia. Therefore, we propose deleting the diagnostic characteristic of a single thin elongated subocular.
The discovery of Pareas baiseensis sp. nov. increases the number of species of the P.chinensis species group to four species (Pareas baiseensis sp. nov., P.boulengeri, P. stanleyi, P.chinensis) in China. Among them, P.boulengeri is the most widely distributed, which is distributed in Guizhou, Sichuan, Yunnan, Chongqing, Henan, Hubei, Hunan, Guangxi, Guangdong, Jiangsu, Zhejiang, Anhui, Jiangxi, Fujian, Shaanxi, and Gansu. P. stanleyi is distributed in Fujian, Zhejiang, Jiangxi, Guizhou, Sichuan, Hunan and Guangxi; The distribution of P.chinensis in China is limited to the western and southern marginal mountains of the Sichuan Basin [6,7,14,15]; Pareas baiseensis sp. nov. is currently known only from the locality investigated, but Baise City is close to the borders of Yunnan and Vietnam, and this species may also occur in these adjacent areas.
The description of Pareas baiseensis sp. nov. from southern China brings the total number of recognized Pareas species to 30, of which 24 occur in China [2,4,14,15]. The genus Pareas has an ancient origin and poor migration ability, and the morphological difference between different species are subtle [9,21,[40][41][42]; a large range of intensive sampling is helpful in discovering cryptic species, especially for some widely distributed types with unclear internal relationships, so sampling should be increased.

Conclusions
A new species of Pareas, Pareas baiseensis sp. nov., is described based on four specimens collected from the Youjiang District, Baise City, Guangxi Zhuang Autonomous Region, China, since Baise City is close to the borders of Yunnan and Vietnam and this species may also occur in these adjacent areas. However, their discovery is largely accidental, which makes it difficult for us to make accurate judgments on the distribution and population status of this new species. Further investigations will be necessary to assess the distribution and population status of this species.

Institutional Review Board Statement:
The study received ethical review and approval from the Animal Ethics Committees at Anhui Normal University (project number AHNU-ET2021025). All sampling and procedures involving live snakes were performed in accordance with the Wild Animals Protection Law of the People's Republic of China and approved by the Animal Ethics Committees at Anhui Normal University (project number AHNU-ET2021025).

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.