Description of a New Species of the Pareas hamptoni Complex from Yunnan, China, with Confirmation of P. hamptoni Sensu Stricto in China (Squamata, Pareidae)

Simple Summary In this paper, a new Pareas species of the P. hamptoni complex is described from southern Yunnan Province, China, based on morphological and molecular evidence. Genetically, the new species is most closely related to P. hamptoni sensu stricto, for which we confirm the distribution in China. Morphologically, the new species can be distinguished from P. hamptoni sensu stricto and all other congeners by a combination of morphological characteristics. The discovery of the new species brings the total number of recognized species of the genus Pareas to 31, of which 25 occur in China. Abstract We describe a new species of the genus Pareas, based on three specimens collected from Guanyinshan Provincial Nature Reserve in Yuanyang County, Honghe Prefecture, Yunnan Province, China. The new species is distinguished from its congeners by one preocular, one postocular or postocular fused with subocular, loreal not bordering the orbit, one row enlarged vertebral scales, five rows keeled mid-dorsal scales at the middle of the body, 189–192 ventral scales and 72–89 subcaudal scales. The dorsal surfaces of the head and body are yellowish red or yellowish brown, and the belly and ventral surfaces of the head and tail are pinkish yellow or yellow with more or less small black spots. Phylogenetic analyses of mitochondrial DNA recovered the new species being the sister taxon to P. hamptoni sensu stricto. The genetic divergences between the new species and P. hamptoni sensu stricto were 4.2% in the Cyt b sequences and 5.0% in the ND4 sequences. In addition, based on specimens collected from Honghe and Wenshan prefectures, we confirmed that P. hamptoni sensu stricto is distributed in China.

The genus Pareas split from its sister genus Aplopeltura during the Oligocene [3,5].Pareas covers a group of highly specialized snakes that only feed on snails or slugs [7][8][9][10][11].This special habit also gives them a significant advantage in the niche, so that they occupied a vast area from Sundaland to northeastern India and southern China and evolved into a large number of different species [5,6,10,[12][13][14][15][16].
Hampton's slug-eating snake, Pareas hamptoni (Boulenger, 1905), was originally described based on a single specimen from Mogok, Myanmar [17].Subsequently, this species was thought to be widely distributed, ranging across mainland Southeast Asia and southern China [18][19][20][21].Thereafter, Wang et al. [6] demonstrated that the populations previously identified as P. hamptoni in southeast China belong to P. formosensis (Van Denburgh, 1909).Ding et al. [11] described the population that had previously been identified as P. hamptoni in south central Yunnan, China as a new species, P. geminatus Ding, Chen, Suwannapoom, Nguyen, Poyarkov & Vogel, 2020.They demonstrated that the populations previously identified as P. hamptoni in northeast, central, and central south Vietnam belong to P. formosensis, whereas the populations in northwest Vietnam belong to P. hamptoni sensu stricto.Liu and Rao [12] described the population that had previously been considered P. hamptoni from southwestern Yunnan, China, as a new species, P. xuelinensis Liu & Rao, 2021.Until now, whether P. hamptoni sensu stricto is distributed in China has been a matter of uncertainty.
During our field research in southern Yunnan Province, China, from 2019 to 2023, we collected a series of Pareas specimens that closely resemble P. hamptoni.However, further comprehensive analyses of their molecular and morphological characteristics have demonstrated that these specimens do not belong to the same species but are from two separate taxa.One corresponds to P. hamptoni sensu stricto, while the other is different from all known congeners.Therefore, we herein confirm the distribution of P. hamptoni sensu stricto in China and describe the unique taxon as a new species.

Sampling
Specimens were caught by hand at night.Photographs were taken in situ before the specimens were collected.Liver tissues were cut and stored in analytical pure ethanol and the snakes were preserved in a 75% concentration of ethanol.Specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Molecular Analyses
Molecular data were generated for nine specimens collected from Honghe and Wenshan prefectures, Yunnan Province, China.Other sequences used in the phylogenetic analyses were obtained from GenBank.All new sequences have been deposited in Gen-Bank.Aplopeltura boa (Boie, 1828) was selected as the outgroup according to Liu et al. [23].Details of all the sequences used in this study can be found in Table 1.Total genomic DNA was extracted from liver tissue samples.The sequences of the mitochondrial genes, cytochrome b (Cyt b) and NADH dehydrogenase subunit 4 (ND4), were amplified by a polymerase chain reaction (PCR) using the primers L14910/H16064 [24] and ND4F/ND4LEUR [25], respectively.The amplification products were purified and sequenced by Tsingke Biotechnology Co., Ltd.(Beijing, China).The sequences were stitched using SeqMan in Lasergene 7.1 [26].
The sequences were aligned using ClustalW [27] that is integrated in MEGA 11 [28] with the default parameters.The genetic divergences (uncorrected p-distance) between species were calculated using MEGA 11 [28].The best substitution models were selected with ModelFinder [29], using the Bayesian information criterion (BIC).Phylogenetic analyses were constructed based on the concatenated sequences of the Cyt b and ND4 gene sequences.Bayesian inference was performed with MrBayes 3.2.6 [30], using the GTR+F+I+G4 model for the first and second codon positions of Cyt b and ND4 and the GTR+F+G4 model for the third codon positions of Cyt b and ND4.Maximum likelihood analysis was performed with IQ-TREE 1.6.12[31], using the GTR+F+I+G4 model for the first and second codon positions of Cyt b and ND4 and the TN+F+R3 model for the third codon positions of Cyt b and ND4.

Morphological Results
Meristic and mensural characteristics were noted for all newly collected specimens (Tables 2 and 3).The morphological characteristics of the specimens collected from Malipo County in Wenshan Prefecture and Hekou and Jianshui counties in Honghe Prefecture agree with the morphological characteristics of Pareas hamptoni sensu stricto given by Ding et al. [11].However, the morphological characteristics of the specimens collected from Yuanyang County, Honghe Prefecture, can be distinguished from those of the specimens collected from Malipo, Hekou, and Jianshui, and all other named species of Pareas.

Molecular Results
Bayesian inference and maximum likelihood analysis resulted in similar topologies; the specimens from Malipo, Hekou, and Jianshui were clustered with Pareas hamptoni sensu stricto from Myanmar and Vietnam with strong supports, which should also be assigned to P. hamptoni sensu stricto, while the specimens from Yuanyang formed a distinct lineage sister to P. hamptoni sensu stricto with strong supports (Figure 1).The genetic divergences (uncorrected p-distance) between the specimens from Yuanyang and P. hamptoni sensu stricto were 4.2% and 5.0% in the Cyt b and ND4 gene sequences, respectively (Tables 4 and 5).Holotype.KIZ 2023038, adult female, collected from Guanyinshan Provincial Nature Reserve, Shuijingwan Village, Ganiang Township, Yuanyang County, Honghe Prefecture, Yunnan Province, China (23 Paratypes.KIZ 2023039-2023040, two adult females, with the same collection data as the holotype.

Class
Diagnosis.SVL 482-540 mm, TL/SVL 0.26-0.30;prefrontal bordering orbit; loreal not bordering orbit; 1 preocular; 1 postocular or postocular fused with subocular; 7-8 supralabials; 6-8 infralabials; infralabial not fused with chin-shield; 3 chin-shield pairs; dorsal scales in 15 rows throughout; 1 row of vertebral scales enlarged; scales not keeled at anterior part of body, 5 rows of mid-dorsal scales keeled at middle part of body, 5-7 rows of mid-dorsal scales keeled at posterior part of body; ventral scales 189-192; subcaudals 72-89, paired; precloacal plate undivided; maxillary teeth 4-5.Dorsal surface of head dark yellowish red or yellowish brown, with dense small black spots; dorsal surface of body yellowish red or yellowish brown; belly and ventral surfaces of head and tail pinkish yellow or yellow, with more or less small black spots; iris reddish yellow or yellow.
Description of the holotype.Adult female, SVL 488 mm, TL 146 mm, TL/SVL 0.30, TL/total length 0.23; body elongated, laterally compressed; head distinct from neck; snout wide and blunt, projecting beyond lower jaw; rostral approximately as wide as high, slightly visible from above; nasal undivided; internasal elongated; prefrontal approximately trapezoidal, bordering orbits; frontal shield-shaped, slightly longer than wide; parietals large, longer than wide, gradually narrower posteriorly, median suture approximately equal to length of frontal; single loreal, not bordering orbit; 1 supraocular, approximately triangular; 1 preocular; 1 postocular and 1 elongated crescent-shaped subocular; 2 anterior temporals, 3 posterior temporals; 7 supralabials on each side, separated from eyes; 7 infralabials on left side and 8 infralabials on right side, anterior-most in contact with its opposite between mental and anterior chin-shields, first 4 in contact with anterior chin-shield on left side and first 5 in contact with anterior chin-shield on right side; 3 chin-shields pairs, first pair and third pair triangle and large, second pair small and elongate, chin-shields interlaced, no mental groove under chin and throat; ventral scales 190; precloacal plate undivided; subcaudals 89, paired; dorsal scales in 15 rows throughout, 1 row of vertebral scales distinctly enlarged, scales not keeled at anterior body, 5 rows of mid-dorsal scales keeled at middle and posterior body; 5 maxillary teeth on left side and 4 maxillary teeth on right side.
Coloration of the holotype in life.Dorsal surface of head dark yellowish red, scattered with dense, small black spots; dorsal surface of body yellowish red; 2 wide black stripes on dorsal neck from occipital region; a black stripe from lower posterior orbit downwards and backwards to junction of last 2 supralabials and a black dot on last supralabial on each side of head; approximately 47 vertical black stripes on each side of body, most stripes on different sides connected to each other on vertebrals; some irregular black stripes on each side of tail; belly and ventral surfaces of head and tail pinkish yellow with many small black spots; iris reddish yellow, pupil black.
Coloration of the holotype in preservative.Yellowish red dorsal surfaces of head and body faded to pinkish brown; black stripes on sides of body and tail still distinct; pinkish-yellow belly and ventral surfaces of head and tail faded to pinkish white; iris changed to grayish black and pupil changed to white.
Variations.Morphometric and meristic data for the type series of the new species are provided in Table 2.The paratypes resemble the holotype, except that the subocular and the postocular are fused on one side of the head.The posterior temporals vary in number from two to four.The dorsal surfaces of the head and body vary from light yellowish red to yellowish brown, the belly and ventral surfaces of the head and tail vary from yellowish white to yellow, the number of vertical black stripes on each side of the body vary from 46 to 53, and the iris varies from reddish yellow to yellow in the paratypes.In addition, the small black spots on the belly are much fewer in one paratype.
Etymology.The specific epithet guanyinshanensis refers to Guanyinshan Provincial Nature Reserve, where the new species was found.
Distribution.This new species is currently known only from Guanyinshan Provincial Nature Reserve in Yuanyang County, Honghe Prefecture, Yunnan Province, China (Figure 5).Habitat.All specimens of the new species were found on small branches or on the ground beside a stream at night, with forest and farmland nearby (Figure 6).
As the new species is phylogenetically the sister to and most resembles Pareas hamptoni sensu stricto, in order to compare the new species with P. hamptoni sensu stricto, we rely on the morphological data given by Ding et al. [11], combined with the original description [17] of this species and the new data in this paper, to obtain a relatively reliable morphological characterization of P. hamptoni sensu stricto.Although the morphological data of P. hamptoni sensu stricto given by Ding et al. [11] are slightly different from the original description [17] of this species, Ding et al. examined five specimens of P. hamptoni sensu stricto, which included the holotype of this species.Therefore, when the morphological data of P. hamptoni sensu stricto given by Ding et al. [11] are inconsistent with the original description [17] of this species, we adopt those given by Ding et al. [11].
Pareas hamptoni was once considered to be widely distributed, from Myanmar, Thailand, Laos, and Vietnam to southern China [18].Wang et al. [6] restricted the distribution of P. hamptoni sensu stricto to Myanmar.Ding et al. [11] demonstrated that P. hamptoni sensu stricto is also distributed in northern Vietnam.Based on the specimens collected from Honghe and Wenshan prefectures in Yunnan Province, we confirmed the distribution of P. hamptoni sensu stricto in China (Figure 5) and describe a new species that is closely related to P. hamptoni sensu stricto.The distribution areas of these two species do not overlap, as the new species is from the southwest of the Red River, while all specimens of P. hamptoni sensu stricto in China were from the northeast of the Red River.But further downstream of the Red River, the situation is different.The record of P. hamptoni sensu stricto in Vietnam is from Lao Cai, which is located southwest of the Red River.In this way, P. hamptoni sensu stricto is distributed on both sides of the lower reaches of the Red River.However, in the relative upstream, P. hamptoni sensu stricto and the new species are distributed on different sides of the Red River, respectively.

Conclusions
A new Pareas species of the P. hamptoni complex (Figure 7) is described in this paper, based on three specimens collected from Guanyinshan Provincial Nature Reserve in Yuanyang County, Honghe Prefecture, Yunnan Province, China.Currently, the new species is known only from its type locality.The local ecological environment is relatively well maintained, and this species is less threatened at present.

Figure 1 .
Figure 1.Bayesian phylogeny tree of Pareas, based on concatenated Cyt b and ND4 fragments.Node numbers before "/" indicate Bayesian posterior probabilities (values below 0.90 are not shown) and numbers after "/" indicate ultrafast bootstrap support for the maximum likelihood analyses (values below 90 are not shown).

Figure 1 .
Figure 1.Bayesian phylogeny tree of Pareas, based on concatenated Cyt b and ND4 fragments.Node numbers before "/" indicate Bayesian posterior probabilities (values below 0.90 are not shown) and numbers after "/" indicate ultrafast bootstrap support for the maximum likelihood analyses (values below 90 are not shown).

Figure 3 .
Figure 3. Close-up views of the head of the holotype (KIZ 2023038) in preservative.(A) Dorsal view; (B) right side view; (C) ventral view.Figure 3. Close-up views of the head of the holotype (KIZ 2023038) in preservative.(A) Dorsal view; (B) right side view; (C) ventral view.

Figure 3 .
Figure 3. Close-up views of the head of the holotype (KIZ 2023038) in preservative.(A) Dorsal view; (B) right side view; (C) ventral view.Figure 3. Close-up views of the head of the holotype (KIZ 2023038) in preservative.(A) Dorsal view; (B) right side view; (C) ventral view.

Figure 6 .
Figure 6.The habitat of Pareas guanyinshanensis sp.nov. at the type locality.Figure 6.The habitat of Pareas guanyinshanensis sp.nov. at the type locality.

Figure 6 .
Figure 6.The habitat of Pareas guanyinshanensis sp.nov. at the type locality.Figure 6.The habitat of Pareas guanyinshanensis sp.nov. at the type locality.

Table 1 .
Samples used for molecular phylogenetic analysis in this study.

Table 2 .
Measurements (in mm) and the scalation data of the type series of Pareas guanyinshanensis sp.nov.For abbreviations, see the Materials and Methods section.

Table 3 .
Measurements (in mm) and scalation data of the specimens of Pareas hamptoni sensu stricto from China.For abbreviations, see the Materials and Methods section.

Table 6 .
[11]arison between Pareas guanyinshanensis sp.nov.andP.hamptoni sensu stricto.Measurements are in mm.The data for P. hamptoni sensu stricto were obtained by combining those of the original description, Ding et al.[11], and this study.