A New Species of Pareas Wagler, 1830 (Squamata, Pareidae) from Northwestern Yunnan, China †

: A new species of Pareas Wagler, 1830 is described from Dulongjiang Township, Gongshan County, Yunnan Province, China. Phylogenetically, the new species is most closely related to Pareas kaduri Bhosale, Phansalkar, Sawant, Gowande, Patel and Mirza, 2020; however, the genetic diver‑ gence (uncorrected p‑distance) of the cyt b gene between the new species and P. kaduri reached 9.4%. Morphologically, the new species can be distinguished from P. kaduri by the absence of preoculars, lo‑ real bordering the orbit, the fusion of subocular and postocular, three rows enlarged vertebral scales, five rows keeled mid‑dorsal scales at the middle of the body, having more subcaudals, and having a relatively longer tail. In addition, the new species can be distinguished from all other congeners by a combination of morphological characteristics. Our work brings the total number of recognized species of the genus Pareas to 29, of which 23 occur in China.

Evolutionarily, although there is practically no fossil record for Pareinae [9], based on the calibration priors from Deepak et al. [5], Poyarkov et al. [8] inferred that Pareinae originated in Sundaland during the middle Eocene and the most recent common ancestor of Pareinae originated in late Eocene. Pareinae colonized mainland Asia in the early Oligocene, when the genera Asthenodipsas and Pareas started to diversify [8].

Materials and Methods
The specimen of the new species was collected by hand at night and was euthanized using isoflurane, injected intraperitoneally with 95% ethanol, and then preserved in 75% ethanol. Liver tissue was collected for molecular analysis and stored in 99% ethanol. The specimen was deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).
Measurements of the body and tail were taken to the nearest 1 mm with a measuring tape and measurements of the head were taken to the nearest 0.1 mm with a digital caliper. Paired meristic characters are given as left/right. The methodology of measurements and meristic counts followed those of Bhosale et al. [16] and Liu et al. [21]. Abbreviations used were as follows: ATem, number of anterior temporals; CL, cephalic length (measured from tip of snout to constriction of neck); DNB, dark nuchal band; DS, dorsal scale rows (counted at one head length behind the head, mid-body, and one head length before the vent); ED, eye diameter (widest diameter of the eye); El, eye to labial height (measured from lowest border of the eye to lower border of the labial); EN, eye to nares distance; ES, eye to snout length; HL, head length (measured from snout tip to the angle of the jaw); HW, head width (measured at the widest part of the head); InfL, infralabials; LoBO, loreal bordering orbit; Max, maxillary teeth; NED, number of enlarged dorsal scale rows at midbody; NKD, number of keeled dorsal scale rows at the middle of the body; NW, neck width (measured at the constriction of the neck); PosO, postoculars; Prec, precloacal plate; PreO, preoculars; PrFBO, prefrontal bordering orbit; PTem, number of posterior temporals; Sc, subcaudals; SPOF, subocular-postocular fused or not; SubO, suboculars; SupL, supralabials; SVL, snout-vent length (measured from tip of snout to posterior margin of cloacal plate); TaL, tail length (measured from posterior margin of cloacal plate to tip of tail); TL, total length; VBTr, vertical dark bars on trunk; Vs, ventrals. For comparison, data for other species were taken from the original and subsequent descriptions [2,6,8,[11][12][13][14][15][16][17][18][19][20][21][25][26][27][28][29][30][31][32][33].
The sequence of the mitochondrial gene fragment cytochrome b (cyt b) was generated for the newly collected specimen. Homologous sequences were obtained from GenBank. The new sequence has been deposited in GenBank. Aplopeltura boa (Boie, 1828) was selected as the outgroup, based on the work of Liu et al. [21]. All the GenBank accession numbers for the taxa used in this study are listed in Table 1. Genomic DNA was extracted from liver tissue; the primers L14910/H16064 [34] were used for DNA sequencing. DNA extraction and amplification conditions followed those of Poyarkov et al. [8]. The amplification products were purified and sequenced by Tsingke Biotechnology Co., Ltd., Beijing, China. The sequence was edited and managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA 11 [35]. Table 1. Sequences (cyt b) used in phylogenetic analysis of this study. "Mt." = mountain, "Is." = island, "Hw." = highway. Sequences were aligned using ClustalW [36] integrated in MEGA 11 [35] with default parameters. Uncorrected pairwise genetic divergences (p-distance) were estimated using MEGA 11 [35]. The best substitution model (GTR + F + I + G4) for Bayesian inference and the best substitution model (GTR + F + R4) for maximum likelihood were selected using ModelFinder [37] in IQ-TREE 1.6.12 [38] under the Akaike Information Criterion (AIC). Bayesian inference was performed in MrBayes 3.2.7 [39]. Four Markov chains were run for 10,000,000 generations sampled every 1000 generations. The first 25% of the sampled trees were discarded as burn-in and the remaining trees were used to calculate Bayesian posterior probabilities. Maximum likelihood analysis was performed using IQ-TREE 1.6.12 [38]. Nodal support was estimated by 1000 ultrafast bootstrap replicates. Nodes with Bayesian posterior probabilities of 0.95 and above were considered strongly supported [40][41][42] and nodes with ultrafast bootstrap values of 95 and above were considered strongly supported [43].

Results
Maximum likelihood analyses and Bayesian inference showed identical topology; the newly collected specimen formed a distinct lineage which is sister to Pareas kaduri with strong support, which means that the newly collected specimen belongs to the subgenus Eberhardtia and is a member of the P. hamptoni species group [8] (Figure 1). The genetic divergence (uncorrected p-distance) between the newly collected specimen and P. kaduri was 9.4%, the genetic divergence (uncorrected p-distance) between the newly collected specimen and other congeners ranged from 10.3% to 25.0% (Table 2).         Diagnosis. Preocular absent; postocular fused with subocular; loreal bordering orbit; prefrontal bordering orbit; infralabials not fused with chin-shield; chin-shields three pairs, first pair triangular, second pair and third pair elongate; dorsal scales 15 rows throughout without apical pits; three rows vertebral scales enlarged; five rows mid-dorsal scales keeled at middle of body; supralabials6-7; infralabials7-9; precloacal plate undivided; ventral scales182; and subcaudals 76, paired. In preservative, dorsal surface of head chocolate brown, from which two brownish-black longitudinal stripes run on each side of the neck, leaving a pale central portion; lateral surface of head dense with brownish black dusting and two short brownish-black streaks, one on lower anterior temporal and another on last supralabial; almost no mottling on ventral surface of head; sparse brown mottling on ventral surfaces of body and tail; and indistinct vertical dark bars on trunk and tail. Diagnosis. Preocular absent; postocular fused with subocular; loreal bordering orbit; prefrontal bordering orbit; infralabials not fused with chin-shield; chin-shields three pairs, first pair triangular, second pair and third pair elongate; dorsal scales 15 rows throughout without apical pits; three rows vertebral scales enlarged; five rows mid-dorsal scales keeled at middle of body; supralabials6-7; infralabials7-9; precloacal plate undivided; ventral scales182; and subcaudals 76, paired. In preservative, dorsal surface of head chocolate brown, from which two brownish-black longitudinal stripes run on each side of the neck, leaving a pale central portion; lateral surface of head dense with brownish black dusting and two short brownish-black streaks, one on lower anterior temporal and Description. Adult female, SVL 373 mm, TL 115 mm, TaL/SVL 0.308, TaL/TL 0.236; body elongated, slightly compressed laterally; body vertebral ridge developed; head distinct from neck; snout wide and blunt, projecting beyond lower jaw. Rostral approximately as wide as high, not visible from above; nasals undivided; internasals wider than long; prefrontals bordering orbits; frontal anteriorly fused with prefrontals; parietals large, much longer than wide; loreal, single, large, approximately rectangular, broadly bordering orbit; preocular absent; supraocular single, slightly longer than wide; subocular and postocular fused into one thin elongated crescent-shaped scale; anterior temporals two on each side, posterior temporals three on left side and two on right side; supralabials six on left side and seven on right side, all separated from eye; infralabials seven on left side and nine on right side, anterior-most in contact with its opposite between mental and anterior chin-shields; infralabials not fused with chin-shield; chin-shields three pairs, first pair triangular, second pair and third pair elongate, and first pair larger than other two; ventral scales182; precloacal plate undivided; subcaudals 76, paired; dorsal scales 15 rows throughout without apical pits, three rows vertebral scales enlarged, and five rows mid-dorsal scales keeled at middle of body.
Coloration. In preservative over seven years, dorsal surface of head chocolate brown, from which two brownish-black longitudinal stripes run on each side of neck leaving a pale central portion; dorsal surface of body brownish-yellow, and approximately 52 indistinct, vertical, slightly billowing dark bars on trunk, and dark bars on tail more indistinct or invisible; lateral surface of head dense with brownish-black dusting and two short brownish-black streaks, one on lower anterior temporal and another on last supralabial; ventral surface of head light yellow with almost no mottling; and ventral surfaces of body and tail light yellow with sparse brown mottling. Coloration in life unknown.
Comparison. Pareas dulongjiangensis sp. nov. can be distinguished from P. andersonii, P. modestus, P. macularius, and P. margaritophorus by the yellow body background color (vs. body background colors being grey, dark grey, brownish-grey, or completely black).
Pareas dulongjiangensis sp. nov. can be distinguished from P. boulengeri and P. stanleyi by the three rows enlarged vertebral scales (vs. vertebral scales not being enlarged).
Pareas dulongjiangensis sp. nov. can be distinguished from P. nigriceps by the absence of preoculars (vs. preoculars being present), loreal bordering the orbit (vs. not bordering), three rows enlarged vertebral scales (vs. one row vertebral scales being enlarged), and having more ventral scales (182 vs. 175).
Pareas dulongjiangensis sp. nov. is most similar to P. kaduri in morphological characteristics and coloration; however, the new species can be distinguished from P. kaduri by the absence of preoculars (vs. preoculars being present), loreal bordering the orbit (vs. not bordering), the fusion of subocular and postocular (vs. the presence of one subocular and two postoculars), three rows enlarged vertebral scales (vs. one row vertebral scales being enlarged), five rows keeled mid-dorsal scales at the middle of the body in the single female (vs. mid-dorsal scales at the middle of the body being smooth in the single female), having more subcaudals (76 vs. 52-70), and having a relatively longer tail (TaL/SVL 0.308 vs. 0.226-0.262) (see Table 3). Table 3. Comparison of morphometric and meristic data for Pareas dulongjiangensis sp. nov. and P. kaduri. The data for P. kaduri were obtained from Bhosale et al. [16]. Etymology. The specific epithet dulongjiangensis refers to the Dulongjiang Township, where the new species was discovered. We propose "Dulongjiang Slug-eating Snake" for the common English name and " 独龙江钝头蛇" (Pinyin: dú lóng jiāng dùn tóu shé) for the common Chinese name of this species.

Pareas dulongjiangensis
Distribution. The new species is currently known only from its type locality in Dulongjiang Township, Gongshan County, Nujiang Prefecture, Yunnan Province, China.
Natural history notes. The specimen of the new species was found at night on the road near a small village by the Dandangluo River, which is a tributary of the Dulongjiang River; there are some rocks, grass, and plantains along the road (Figure 4) Natural history notes. The specimen of the new species was found at night on the road near a small village by the Dandangluo River, which is a tributary of the Dulongjiang River; there are some rocks, grass, and plantains along the road (Figure 4). More ecological information about this species is unknown. Other herpetological species observed near the type locality of the new species include Elaphe carinata (Günther, 1864); Nanorana chayuensis (Ye, 1977); Odorrana dulongensis Liu, Che & Yuan, 2021; Ovophis zayuensis (Jiang, 1977); Raorchestes dulongensis Wu, Liu, Gao, Wang, Li, Zhou, Yuan & Che, 2021; Rhabdophis leonardi (Wall, 1923); and Zhangixalus burmanus (Andersson, 1939).

Discussion
Vogel et al. [19] indicated a potential new species of Pareas from Putao District, Kachin State, northern Myanmar, which lies just between the type localities of P. kaduri and Pareas dulongjiangensis sp. nov. ( Figure 5); however, due to the lack of available molecular data for this potential new species, it was not formally described. In terms of morphology, this potential new species also resembles P. kaduri and Pareas dulongjiangensis sp. nov.; nevertheless, it differs from P. kaduri as the loreal contacts the eye (vs. not touching the eye) (see Figure 6), the dorsal scales are keeled in the single female (vs. smooth dorsal scales in the single female), it has slightly more subcaudals (71 vs. 65-70), and has a slightly, relatively longer tail (TaL/TL 0.210 vs. 0.184-0.207); it differs from Pareas dulongjiangensis sp. nov. by the presence of preoculars (vs. preoculars being absent), having one postocular (vs. postocular being fused with subocular), having one row enlarged median vertebral scales (vs. three rows), having fewer subcaudals (71 vs. 76), having a relatively shorter tail (TaL/TL 0.210 vs. 0.236), and having more vertical dark bars on the trunk (60 vs. 52). Although P. kaduri and Pareas dulongjiangensis sp. nov. have the closest phylogenetic relationship, the genetic distance between them is still relatively large (9.4%). We speculate that this potential new species is probably a species sandwiched between P. kaduri and Pareas dulongjiangensis sp. nov. As there is still no molecular data available for this potential new species, this speculation needs to be verified. slightly, relatively longer tail (TaL/TL 0.210 vs. 0.184-0.207); it differs from Pareas dulongjiangensis sp. nov. by the presence of preoculars (vs. preoculars being absent), having one postocular (vs. postocular being fused with subocular), having one row enlarged median vertebral scales (vs. three rows), having fewer subcaudals (71 vs. 76), having a relatively shorter tail (TaL/TL 0.210 vs. 0.236), and having more vertical dark bars on the trunk (60 vs. 52). Although P. kaduri and Pareas dulongjiangensis sp. nov. have the closest phylogenetic relationship, the genetic distance between them is still relatively large (9.4%). We speculate that this potential new species is probably a species sandwiched between P. kaduri and Pareas dulongjiangensis sp. nov. As there is still no molecular data available for this potential new species, this speculation needs to be verified.

Figure 5.
Map showing the type locality (black star) of Pareas dulongjaingensis sp. nov., the type locality (black dot) of P. kaduri, and the locality (black square) of the potential new species in Vogel et al. [19]. Map showing the type locality (black star) of Pareas dulongjaingensis sp. nov., the type locality (black dot) of P. kaduri, and the locality (black square) of the potential new species in Vogel et al. [19].
Pareas dulongjiangensis sp. nov. is phylogenetically closely related to P. kaduri. Pareas kaduri is distributed near the Lohit River bank, which belongs to the Brahmaputra River system, whereas Pareas dulongjiangensis sp. nov. is distributed near the Dulongjiang River bank, which belongs to the Irrawaddy River system. There are many high mountains between the two river systems [44], which would lead to geographical isolation. Therefore, biogeography also supports that Pareas dulongjiangensis sp. nov. and P. kaduri belong to two different species.
At present, Pareas dulongjiangensis sp. nov. is known only from a single specimen, and nothing is known about the actual distribution range and ecological data of this species. Further research is required to clarify the extent of its distribution, population trends, and conservation status. Moreover, investigation of species diversity in the China-Myanmar border region is far from complete due to the relatively inconvenient traffic conditions; more survey efforts are required to achieve a better understanding of the diversity of Pareas in this region. Pareas dulongjiangensis sp. nov. is phylogenetically closely related to P. kaduri. Pareas kaduri is distributed near the Lohit River bank, which belongs to the Brahmaputra River system, whereas Pareas dulongjiangensis sp. nov. is distributed near the Dulongjiang River bank, which belongs to the Irrawaddy River system. There are many high mountains between the two river systems [44], which would lead to geographical isolation. Therefore, biogeography also supports that Pareas dulongjiangensis sp. nov. and P. kaduri belong to two different species.
At present, Pareas dulongjiangensis sp. nov. is known only from a single specimen, and nothing is known about the actual distribution range and ecological data of this species. Further research is required to clarify the extent of its distribution, population trends, and conservation status. Moreover, investigation of species diversity in the China-Myanmar border region is far from complete due to the relatively inconvenient traffic conditions;