A new species of the genus Pareas (Squamata, Pareidae) from Yunnan, China

Abstract A new species of Pareas is described from Yunnan Province, China, based on morphological comparisons and molecular data. Genetically, the new species is most closely related to the recently-described Pareas geminatus, for which we present new topotypic findings. The genetic divergence (uncorrected p-distance) of the cytb gene between the new species and congeners ranged from 6.14% to 24.68%. Morphologically, the new species can be distinguished from P. geminatus and all other congeners. Our work brings the total number of species within the genus Pareas to 22.


Introduction
The family Pareidae Romer encompasses two subfamilies and four genera (Deepak et al. 2018;Uetz et al. 2020) and was once considered as a subfamily (called Pareatinae) of Colubridae (Smith 1943;Zhao et al. 1998;Zhao 2006). The taxonomy of the genus Pareas Wagler remains in a state of flux and species complexes with wide distributions and several lineages with an unclear taxonomic status remain especially challenging (Guo et al. 2011;Vogel 2015;You et al. 2015;Vogel et al. 2020;Wang et al. 2020). You et al. (2015) re-evaluated the taxonomic status of the P. hamptoni-formosensis complex from Taiwan, China, Ryukyus and adjacent regions, but the taxonomic status of the complex from mainland Chian was not involved. Wang et al. (2020) described P. menglaensis Wang, Che, Liu, Li, Jin, Jiang, Shi and Guo in the P. carinatus-nuchalis complex from Yunnan, China, but without any comments on the distribution of P. carinatus (Wagler) and described P. mengziensis Wang, Che, Liu, Li, Jin, Jiang, Shi and Guo without first resolving the historically taxonomic confusions of P. yunnanensis (Vogt) and P. niger (Pope). Vogel et al. (2020) re-assessed the taxonomy of the P. margaritophorus-macularius complex, re-validated two species and further reconfirmed the full species status of P. macularius Theobald in the complex.  underlined that the taxonomy of the genus Pareas has not yet been fully assessed, especially in widely-distributed taxa often representing complexes of cryptic or morphologically-similar species.
During our field research in Yunnan Province, China, from 2019 to 2020, some small and slender arboreal nocturnal snakes with blunt snouts and no mental groove and no teeth on the anterior part of maxillary, which could be assigned to the genus Pareas, were collected from Lancang County, Jiangcheng County and Kunming City. Morphological comparison and molecular analyses indicated that the specimens from Lancang County are distinct from all named species of the genus Pareas and we consequently described them as a new species.

Materials and methods
Specimens were collected in the field. Photographs were taken to document the colour pattern in life prior to euthanasia. Liver tissues were stored in 99% ethanol and snakes were preserved in 75% ethanol. Specimens were deposited at Kunming Natural History Museum of Zoology, Kunming Institute of Zoology, Chinese Academy of Sciences (KIZ).

Morphometrics
Measurements were taken to the nearest 1 mm with digital calipers. Paired meristic characters are given as left/right. The methodology of measurements and meristic counts followed Wang et al. (2020). The abbreviations of measurements and meristic counts are given below:

DS
dorsal scale rows (counted at one head length behind head, at mid-body and at one head length before vent, respectively); InfL infralabials; LoBO loreal bordering orbit; Max maxillary teeth; NED number of enlarged dorsal scale rows at mid-body; NKD number of keeled dorsal scale rows at anterior/middle/posterior of body; PosO postoculars; PreO preoculars; PrFBO prefrontal bordering orbit; Sc subcaudals; SPOF subocular-postocular fused or not; SubO suboculars; SupL supralabials; SVL snout-vent length (from tip of snout to posterior margin of cloacal plate); Tem temporals; TL tail length (from posterior margin of cloacal plate to tip of tail); Vs ventrals.

Phylogenetic analyses
Molecular data were generated for two specimens from Lancang County, two specimens from Jiangcheng County and one specimen from Kunming City. Homologous sequences were obtained from GenBank. All new sequences have been deposited in GenBank. Aplopeltura boa (Boie), Asthenodipsas laevis (Boie) and Xylophis captaini Gower and Winkler were selected as outgroups, based on Wang et al. (2020). All the GenBank accession numbers for taxa used in this study are listed in Table 1.
Total genomic DNA was extracted from liver tissues using the OMEGA DNA Kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The sequences of the mitochondrial gene fragment, cytochrome b (cytb), were amplified by polymerase chain reaction (PCR) using primers L14910/H16064 (Burbrink et al. 2000). The double-stranded products were purified and sequenced at Genewiz Co. (Suzhou, China). Sequences were edited and manually managed using SeqMan in Lasergene 7.1 (DNASTAR Inc., Madison, WI, USA) and MEGA 7 (Kumar et al. 2016).
Sequences were aligned using ClustalW (Thompson et al. 1994) integrated in MEGA 7 with default parameters (Kumar et al. 2016). The genetic divergence (uncorrected p-distance) between species was calculated in MEGA 7 with the parameters Transitions + Transversions, Uniform rates and Pairwise deletion (Kumar et al. 2016). The substitution model GTR+I was selected in MODELTEST 3.7 (Posada and Crandall 1998). Bayesian Inference was performed in MrBayes 3.2.6 (Ronquist et al. 2012), based on the selected substitution model. Two runs were performed simultaneously with four Markov chains starting from a random tree. The chains were run for 1,000,000 generations and sampled every 100 generations. The first 25% of the sampled trees was discarded as burn-in after the standard deviation of split frequencies of the two runs was less than a value of 0.01 and then the remaining trees were used to create a 50% majority-rule consensus tree and to estimate Bayesian posterior probabilities. Maximum Likelihood analysis was performed in RaxmlGUI 2.0 (Silvestro and Michalak 2012) and nodal support was estimated by 1,000 rapid bootstrap replicates.

Results
Meristic and mensural characters were noted for all examined specimens (Tables 2, 3). The three specimens collected from Lancang County could be distinguished from all other congeners. Morphological characters of the two specimens from Jiangcheng  0-0-0 0-5-5 0-5-5 0-0-3 0-5-5 Max 6/6 8/7 6/6 6/6 6/7 County agreed with Pareas geminatus Ding, Chen, Suwannapoom, Nguyen, Poyarkov and Vogel and morphological characters of the specimen collected from Kunming City agreed with the topotypic specimens of P. niger. Maximum Likelihood analyses and Bayesian Inference showed similar results, the specimen form Kunming City clustered with Pareas mengziensi, the specimens from Jiangcheng County clustered with P. geminatus and the specimens from Lancang Coun- ty formed a distinct lineage which is sister to P. geminatus with strong support (Fig. 1). The genetic divergence (uncorrected p-distance) between the lineage from Lancang County and P. geminatus was 6.14% (Table 4).   Paratypes. KIZ XL2, adult male and KIZ XL3, adult female, the same collection data as the holotype.
Diagnosis. Single preocular; postocular fused with subocular; loreal not bordering orbit; prefrontal bordering orbit; fourth or fifth infralabial fused with second chinshield; three chin-shield pairs; dorsal scales in 15 rows throughout; vertebral scales not enlarged; scales not keeled at the anterior part of the body, three rows of mid-dorsal scales keeled at the middle of the body, five rows of mid-dorsal scales keeled at the posterior of body; seven supralabials; seven or eight infralabials; cloaca undivided; ventral scales 182-188; subcaudals 87-93, paired.
Description of holotype. Male, SVL 403 mm, TL 132 mm, TL/total length 0.25; body elongated; head distinct from neck; snout wide and blunt, projecting beyond lower jaw; body laterally compressed, vertebral ridge poorly developed. Rostral approximately as wide as high, almost invisible from above; nasals undivided; internasals elongated, much wider than long; prefrontals triangular, wider than long, bordering orbits; frontal shield-shaped, longer than wide; parietals large, longer than wide, median suture longer than frontal; single loreal, separated from eyes; single preocular; one relatively small supraocular, longer than wide; subocular and postocular fused into one thin elongated crescent-shaped scale; temporals 2+2+2 on both sides; seven supralabials on both sides, separating from eyes; seven infralabials on both sides, anterior-most in contact with its opposite between mental and anterior chin-shields, first four in contact with anterior chin-shields; fourth fused with second chin-shield; three chinshields pairs, the first pair and the third pair triangle and almost equal size, the second pair elongate; ventral scales 188; cloaca undivided; subcaudals 89, paired; dorsal scales in 15 rows throughout, vertebral scales not enlarged, scales not keeled at anterior of body, three rows of mid-dorsal scales keeled at middle of body, five rows of mid-dorsal scales keeled at posterior of body; seven maxillary teeth on left side and six maxillary teeth on right side; hemi-penis in situ extending to the 19 th subcaudal.
Colouration in life. Dorsal surface of head and body reddish-yellow with many black tiny spots on each scale; a thin black discontinuous postorbital stripe extending from postocular to neck, which is connected with its fellow on the opposite side by a thick black line which curves forward so as to almost touch the parietals; two thick black discontinuous stripes on neck followed the black curves forward line; many irregular longitudinal black stripes on the sides of body and tail, the stripes on different sides not connected to each other on the vertebrals; belly and ventral surface of head and tail yellow with sparse small black spots; iris reddish-yellow, pupil black.
Colouration in preservative. The reddish-yellow dorsal surface of the head and body faded to yellowish-white; the yellow belly and ventral surface of head and tail faded to pale white; the iris changed to greyish-black from reddish-yellow and the pupil changed from black to white. Variations. Morphometric and meristic data for the type series are provided in Table 3. The paratype KIZ XL2 has 2+3+2 temporals on the left side and 2+2+3 temporals on the right side. The paratype KIZ XL3 has eight infralabials on both sides, first five being in contact with anterior chin-shields, fifth fused with second chin-shield.
Etymology. The specific epithet xuelinensis refers to Xuelin Township, the type locality of the new species.
Distribution. This species is currently known only from the type locality Xuelin Township, Lancang County, Pu'er City, Yunnan Province, China. It is expected to be found in Myanmar.
Habitat. Both the holotype and paratypes were found on the bushes beside a small road at night, surrounded by forest and farmland, with no river or stream nearby.
Pareas xuelinensis sp. nov. can be distinguished from P. geminatus by vertebral scales not enlarged (vs. vertebral scales enlarged), three rows of mid-dorsal scales keeled at middle of body (vs. five rows of mid-dorsal scales keeled at middle of body), fourth or fifth infralabial fused with the second chin-shield (vs. infralabials not fused with chinshield), temporals 2+2+2 or 2+3+3 (vs. 1+2+1 or 1+1+1) and no black spot on each side of head (vs. having two black spots on each side of head).
Pareas xuelinensis sp. nov. can be distinguished from P. niger by having more ventral scales (182-188 vs. 154-172), more subcaudals (87-93 vs. 55-66) and the dorsal surface of head and body reddish-yellow (vs. the dorsal surface of head and body solid black). Pareas xuelinensis sp. nov. can be distinguished from P. yunnanensis by the loreal separating from the eye (vs. the point of the large loreal touching the eye), vertebral scales not enlarged (vs. vertebral scales enlarged), and 0-5 rows of mid-dorsal scales keeled (vs. six rows of dorsal scales keeled).

Discussion
Amblycephalus niger (now Pareas niger) was described by Pope (1928) from Yunnanfu (now Kunming). Pareas niger was considered as synonyms of P. chinensis, P. yunnanensis and P. komaii, successively (Sichuan Institute of Biology 1977; Rao and Yang 1992;Uetz et al. 2020). We compared the morphometric and meristic data of P. niger, P. chinensis, P. komaii and P. yunnanensis (Table 5) and found that P. niger can be distinguished from P. chinensis, P. komaii and P. yunnanensis, so we consider P. niger to represent a valid species. During August 2019, we collected one specimen (voucher: KIZ 059339; Fig. 7) of Pareas with a solid black dorsal surface on the head and body and no enlarged mid-dorsal scales from Changchong Mountain in Kunming City, its morphological characters agreeing with the original description of P. niger, except that it has nine infralabials, which agreed with the original description of P. mengziensis. Molecularly, the specimen from Kunming was clustered together with P. mengziensis and the genetic divergence (uncorrected pdistance) between the specimen from Kunming and P. mengziensis was only 0.29%. After checking the topotypic specimens of P. niger preserved in KIZ, we found that the infralabials of P. niger range from seven to nine and one or three rows of middorsal scales of several individuals are slightly enlarged. This means that the specimen (KIZ 059339) from Kunming should belong to P. niger and it is not appropriate to distinguish P. mengziensis and P. niger either morphologically or molecularly; therefore, we consider P. mengziensis and P. niger as the same species, P. mengziensis being a synonym of P. niger. Table 5. Comparisons of morphometric and meristic data for Pareas niger, P. chinensis, P. komaii and P. yunnanensis. The data for P. chinensis, P. komaii and P. yunnanensis were obtained from the original descriptions and the subsequent descriptions of the type specimens (Barbour 1912;Vogt 1922;Maki 1931;Pope 1935). "?" = data not available.