A New Species of the Genus Elaphe (Squamata: Colubridae) from Zoige County, Sichuan, China

Asian Herpetological Research 2012, 3(1): 38 45 DOI: 10.3724/SP.J.1245.2012.00038 A New Species of the Genus Elaphe (Squamata: Colubridae) from Zoige County, Sichuan, China Song HUANG 1, 2, 3*, Li DING 4*, Frank T. BURBRINK 5, 6, Jun YANG 7, Jietang HUANG 3, Chen LING 8, Xin CHEN 5, 6 and Yaping ZHANG 2** 1 College of Life and Environment Sciences, Huangshan University, Huangshan 245041, Anhui, China 2 State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China 3 Huangshan Institute of Ophiology, Huangshan 245000, Anhui, China 4 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, Sichuan, China 5 Department of Biology, the College of Staten Island, the City University of New York, 2800 Victory Blvd, Staten Island, NY 10314, USA 6 Department of Biology, the Graduate Center, the City University of New York, 365 Fifth Avenue, New York, NY 10016-4309, USA 7 School of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, China 8 College of Forestry, Southwest Forestry University, Kunming 650224, Yunnan, China Abstract Previous phylogenetic analyses of DNA sequence data reveal a likely new species of Elaphe Fitzinger from China, which forms the sister group of all extant Elaphe. One of the three original specimens is crushed and the other two are neonates, which precludes a morphological analysis. Three adult snakes (2 females, 1 male) collected from Jiangzha Hot Spring in Zoige County, Sichuan, China on 26 July 2010 now facilitate the species description. Mitochondrial DNA nucleotide sequences of cytochrome b (cyt b), NADH dehydrogenase subunit 4 (ND4), 12S ribosomal RNA (12S rrna), and cytochrome oxidase subunit 1 (CO1) from the adults are identical to sequences from the crashed specimen. Interspecific uncorrected p-distances of partial CO1 gene of 26 sequences from all 11 species of Elaphe, including this new species, are large (8.5 15.2%). This new species is morphologically distinguished from all other species of Elaphe by several characters: M -shaped marking on the supraoculars and adjacent frontal; four parallel series of red-brown spots on the dorsum extending from the neck to the vent; four longitudinal stripes associated with the spots; each spot is usually composed of a whole (or incomplete) red-brown scale with puce borders; spots of each stripe are similar in shape, they occur at relatively regular intervals of one or two scales, and they are slightly red-brown in color; and it has three preoculars, whereas all other species have one or two. Now Elaphe contains 11 species and this discovery highlights the need for continued exploration of temperate regions. Keywords new species, mitochondrial DNA, DNA barcoding, Zoige ratsnake 1. Introduction Nowadays, taxonomic revisions of major groups of vertebrates are common due, in part, to the revolution in * Both authors contribute equally to this work. ** Corresponding author: Prof. Yaping ZHANG, from Kunming Institute of Zoology, Chinese Academy of Sciences, with his research focusing on molecular phylogeny and evolution of vertebrates. E-mail: zhangyp1@263.net.cn Received: 5 January 2012 Accepted: 27 Feburary 2012 molecular phylogenetics and increased taxon sampling. These changes are important not only for classification, but also for comparative biology, which requires credible phylogenetic estimates and accurate assessments of extant taxa for hypothesis testing (Felsenstein, 1988; Díaz-Uriarte and Garland, 1996, 1998). Advanced snakes (Caenophidia), representing about 3 000 species, are the subject of several recent massive phylogenetic studies that propose numerous changes in classification (Pyron

No. 1 Song HUANG et al. New Species of the Genus Elaphe from Zoige, China 39 et al., 2011; Burbrink and Crother, 2011) as well as provide important tests of major hypotheses in evolution (Burbrink and Pyron, 2010). Within the advanced snakes, the ratsnakes are one of the most-well-studied groups. Recent major taxonomic changes occur at generic and species levels (Utiger et al., 2002; Burbrink and Lawson, 2007; Pyron and Burbrink, 2009a). Several studies demonstrate that the ratsnake genus Elaphe (sensu lato) is likely paraphyletic, particularly with respect to New World taxa that are more closely related to kingsnakes, pinesnakes and their allies (e. g., Lampropeltis, Pituophis, Rhinocheilus, Cemophora). The genus Elaphe, with the type species Coluber sauromates Pallas 1811 from Europe, is now restricted to the following ten taxa found throughout Eurasia: E. anomala (Boulenger), E. bimaculata Schmidt, E. carinata (Günther), E. climacophora (Boie), E. davidi (Sauvage), E. dione (Pallas), E. quadrivirgata (Boie), E. quatuorlineata (Lacépède), E. sauromates (Pallas) and E. schrenckii (Strauch) (Schulz, 1996; Helfenberger, 2001; Utiger et al., 2002; Burbrink and Lawson, 2007). Phylogenetic support for the monophyly of Elaphe is strong and placement of species within in this genus is confirmed by both nuclear and mtdna genes (Utiger et al., 2002; Burbrink and Lawson, 2007). Geographically, the highest diversity is centered in East Asia, although the range of the genus extends as in the far north to Russia (E. dione, E. schrenckii), in the south to Vietnam (E. carinata), east to the China Sea, Korean Peninsula and Japan (E. anomala, E. bimaculata, E. carinata, E. davidi, E. climacophora, E. quadrivirgata), and west to Italy (E. quatuorlineata). Molecular data provide a powerful means of identifying cryptic diversity, such as that within Elaphe (E. quatuorlineata) and the related genera Zamenis (Z. longissimus), Pantherophis (P. guttatus, P. obsoletus), and Lampropeltis (L. getula), (Lenk and Joger, 1994; Lenk et al., 2001; Burbrink et al., 2000; Burbrink, 2001, 2002; Pyron and Burbrink, 2009b, c). The rapid radiation of these advanced snakes may be responsible for the absence of morphological differentiation. For example, both E. dione and E. bimaculata are morphologically quite similar with respect to scutellation and color pattern, and both occur in China. Based on molecular phylogenetic analyses of nucleotide sequences from three mitochondrial DNA genes (cyt b, ND4 and 12S rrna), Ling et al. (2010) suggest that three specimens collected from Zoige County, Sichuan, China likely represent a new species of Elaphe. The matrilineal genealogy, which contains members from all genera of Old World ratsnakes and representatives from the New World strongly indicates that the three specimens represent the sister species to all other taxa in the genus Elaphe. This relationship suggests an undescribed species of Elaphe but it is not described because the absence of adult specimens precluded a morphological description. On 26 July 2010, we captured three adult snakes (2 females, 1 male) at Jiangzha Hot Spring, Zoige County, Sichuan, China. Based on molecular data from cyt b, ND4 and 12S rrna, the new specimens were determined to belong to Elaphe and to be conspecific with those reported by Ling et al. (2010). 2. Materials and Methods Three adult snakes (2 females, 1 male) were sampled at the side of a stream (Figure 1) by Li DING within 40 m of the mouth of Jiangzha Hot Spring at 12:00 hr on 26 July 2010. Several days later, two females laid 11 eggs, respectively (Figure 2) in field terrariums. The eggs were smooth-shelled and adhesive. The holotype (Collection number: HS201014) and paratypes (HS201015 16) were preserved and deposited in the Museum of Huangshan University (Voucher numbers: HUM20110001 3). Tissues from the distal ends of ventral scales were sampled from all the new specimens. Partial mtdna sequences were obtained by polymerase chain reaction (PCR) and direct sequencing using the primers and methods described in Arévalo et al. (1994), Burbrink et al. (2000), and Huang et al. (2009) for cyt b (1 015 bp) and ND4 (696 bp), and Utiger et al. (2002) for 12S rrna (428 bp) and CO1 (513 bp). The new cyt b, ND4 and 12S sequences were identical to those reported by Ling et al. (2010) (GenBank accession number: HQ330525, HQ330527 and HQ330523, respectively). The absence of variation indicated that the six snakes were conspecific. Because sequences from COI DNA barcoding have enabled the discrimination of closely allied species (Hebert et al., 2003) and provided a standardized tool to inventory biodiversity (Naro-Maciel et al., 2010), we sequenced the gene for the three new specimens (GenBank accession numbers: JF510474 76). The uncorrected p-distances of partial CO1 gene of 26 sequences from all the 11 species of Elaphe (23 from Utiger et al., 2002; 3 from this study) were calculated using the program MAGA 4 (Tamura et al., 2007). COI p-distances ranged between 8.2 15.0 among species and between 0 2.5 within species (Table 1). All measurements and observations were conducted

40 Asian Herpetological Research Vol. 3 Table 1 Matrix of uncorrected P-distances of partial CO1 gene (513bp) for 26 sequences from 11 species of Elaphe. GenBank accession numbers follow species names. [ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 ] [ 1] HUM20110001 JF510474 0 [ 2] HUM20110003 JF510475 0.4 0 [ 3] HUM20110002 JF510476 0.4 0.4 0 [ 4] E. climacophora AY122687 14.0 13.6 13.6 0 [ 5] E. climacophora AY122686 14.0 13.6 13.6 0.0 0 [ 6] E. climacophora AY122688 13.5 13.1 13.1 0.6 0.6 0 [ 7] E. bimaculata AY122753 13.5 13.5 13.1 13.8 13.8 13.5 0 [ 8] E. bimaculata AY122683 13.5 13.5 13.1 13.5 13.5 13.1 0.4 0 [ 9] E. bimaculata AY122684 13.5 13.5 13.1 13.1 13.1 12.7 1.8 1.4 0 [10] E. dione AY122750 12.9 12.9 12.5 11.5 11.5 11.3 9.6 9.6 9.6 0 [11] E. dione AY122748 12.9 12.9 12.5 11.5 11.5 11.3 9.6 9.6 9.6 0.0 0 [12] E. dione AY122747 13.1 13.1 12.7 10.9 10.9 10.7 9.2 9.2 8.8 1.4 1.4 0 [13] E. dione AY122746 12.9 12.9 12.5 10.7 10.7 10.5 9.0 9.0 8.6 1.2 1.2 0.2 0 [14] E. dione AY122745 13.5 13.5 13.1 11.3 11.3 11.1 9.7 9.4 9.4 1.9 1.9 1.9 1.8 0 [15] E. dione AY122749 13.1 13.1 12.7 11.3 11.3 11.1 9.4 9.4 8.6 2.1 2.1 1.6 1.4 2.3 0 [16] E. davidi AY122691 13.3 12.9 12.9 11.3 11.3 11.9 12.1 11.9 10.7 10.9 10.9 10.5 10.3 11.1 10.3 0 [17] E. carinata AY122755 13.5 13.1 13.3 12.1 12.1 12.1 12.9 12.5 12.3 11.9 11.9 11.1 10.9 11.9 12.3 11.5 0 [18] E. carinata AY122754 14.4 14.0 14.2 11.9 11.9 11.9 12.7 12.3 12.5 11.3 11.3 10.5 10.3 11.3 11.7 11.9 2.5 0 [19] E. quadrivirgata AY122710 11.5 11.5 11.1 11.9 11.9 11.9 11.3 10.9 11.5 10.7 10.7 10.5 10.3 10.5 10.9 9.9 8.6 8.6 0 [20] E. quadrivirgata AY122709 11.5 11.5 11.1 11.9 11.9 11.9 11.3 10.9 11.5 10.7 10.7 10.5 10.3 10.5 10.9 9.9 8.6 8.6 0.0 0 [21] E. schrenckii AY122720 12.7 12.7 12.9 12.5 12.5 12.5 12.5 12.9 13.1 11.5 11.5 11.5 11.3 11.9 11.5 12.1 10.7 10.3 8.2 8.2 0 [22] E. anomala AY122719 12.7 12.7 12.9 12.5 12.5 12.5 12.5 12.9 13.1 11.5 11.5 11.5 11.3 11.9 11.5 12.1 10.7 10.3 8.2 8.2 0.0 # 0 [23] E. quatuorlineata AY122714 13.5 13.5 13.5 15.0 15.0 14.6 13.1 13.1 12.9 12.9 12.9 12.7 12.5 13.1 12.3 13.1 12.1 11.9 11.5 11.5 12.5 12.5 0 [24] E. quatuorlineata AY122712 13.5 13.5 13.5 15.0 15.0 14.6 12.5 12.9 13.1 12.9 12.9 12.7 12.5 12.7 12.3 13.3 12.1 11.9 11.5 11.5 11.7 11.7 0.8 0 [25] E. sauromates AY122713 13.1 13.5 13.1 12.5 12.5 12.1 13.8 13.8 14 10.7 10.7 11.3 11.1 11.7 11.3 13.1 11.3 11.1 10.9 10.9 12.3 12.3 8.8 8.8 0 [26] E. sauromates AY122711 13.1 13.1 13.1 12.9 12.9 12.7 13.1 13.5 13.5 11.9 11.9 11.9 11.7 12.3 12.3 13.1 13.5 12.9 12.1 12.1 11.9 11.9 9.2 8.4 7.6 * 0 Conspecific distances (0.0% 2.5%) are shadowed in gray. * : A distance of 7.6% occurs between two individuals of E. sauromates [25] vs. [26]; the species was previously was known as E. quatuorlineata sauromates. Their taxonomic status may need further study. Interspecific distances (8.2% 15.0%) are not shadowed. # : The distance of 0.0% between E. anomala [22] and E. schrenckii [21] suggests they are either conspecific or the former is a subspecies of the latter (Pope, 1935; Utiger et al., 2002; Ling et al., 2010). Numbers in grid show the distances between E. zoigeensis sp. nov. and all other ten species of Elaphe (11.1% 14.4%).

No. 1 Song HUANG et al. New Species of the Genus Elaphe from Zoige, China 41 Figure 1 Sampling site and type locality of Elaphe zoigeensis sp. nov. A: The type locality of Elaphe zoigeensis sp. nov. is indicated by an asterisk, located at Jiangzha Hot Spring, Zoige County, Sichuan, China (N34º12 18, E102º42 48, 3200 m asl); B: Habitat of type locality of the new species. Photo by S. Huang. Figure 2 Clutch of eggs of HUM20110001 (holotype) and HUM20110002 (paratype). A: Eleven eggs laid by HUM20110002 on 31 July 2010, with egg length being 29.45 ± 1.35 mm, egg diameter 16.74 ± 0.99 mm, and mean egg weight 5.50 g; B: Eleven eggs laid by HUM20110001 on 29th July 2010, with egg length being 29.35 ± 1.80 mm, egg diameter 16.46 ± 0.78 mm, and mean egg weight 4.59 g. Photos by S. Huang. on live snakes and the description of eggs was taken at 90% humidity. Terminology, methods, and characters for the description of the new taxon mainly followed Schulz (1996). Herein, the term subpreocular was used to describe the lowest small preocular. The measurements of length and weight were taken using a digital vernier caliper and electronic balance to the nearest 0.1 mm and 0.1 g, respectively, except for snout-vent and tail lengths, which were recorded with a measuring tape to the nearest 1 mm. 3. Results Elaphe zoigeensis sp. nov. (Figures 3 5) Zoige Ratsnake, Ruoergai Jinshe (Bopomofo) Holotype: HUM20110001, adult female captured at 12:00 on 26 July 2010. Specimen was basking on a large rock about 20 m from the mouth of Jiangzha Hot Spring (3200 m elevation, N34º12 18, E102º42 48 ) in Zoige County, Sichuan, China. Paratypes: Captured on gravel by a stream (HUM20110002, adult female, about 40 m from the mouth of Jiangzha Hot Spring) and in a small soil hole by the same stream (HUM20110003, adult male, about 30 m from the mouth of Jiangzha Hot Spring), same place and time as the those of the holotype. Diagnosis: Elaphe zoigeensis sp. nov. is distinguished from all other species of Elaphe by several sets of characters involving color and scutellation. Color pattern

42 Asian Herpetological Research Vol. 3 Figure 3 Body of holotype (HUM20110001, adult female). A: Dorsolateral; B: Abdomen; C: Back. Photos by S. Huang. Figure 4 Head of holotype (HUM20110001, adult female). A: Dorsal; B: Ventral; C: Right; D: Left. Po: Preocular; Sp: Supralabial; Sp*: Supralabial in contact with the eye; At: Anterior temporal; Pt: Posterior temporal. Photos by S. Huang. is distinct in having a red brown, M shaped marking on the back of the head at the margins of the supraoculars and adjacent posterior frontal and the presence of 4 longitudinal, parallel rows of spots on the dorsum extending from the neck to vent. Each spot usually encompasses an entire scale and is red brown, while the edges of the spot and bordering scales are puce. The spots from each of the parallel rows are similar in size and shape, and rank at a relatively regular interval of one or two scales of slightly red brown color. Additionally, this species has 3 preoculars, rather than 1 2 found in all other species (Schulz, 1996). Description of the holotype and variation: When differing from the holotype, features of the paratypes follow in parentheses (HUM20110002, HUM20110003 respectively). Holotype an adult female with body weight 97.96 g (103.68 g, 124.29 g), snout-vent length 722 mm (695 mm, 64 mm), tail length 158 mm (150 mm, 175 mm). Scutellation: two postoculars; 2 + 3 temporals (anterior temporal fused); 3 preoculars (HUM20110003

No. 1 Song HUANG et al. New Species of the Genus Elaphe from Zoige, China 43 Figure 5 Heads of the paratypes, showing the lateral scales on both sides. A, B: Right, left side of HUM20110002 (adult female); C, D: Right, left side of HUM20110003 (adult male). Abbreviations are same as those in Figure 4. Photos by S. Huang. left includes a subpreocular); 7 supralabials, the 3 rd and 4 th bordering the eye (HUM20110003 left 8 supralabials, the 4 th and 5 th bordering the eye); 9 infralabials; 1 loreal; 21 19 17 forebody-midbody-hindbody transverse dorsal scale rows (21 21 17), weakly keeled on the back; 205 ventrals (212, 202); 69 pairs subcaudals (68, 79); anal plate divided. Body and headshape: body relatively slender; venter round; head elongated and flattened, and slightly wider than the neck; pupils round. Coloration in life: body ground color off-white; clear red brown M shaped marking with black edges formed at the margins of the supraocular that borders the posterior margins of the frontal; a red brown postocular stripe with black edge continuing to the last supralabial; four series of red brown spots placed next to each other on back of body that range from posterior of the neck to vent and form four longitudinal rows; dorsal spots are usually composed of an entire (or incomplete) red brown scale and puce edges at the borders of scales; spots of each stripe are similar in shape and range at relatively regular intervals of one or two scales with slightly red brown color; ventrals, basal part (close to trunk) black and distal ends non-uniform gray, making its belly look like having many black rings. The holotype laid 11 eggs on 29 July 2010 and the eggs are white, smooth-shelled, and adhesive. Ecology: The species is a diurnal terrestrial oviparous snake, laying eggs around the end of July, with a clutch size of at least 11 eggs. Eggs are white, smooth-shelled, and adhesive. From the day of capture until 3 August 2010, almost every day, some short soft black hairs were found in their excrement, suggesting that they ate rodents or insectivores. Etymology: The specific name refers to the type locality, Zoige County, Sichuan, China. 4. Discussion Whereas traditional morphological approaches fail to define cryptic species, molecular data often succeed (Burbrink and Castoe, 2009). Because of the relatively high rate of mutation, largely neutral evolution, and large suite of characters, analyses of molecular data augment those of morphological data and synergistically they identify morphologically cryptic lineages. When morphological differences are found, the data may be coded for phylogenetic analyses and combined molecular data to obtain a much more convincing conclusion regarding species history, identification and diagnosis (Burbrink, 2001; Ling et al., 2010). Ling et al. (2010) demonstrate that Elaphe zoigeensis sp. nov. is a new species and likely the sister taxon to all other extant Elaphe. While most species of Elaphe found in China are easily diagnosed, E. zoigeensis sp. nov., E. dione, and E. bimaculata superficially resemble each other with respect to basic coloration (e. g., similar striping pattern) and aspects of scutellation. Utiger et al. (2002) confirm that E. dione and E. bimaculata are likely valid species based on an analysis of genetic distances. Elaphe

44 Asian Herpetological Research Vol. 3 zoigeensis sp. nov. is morphologically unique in its dorsal striping, pattern of spotting, and number of preoculars. Some specimens from Zoige County and surrounding areas formerly identified as E. dione or E. bimaculata may be E. zoigeensis sp. nov. Previously collected specimens designated as E. dione or E. bimaculata should be carefully reexamined. This activity is likely to better determine the distribution of E. zoigeensis sp. nov., as well as characterize its variation more precisely. The discovery of E. zoigeensis sp. nov. represents the first new species of Elaphe in 87 years, the last being E. bimaculata (Schmidt, 1925). This discovery highlights the importance of continued exploration for new species of snakes even in temperate regions of the globe. This new species roots at the basal node of the tree for Elaphe. 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