A new ovoviviparous rat snake species of the genus Elaphe (Squamata: Colubridae) from western Sichuan, China

The genus Elaphe is a highly diverse snake group with wide distribution in Eurasia. Here, based on morphological comparisons and molecular data, we describe a new species of this genus from western Sichuan, China. Bayesian inference and maximum-likelihood analyses of two mitochondrial DNA fragments (CO1 and 12S) showed that the new taxon differs from its congeners (CO1-based p-distance ≥ 4.1%). Morphologically and ecologically, the new species can be diagnosed from other species by a combination of the following characters: 1) medium body size, < 780 mm in total length; 2) midbody dorsal scales in 23 rows generally, not keeled; 3) ventrals 169–180 and subcaudal pairs 56–63; 4) first preocular docked with frontal; 5) internasal scales approximately trapezoidal; 6) length of gap between internasals more than 3/5 that between prefrontal; 7) length of tip of snout to frontal slightly larger than length of frontal; 8) ovoviviparous. Currently, the new species is known only from the Sichuan and Shaanxi provinces in China. This new species brings the total number of species in the genus Elaphe to 18.

The genus Elaphe Fitzinger in Wagler, 1833 (i.e., rat snakes) was originally erected based on the species Coluber quatuorlineatus Lacépède, 1789. The genus currently contains were sequenced by a commercial company (Sangon, Chengdu, China). Sequences were edited manually using SeqMan in Lasergene v15.1 (DNASTAR, Inc., Madison, Wis.), aligned in MEGA v7 using the ClustalW algorithm with default parameters (Thompson et al. 2003;Kumar et al. 2016), and checked by eye for ambiguous alignments. We translated the protein-coding gene (CO1) into an amino acid sequence using MEGA v7 to evaluate sequence quality (Kumar et al. 2016). The newly generated sequences were submitted to GenBank, and composed the dataset (Table 1).
We downloaded 88 sequences from 18 Elaphe species from GenBank, with Euprepiophis mandarinus used as the outgroup (Qi et al. 2021). Details on sample information are shown in Table 1.
Bayesian inference (BI) and maximum-likelihood (ML) analyses were executed using MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) and IQ-tree v1.6.12 (Lam-Tung 2015) respectively based on the combined CO1 and 12S dataset. Prior to analysis, best-fit nucleotide substitution models were chosen using PartitionFinder v2.1.1 under Akaike information criteria (Lanfear et al. 2017). For BI, all searches consisted of three heated chains and a single cold chain. Three independent iterations were performed, each comprised of two runs of 1×10 7 generations, with sampling every 1 000 generations. The parameter estimates were plotted against the generations and the first 25% were discarded as burn-in. ML analysis was performed with 1 000 fast bootstrap repeats.
Uncorrected p-distances based on the partial CO1 fragment were calculated using MEGA v7 (Kumar et al. 2016).

Morphological comparison
In total, 37 specimens, including three unidentified specimens and 34 specimens from E. dione, were examined morphologically (Appendix I). All specimens examined are preserved at Yibin University (YBU) or the Chengdu Institute of Biology, Chinese Academy of Sciences (CIB, CAS).
Snout-vent length (SVL, tip of snout to vent) and tail length (TaL) were measured with a tape measure to the nearest 1 mm. Symmetrical head characters were measured on both sides and given in left/right order. Number of dorsal scale rows is given at two head lengths posterior to head, at midbody (corresponding to half total ventral number), and at two head lengths anterior to anus. Ventral scales were counted according to Dowling (1951).
Coloration patterns were based on preserved specimens. Abbreviations of body characters include: DS: dorsal scale rows, counted at two head length behind head, at midbody, and at two head length before vent; Sup: supralabials; In: infralabials; Pr: preoculars; Pt: postoculars; Vs: ventral scales; Sc: subcaudals; IgL: length of gap between internasals; PgL: length of gap between prefrontal; FL: maximal length of frontal; FAW: anterior width of frontal; FPW: posterior width of frontal; RFL: distance between anterior frontal and rostral; PrF: preocular touching frontal or not.

Reproductive observations
In August 2020, we collected two pregnant female snakes in Toba Township, Ganzi County, Ganzi Tibetan Autonomous Prefecture, Sichuan, China. They were bred in the laboratory and their reproduction was observed and recorded.

Phylogenetic analyses
The final alignment of mitochondrial gene fragment consisted of 941 nucleotide base pairs (bp) (CO1: 513 bp, 12S: 428 bp). The best-fit model for each coding position in both sequences is listed in Table 2. The BI and ML results were generally identical in topology, with only slight disagreement in some nodal support ( Figure 2). Both trees indicated that all putative species of Elaphe formed a highly supported lineage (1.00 posterior probability (PP) in BI and 100% bootstrap support (BS) in ML) ( Figure 2). The two unidentified specimens from Sichuan were nested within the genus and formed a clade with three specimens previously identified as E. dione with high support (1.00 PP and 96% BS) and were sister to a clade including the other recognized specimens of E. dione.
The uncorrected P-distance of CO1 gene (513bp) for 31 sequences from 18 species of Elaphe, its congeners was 4.1%-13.0% and conspecific divergence was 0.2%-0.8% (Table 3). frontal slightly larger than length of frontal; 7) length of tip of snout to frontal slightly larger than length of frontal; 8) ovoviviparous. Zigzagging irregular brown stripe extending from top of head to nape; dark stripe extending from eye to corner of mouth. Head below milky white without spots normally.

Description
Body light brown with dark brown transverse bands on back of body; three dark, longitudinal stripes, two of which extend onto tail; abdomen milky white with scattered dark spots. Elaphe ganziensis sp. nov. is very similar to E. dione in morphology and genetics.

Infraspecific morphological variation.
However, in addition to the characters mentioned above, these species can be differentiated Distribution. This species is currently known only from the Sichuan (Ganzi, Hongyuan, and Kangding) and Shaanxi provinces in China.
Natural history. The specimens were found between 9:30 am and 10:30 am on grass between the road and hill at an elevation > 3 000 m ( Figure 9). The area has a plateau mountain climate characterized by a short spring and summer and long autumn and winter.
The new species is ovoviviparous, and gives birth from August to September, with 3-7 neonates each time. Newborns weigh 4.05-6.81 g (body mass) and are 19.20-23.70 cm in total body length (Table 4). No information on diet is available.

Discussion
Oviparity and ovoviviparity are two reproduction models in snakes, though most colubrid snake species are oviparous (Zhao 2006). As far as we know, all previously described species of Elaphe are oviparous (Zhao 2006;Uetz et al. 2021). In this study, based on genetics, morphology, and ecology, we described a new species with unique reproduction from the Hengduan Mountains and adjoining regions. Morphologically, the new species is very similar to E. dione, and both species are close in genetic distance. However, Elaphe ganziensis sp. nov. differs from E. dione based on its ovoviviparous reproduction and several morphological characters.
Elaphe dione has been recorded from China, Russia, Korea, and Central Asia (Uetz 2021). Previously collected specimens from western Sichuan were designated as E. dione (Zhao et al. 1998(Zhao et al. , 2003Zhao 2006