Amphibia-Reptilia 38 (2017): 517-532

A new moth-preying alpine from Qinghai-Tibetan Plateau (, Crotalinae)

Jingsong Shi1,2,∗, Gang Wang3, Xi’er Chen4, Yihao Fang5,LiDing6, Song Huang7,MianHou8,9, Jun Liu1,2, Pipeng Li9

Abstract. The Sanjiangyuan region of Qinghai-Tibetan Plateau is recognized as a biodiversity hotspot of alpine but a barren area in terms of amphibians and . Here, we describe a new pit viper species, rubromaculatus sp. n. Shi, Li and Liu, 2017 that was discovered in this region, with a brief taxonomic revision of the Gloydius.The new species can be distinguished from the other congeneric species by the following characteristics: cardinal crossbands on the back, indistinct canthus rostralis, glossy dorsal scales, colubrid-like oval head shape, irregular small black spots on the head scales, black eyes and high altitude distribution (3300-4770 m above sea level). The mitochondrial phylogenetic reconstruction supported the validity of the new species and furthermore reaffirms that G. intermedius changdaoensis, G. halys cognatus, G. h. caraganus and G. h. stejnegeri should be elevated as full species. Gloydius rubromaculatus sp. n. was found to be insectivorous: preying on moths (Lepidoptera, Noctuidae, Sideridis sp.) in the wild. This unusual diet may be one of the key factors to the survival of this species in such a harsh alpine environment.

Keywords: Gloydius rubromaculatus sp. n., insectivorous, new species, Sanjiangyuan region.

Introduction leopards (Uncia uncia), wild yaks (Bos grun- niens) and Tibetan antelopes (Pantholops hodg- The Sanjiangyuan region (the Source of Three sonii) (Shen and Tan, 2012). However, the her- Rivers region) lies in the southern part of Qing- petological diversity here is quite low due to hai Province, along the eastern part of Qinghai- the harsh conditions for sustaining life (e.g. Tibetan Plateau with an area covering 0.36 mil- low temperatures, low oxygen levels, and in- lion km2. It encompasses the headwaters of tense solar radiation). To date, only ten rep- the Yellow River, the Yangtze River, and the tile species have been recorded (Li et al., 1989; Mekong River. The Sanjiangyuan region is rich Zhao et al., 1998), within which are three in biodiversity of alpine mammals, such as snow species (Gloydius strauchi, Gloydius cognatus and Elaphe dione). In this study, a new species

1 - Key Laboratory of Vertebrate Evolution and Human of Asian pit viper (Gloydius) has been discov- Origins of Chinese Academy of Sciences, Institute of ered along the Tongtianhe River at the elevation Vertebrate Paleontology and Paleoanthropology, Chi- up to 4770 m. nese Academy of Science, 100044 Beijing, Asian pit vipers are small venomous 2 - University of Chinese Academy of Sciences, 100044 Beijing, China distributed mainly in . They are widely 3 - Chengdu Normal University, 611130 Chengdu, China recognized to be one of the most successfully 4 - College of Life Sciences, Peking University. 100871 evolved snake groups, which radiated into vari- Beijing, China ous habitats, such as subfrigid forests (G. halys), 5 - Institute of Eastern-Himalaya Biodiversity Research, Dali University, 671003 Dali, China alps or plateaus (G. strauchi, G. himalayanus 6 - Chengdu Institute of Biology, Chinese Academy of Sci- and G. monticola), islands (G. shedaoensis ences. 610041 Chengdu, China and G. changdaoensis) and deserts (G. cogna- 7 - Huangshan University, 245000 Huangshan, China tus) (Hoge and Romano-Hoge, 1981). Asian 8 - Sichuan Normal University, 610101 Chengdu, China 9 - Institute of , Shenyang Normal University, pit vipers hold the record for the highest alti- 110034 Shenyang, China tude distribution within venomous snakes (G. ∗Corresponding author; e-mail: [email protected] himalayanus, above 4880 m; Sharma et al., Downloaded from Brill.com10/07/2021 08:28:56AM via free access © Koninklijke Brill NV, Leiden, 2017. DOI:10.1163/15685381-00003134 518 J. Shi et al.

2013) and the highest population density within and G. liupanensis. Thus, a further investigation the suborder of Serpentes (G. shedaoensis, is required to clarify the taxonomic relationship 20 281 snakes within 0.73 km2, express as about between the different taxa of this complex. 0.028/m2, based on the pit viper population sur- vey conducted by the Snake Island National Na- ture Reserve; Li et al., 2007). Taking advan- Material and methods tage of their heat sensitive pits, most Asian pit We examined preserved specimens from Chengdu Insti- vipers tend to prey on small endotherms. As tute of Biology (CIB), Northwest Institute of Plateau Bi- well, some of them are reported to be insectivo- ology (NWIPB) and Kunming Institute of Zoology (KIZ). Newly obtained specimens collected were preserved in 75% rous (Gloyd and Conant, 1990; Zhao, 2006). ethanol and deposited at Institute of Zoology (IOZ), North- Based on previous (Orlov and west Institute of Plateau Biology (NWIPB) (table 1). Barabanov 1999; Xu et al., 2012; Shi et al., 2016; Wagner et al., 2016), the genus Gloydius Institutional abbreviations could be preliminarily divided into the follow- IVPP: Institute of Vertebrate Paleontology and Paleoanthro- ing complexes (groups): pology; CIB: Chengdu Institute of Biology; IOZ: Institute 1. Gloydius halys-intermedius complex (G. of Zoology; NWIPB: Northwest Institute of Plateau Biol- ogy; SYNU: Shenyang Normal University; KIZ: Kunming halys, G. intermedius, G. changdaoensis, Institute of Zoology. (IVPP, CIB, IOZ and NWIPB are be- G. rickmersi, G. cognatus, G. stejnegeri longing to Chinese Academy of Science.) and G. shedaoensis). 2. Gloydius blomhoffii complex (G. blomhof- Morphology fii, G. brevicaudus, G. tsushimaensis and Measurements were taken with vernier calliper (Guanglu, G. ussuriensis). 0-200 mm, Made in China). Snout-vent length (SVL), tail length (TL) and total length (TTL = SVL + TL) are 3. Gloydius strauchi complex (G. strauchi, measured to the nearest 0.1 mm; head length (HL, from the G. monticola, G. qinlingensis G. liupanen- tip of snout to the posterior margin of mandible), head width sis and G. himalayanus). (HW, from the posterior jaw, which is the widest part of the head), head height (HH, the highest part of the head), eye The members of Gloydius strauchi complex diametre (ED, horizontal distance), interorbital space (IOS), are generally described as an alpine group with and internasal space (INS). We took counts of supralabials 21 dorsal scale rows (except for 19 rows in G. (SPL), infralabials (IFL), dorsal scales (DS), ventral scales (V) and subcaudal scales (Sc). Dimensions and scale data monticola) and three palatine teeth, distributed are listed in table 2. along the north of the Hengduanshan Moun- tains (Zhao and Yang, 1997). The taxonomy on X-ray micro-computerized tomography this group is still controversial: some regard G. The scanning was carried out with the 225 kV micro- monticola as a full species (Gloyd and Conant, computerized tomography (developed by the Institute of 1990; Wagner et al., 2016), while others suggest High Energy Physics (IHEP), Chinese Academy of Sciences that G. monticola should be attributed to one (CAS)) at the Key Laboratory of Vertebrate Evolution and Human Origins, CAS. Specimens were scanned at 140 kV of the of G. strauchi and deny the with a flux of 100 μA at a resolution of 42.3 μmperpixel validity of qinlingensis and liupanensis (Zhao, using a 360° rotation with a step size of 0.5° and an unfil- 1998; Zhao, 2006). Xu et al. (2012) conducted tered aluminium reflection target. A total of 720 transmis- sion images were reconstructed into the 2048 × 2048 ma- the molecular phylogeny of the genus Gloydius trix of 1536 slices using a two-dimensional reconstruction and suggested that G. qinlingensis and G. li- software developed by IHEP, CAS. upanensis should be regarded as full species. However, the topological structures of the max- Laboratory protocols imum likelihood (ML), maximum parsimony Specimens were fixed in 95% ethanol or 10% formalin. (MP) and Bayesian inference (BI) trees differ Shed skin and scale tissues were preserved in 98% ethanol for molecular study. Genomic DNA was extracted with significantly, with primary differences indicated Miniprep Kit (Axygen). Samples included in this study are by the positions of G. qinlingensis, G. strauchi listed in table 1. Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 519 12s 16s Cytb ND4 KY040527KY040528 KY040559 KY040560 KX063803 KY040618 KX063776 this KY040639 study this study KY040531 KY040563 KY040621 KY040642 this study Heilongjiang Inner Mongolia Litang, Sichuan KY040544 KY040576 KY040630 KY040651 this study Greater Hinggan Mts., Heilongjiang KM434236 KM434236 KM434236 KM434236 Xu et al. (2012) NingxiaNingxiaNingxia / / / / / / JQ687491 JQ687492 JQ687493 JQ687472 JQ687473 JQ687474 Xu et al. (2012) Xu et al. (2012) Xu et al. (2012) Kangting, Sichuan KY040543 KY040575 KY040629 KY040650 this study Lingyuan, LiaoningXilinhot, Inner KY040526 KY040558 KX063802 KX063775 this study Taibai, ShaanxiZhouzhi, Shaanxi / / / / KF997922 JQ687490 KF997981 JQ687471 this study Xu et al. (2012) Liupanshan, NingxiaTongchuan, ShaanxiLinfen, ShanxiMentougou, Beijing EF012814Mentougou, Beijing KY040536 KY040568 KY040538 / KY040537 KY040539 KX063817 KY040570 KY040569 KY040571 KX063790 KX063815 KX063818 KY040625 this / KX063788 study KX063791 KY040646 this study this study this study EF012795 Zhou et al. (2006) Zoige, SichuanSonit Right Banner, KY040529 KY040561 KY040619 KY040640 this study Zhuanghe, LiaoningAnshan, Liaoning KY040524 KY040556 JX661216 KY040617 KY040638 / thisYinchuan, study Ningxia JX661205 KY040532 JX661228 KY040564 Wu et al. (2015) KY040622 KY040643 this study Lianyungang, Jiangsu KY040521Wafangdian, Liaoning KY040553 KY040525 KX063821 KY040557 KX063794 KX063793 this study KX063820 this study Wuzhong, NingxiaXunyangba, Shanxi KY040533 KY040534 KY040565 KY040566 KX063809 KY040623 KX063782 KY040644 this study this study Changdao, ShandongLvshun, Liaoning KY040522 KY040554 KY040523 KX063823 KY040555 KX063796 KX063819 this KX063792 study this study sp. n.sp. n. Yushu, Qinghai Yushu, Qinghai KY040546 KY040547 KY040578 KY040579 KY040632 KY040633 KY040653 KY040654 this study this study G. rubromaculatus G. rubromaculatus G. intermedius G. liupanensis G. liupanensis G. liupanensis G. strauchi G. h. halys G. qinlingensis G. qinlingensis G. liupanensis G. stejnegeri G. stejnegeri G. stejnegeri G. stejnegeri G. strauchi G. h. halys G. cognatus G. cognatus G. changdaoensis G. intermedius G. intermedius G. h. halys G. cognatus G. changdaoensis G. intermedius G. cognatus G. qinlingensis G. shedaoensis Y2 Y4 S4 G3 QLS C1 D2     ∗∗ ∗  Details of the molecular samples for this study. IOZ032317 GP198 GP198 GP206GP215NNU95043JSSD1409S3JSSD1508S4 SYNU1510145 GP206 95043 JSSD151054 GP215 S3 JS1501G3 53 54 JS1508G4 G4 SYNU1301908 46 JS1407H9CIBQY224JSSD13109I3 H9 QY224 I3 –SX1 JSSD1508X3 X3 Table 1. Museum voucher CodeJSSD1408Z1 Taxa Z1 Locality GenBank Accession Numbers Reference JSSD1510C1 HS34GP197 HS34 GP197 IOZ032318 JSSD11110D2 JS150622JSSD1110Q4QS002 Q4 22 QS002 JS131147JSSD1504N6JS1505QL1 N6 47 Downloaded from Brill.com10/07/2021 08:28:56AM via free access 520 J. Shi et al. DQ343647 DQ343647 DQ343647 DQ343647 Yan et al. (2008) LiaoningHeilongjiang KP262412 KY040552 KP262412 KY040584 HQ528467 KP262412 HQ528303 KP262412 this study Xu et al. (2012) //Sichuan KT2668 / / KT2668 KT2668 / / KT2668 MF490455 Zhu et al. (2015) MF490456 MF490453 MF490454 this study this study Dali, KY040549 KY040581 KY040635 MG025935 this study Dali, YunnanLijiang, Yunnan KY040550 JX661213 KY040582 KY040636 / MG025936 this JX661200 study JX661243 Wu et al. (2012) JapanJapan AY352780 JN870186 AY352719 JN870196 AY352751 JN870203 AY352814 Malhotra JN870211 et al. (2003) Fenwick et al. (2011) / / / / / / KM096379 / KM078594 Kyrgyzstan / / / KM078592 Wagner et al. (2016) sp. n. Yushu, Qinghai KY040548 KY040580 KY040634 KY040655 this study G. brevicaudus G. ussuriensis G. caraganus G. caraganus T. sichuanensis D. acutus G. rubromaculatus G. tsushimaensis G. caraganus G. monticola G. monticola G. monticola G. blomhoffii G. rickmersi G. rickmersi , topotype. Mts: Mountains.  GY001 , paratype; ∗ (Continued.) , holotype;  ∗∗ ISEA R290 R290 JS1607DL1 DL1 Table 1. JS1607Y5 Y5 Note: JS1607DL2 DL2 GY001 DL70U1 B1 U1 MHNG 2752.70 R2 CR1CR2KT2668Acutus CR1 K CR2 A B524 B524 Ts1MHNG 2752.69 R1 Ts1 Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 521 V: ventral scales, Sc: subcaudal scales. TL), HL: head length, HW: head wide, HH: head height, SL: snout length, + 352 408 56 21.2 14.6 7 / / / / 154 43 21-21-22 7/7 10/11 452 521 69 22.2 14.2 7.2 / / / / 160 40 21-21-15 7/7 / 436 498 62 19.8 15.2 7.2 / / / / 163 42 21-21-16 7/7 10/10 473 554 81452 24.6 529 15.8 77354 7.4 24.8 414 7.8 15.4 3.1 60 7.8 8.2 19.4 7.6 11.6 4.6 3 6 158 43 9.2 5.3 21-21-15 5.4 2.6 146 6 7/8 41 21-21-15 3.7 153 7/8 43 10/11 21-21-15 8/8 11/9 446 10/11 509 63 23.1 15.2 7.4 / / / / 162 35 21-21-15 7/7 9/10 450372 505 55 436427 20.9 64 504 12.4 20.3 77 7.2 12.7 24.6 6.5 / 15.6 5.9 8.2 1.9 2.1 / 7.8 8 6 2.7 4.3 9.9 160 148 5.3 33 42 151 21-21-15 21-21-15 40 21-21-16 7/7 7/7 7/7 10/10 / 10/10 384 438 54 22.4 12.4 7.9 / 2.4 8.4 5.6 158 35 21-21-15 7/7 / 347 412 65 19.9 12.1 8.7 / 2.2 7.8 3.7 146 41 21-21-15 7/7 / 338 404 66 19.4 11.8 6.2 / 2.1 7.7 4.2 151 38 21-21-16 7/7 / 274 308 34 18.1 9.5 6.4 / 1.5 6.9 4.7 145 30 21-21-15 6/6 9/10 436 492 56 21.7 13.3 7.9 / 1.9 8.2 5.8 151 28 21-21-15 / / 422 493 71 25.3 15.2 9.8 / 2.4 9.6 6.7 140 34 21-21-15 6/6 / 407 482 75 21.5 13.4 7.8 / 2.8 9.3 4.4 144 45 21-21-15 7/7 10/10                  snakes for this study. Gloydius Chengduo, Qinghai Jiangda, Tibet Jyekundo, Qinghai Qumarleb, Qinghai Jyekundo, Qinghai Qumarleb, Qinghai Chengduo, Qinghai Kangding, Sichuan Litang, Sichuan Litang, Sichuan Kangding, Sichuan Kangding, Sichuan Kangding, Sichuan Zhongdian, Yunnan Zhongdian, Yunnan Zhongdian, Yunnan Kangding, Sichuan , topotype. SVL: snout-vent length, TL tail length, TTL: total length (SVL  sp. n. sp. n. sp. n. G. strauchi G. strauchi G. strauchi G. rubromaculatus sp. n. G. rubromaculatus sp. n. G. rubromaculatus sp. n. G. rubromaculatus sp. n. G. strauchi Taxon Locality Sex SVL TTL TL HL HW HH SL ED IOS INS V Sc DS SPL (L/R) AFL (L/R) G. strauchi G. rubromaculatus G. rubromaculatus G. rubromaculatus G. strauchi G. monticola G. monticola G. monticola G. strauchi ∗ ∗∗ ∗ ∗      , paratype; ∗ voucher Dimensions (mm) and scale data of the specimens of , holotype; ∗∗ SYNU JS1508G4 NWIPB NWIPB 790056 NWIPB NWIPB 790058 CIB CIB14359 NWIPB NWIPB 0512 CIBNWIPB NWIPB 630064 CIB78588 CIB CIB14358 CIB CIB14357 CIB CIB14356 CIB CIB72551 CIB CIB72553 IOZSYNU IOZ002318 JS1607Y3 SYNU JS1410G3 CIB CIB12948 Table 2. Preserve Museum IOZ IOZ002317 Note: ED: eye diametre (horizontal distance), IOS: interorbital space, INS: internasal space, SPL: supralabials, IFL: infralabials, DS: dorsal scales, Downloaded from Brill.com10/07/2021 08:28:56AM via free access 522 J. Shi et al.

Molecular phylogenetic analysis Etymology. The specific name of the new

Four fragments of mitochondrial genome are specifically species is made up of the Latin word “rubro” amplified in this study: 12s rRNA (12S), 16s rRNA (16S), (red) and “maculatus” (spot), indicating cardi- cytochrome b (Cytb) and NADH dehydrogenase subunit 4 nal crossbands on the body. Its common name is (ND4). The standard PCR protocol is performed in 20 μl of reactant with at least 20 ng of template DNA and suggested to read “Red-spotted alpine pit viper” 10 pmol of primers. The PCR conditions: initial denat- or “Tongtianhe pit viper” in English and “Hóng uration for 3 min at 94°C, followed by 35 cycles: de- Ban¯ Gao¯ Shan¯ Fù ( )” in Chinese. naturation at 94°C for 30 s, 30 s of annealing at differ- ent temperatures (56°C for ND4, 48°C for Cytb, 54°C for 16S, and 52°C for 12s), and then elongation at 72°C Holotype and paratypes. Holotype: IOZ032 for 60 s, then finalized with elongation step of 10 min 317, adult male, collected by Jingsong Shi at 72°C. See online supplementary table S1 for primer se- (JS) and Xi’er Chen (XC) from the mid-upper quences and modifications of the standard PCR protocols. Sequencing is conducted by Beijing Genomics Insti- reaches of the Tongtianhe River, Qumarleb, tute. Sequence data are uploaded to GenBank and are Qinghai Province, on 9 July 2016. Paratypes: available under accession numbers showed in table 1. NWIPB790058 (allotype, adult female), IOZ Data are aligned by Mega 6.0 (Tamura et al., 2013). A dataset with a total of 3129 basepairs containing 44 032318 (adult male), JS1607Y3 (subadult fe- specimens is analyzed in this study: 42 of the sam- male), NWIPB790056 (adult female), NWIPB ples are belonging to the genus Gloydius, while three of 630064 (adult female), and NWIPB0512 (adult which are identified as G. rubromaculatus sp. n. (Y2, Y4 and Y5). Two samples, Deinagkistrodon acutus and male). Referred specimens: NWIPB 17092:1, Trimeresurus sichuanensis are recognized as outgroups. from Jyekundo, Qinghai Province; NWIPB With respect to the different evolutionary characteristics 790 060-790 067, from Zhiduo, Qinghai Pro- of each molecular marker, the dataset is split into 8 par- titions by gene and codon positions, then combined into vince. See table 2 for detailed information. 4 ones taking advantage of PartitionFinder 2.1.1 (Lanfear Note: some of the specimens of the old species et al., 2012) (online supplementary table S2). General time- are labelled with “G. strauchi” which are iden- reversible (GTR) model, the most probable substitution model for the corrected ND4 p-distance matrix is calculated tified as G. rubromaculatus sp. n. in this study. by PAUP 4.0 (https://people.sc.fsu.edu/~dswofford/paup_ test/). Bayesian phylogenetic analyzis is performed with Diagnosis. The above mentioned specimens MrBayes 3.1.2 (Ronquist et al., 2011). All searches consist were identified as the members of Gloydius of three heated chains and a single cold chain. Three inde- pendent iterations each comprising two runs of 20 000 000 judging on their small body size, bilateral generations are performed, sampling every 1000 genera- pits and divided subcaudal scales (Hoge and tions, parameter estimates are plotted against generation, Romano-Hoge, 1981), while differ from other The first 25 percents of the samples are discarded as burnin. Maximum likelihood analyzis (sharing the same partition congeneric species in the following characteris- as Bayesian phylogenetic analyzis) is conducted in RAxML tics: 1. two rows of cardinal crossbands on the 7.0.4 (Stamatakis, 2006), with 1000 fast bootstrap repeats back, regularly spaced along the body; 2. glossy (see Part 3 of the supplementary material for the command blocks). dorsal scales, compared to matte scales in other members of Gloydius; 3. colubrid-liked dome shaped head in lateral view and oval shaped Results in dorsal view, compared to flat-shaped head in lateral view and triangular in dorsal view in Systematic position other Gloydius; 4. irregular small black spots Viperidae Gray, 1825 dispersed on the head scales; 5. inconspicuous Gloydius Hoge and Romano-Hoge, 1981 canthus rostralis; 6. dark brown eyes with black Gloydius rubromaculatus sp. n. Shi, Li pupils (figs 1 and 2). and Liu, 2017 The new species is distinct from species ZooBank accession: BF478EA7-C2D6-4F in the Gloydius blomhoffii complex by hav- 0E-B133-F4D624C3EA2A ing three palatine teeth (versus four palatine Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 523

Figure 1. Photos of Gloydius rubromaculatus sp. n. in the habitat. (A) holotype (IOZ032317); (B) paratype, subadult female (JS1607Y4); (C) another subadult sympatry with the holotype (by Jian-sheng Peng, released). teeth); from the species in the Gloydius halys- See table 3 and fig. S1 for the morphological intermedius complex by having 21 rows of mid- comparisons within the Gloydius strauchi com- body dorsal scales (versus 23-rows). Thus, the plex. new species is suggested to belong to the Gloy- The validity of this new species is also sup- dius strauchi complex. ported by molecular genetics. Three samples Within the Gloydius strauchi complex, G. of G. rubromaculatus sp.n.(Y2,Y4andY5) rubromaculatus sp. n. is distinct from G. mon- form a strongly supported monophyletic group. ticola by its 21-rows mid-body scales (versus In addition, the corrected ND4 p-distance be- 19-rows mid-body scales and 6 supralabials); tween G. rubromaculatus sp. n. and other from G. himalayanus by its indistinct canthus species is greater than the ones between most of the other species (8.6-12.4 percent, see rostralis (versus very distinct canthus rostralis); online supplementary table S3). from G. qinlingensis and G. liupanensis by its oval head (versus triangular head), regular Description of the holotype. Adult male, a crossbands (versus irregular crossbands) and the small slender pit viper with a total length of lack of the white line on each side of body (ver- 554 mm (body length 473 mm and tail length sus obvious white line); from G. strauchi by its 81 mm), preserved in 75% ethanol with its brownish black eyes (versus light brown eyes), bilateral hemipenis extruded. and two rows of regular round crossbands (ver- The head is spoon-shaped in dorsal view and sus four irregular longitudinal strips). dome-shaped in lateral view, 24.6 mm in length, Downloaded from Brill.com10/07/2021 08:28:56AM via free access 524 J. Shi et al.

Figure 2. Head illustration of G. rubromaculatus sp. n. (holotype, IOZ 032317, Y2, by Tingting Zhang and Jingsong Shi); (A) ventral view; (B) dorsal view; (C) lateral view.

15.4 mm in width and 7.4 mm in height. Rostral throughout the inferior temporal and the last scale slightly turns up to the upper side of the two supralabials. Lots of irregular, uneven-sized head. Seven bilateral supralabials are presented: black spots dispersed on most of the head scales, the second one smallest, not reaching the pit; except for the temporals (figs 1 and 2). Mouth the third and fourth largest, with the former ex- lining is pink in life. The anterior part of the tending to the bottom of orbit. Three preocu- tongue is black while the base is pink. lars, two postoculars, and two rows of temporals The body colour is light greyish yellow, with (2 + 4). Ten infralabials are on the left while a column of complete regular round cardinal crossbands on each side of the back, black bor- nine on the right, of which the first pair con- dered and shallow inside, in pairs or interfaces, tact behind the mental, the second and third ones about three to four scales in length, and four meet the chin shield. Mental groove is made up to six scale rows in width, separated by blank of paired parallelogram chin shields, which ex- areas one or two scales in width, extending tend to the mental. The canthus rostralis are not down to lateral one or two ventral scales. Cross- distinct. The head has a clear border with the bands range from the neck to the tip of the tail, neck. The eyes are brownish black, with verti- 49/46 on body and 12/16 on the tail (left/right). cal spindly, light brown margined black pupil. A range of triangular or irregular black ventro- A thick, black bordered yellowish red cheek lateral blotches (irregular one is made up of the stripe extends from the posterior side of each adjacent two or three ones) range on the bound- eye (separated from the orbit by the inferior ary between back scales and ventrals on each postocular) to the first pair of neck crossbands, side of the body, divided by one or two ven- Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 525 qinlingensis two columns of irregular dark brown crossbands, in contact on the axis of the back blackwish markings on a dark ground colour variable dark markings with dark edges similar with four longitudinal zigzag strips, interrupted at intervals, sometimes curving and coalescing two columns of irregular dark brown crossbands Body colouration Body pattern dark brown dark grey or dark brown brown, reddish brown or dark grey greenish brown, yellowish brown or nut-brown khaki of yellowish brown or dark grey (female), white stripe on the body side Canthus rostralis obvious sharp obvious obvious complex. 19 not 21 very 21 Not 21 Not dorsal scale Gloydius strauchi the labials along the mouth line triangular spot between the third and forth between the second, third and forth (G4) or none (G3) black spots sp. n. and the remaining species of the Snout Internasal Prefrontal Supralabials spot Mid-body G. rubromaculatus Lateral heal head rounded dome rounded convex convex irregular small triangular flat sharp flat flat none 21 obvious yellowish brown or rounded flat sharp flat convex whitish borders of rounded dome rounded convex convex large brown triangular flat sharp flat flat none 21 obvious yellowish brown (male) triangular flat sharp flat flat usually a Detailed comparison between sp. n. Table 3. SpeciesG. qinlingensis Dorsal G. liupanensis G. monticola G. himalayanus G. strauchi G. rubromaculatus Downloaded from Brill.com10/07/2021 08:28:56AM via free access 526 J. Shi et al. tral scales (fig. 1). Ventral scales are opales- the base in Gloydius halys complex; Gloyd and cent, with mottled irregular black blotches. The Conant, 1990). The spines gradually increase in black blotches are concentrated on the middle size distally. More spines occur on the asulcate of scales. The edge and the junction of bilateral side on than the sulcus side. Spines and sulcus subcaudals are trimmed in black thread. One merge with the calyces on the distal side. No gi- semicircular black blotch is present on the lat- ant spines present on the basal of the hemipenis eral edge of each subcaudal scale. A black stripe as Gloydius halys-intermedius complex (fig. 4). is present on each side of the boundary of the Infraspecific morphological variation. Some two bilateral ventrals. The tip of the tail is black morphological variations are present within the and bony. specimens for this study: some of the G. rubro- Dorsal scales are in 21-21-15 rows (reduce maculatus sp. n. have large, complete scar- from 21 to 15 rows beginning with the ventral let crossbands, while other have small irreg- 92/93), keeled (excluding the ones bordering the ular ones (e.g., Y4), or large brown regular ventral scales) and glossy. Ventrals 158 (exclud- crossbands (e.g., Y7) (fig. 1). Body colour is ing three preventral scales). Anal plate is com- mostly grayish white, sometimes brownish yel- plete. Subcaudals divided in pairs (43 pairs). low (Y9). Spots are mostly present on the head See table 2 for the detailed measurements of scales, but some have none or only a few the specimens examined. small ones. Most have seven supralabials (rarely Skull. The frontal of G. rubromaculatus sp. eight) and 10 infralabials (rarely 9 or 11, ta- n. is inverted triangular in dorsal view, con- ble 2). Ventrals range from 146 to 158 in males trast to round in G. strauchi, and “T”-shaped (mean 152.3, n = 3) while 153 to 163 in fe- in G. shedaoensis. The lateral margin of nasal males (mean 159.5, n = 4). Subcaudals range is rounded, without any process, while in G. from 41 to 43 in males (mean 42.3, n = 3), and strauchi the lateral process is distinct. The fang 35 to 43 in females (mean 40, n = 4). See ta- is quite short, approximately one third length ble 2 for the detailed measurements of the spec- of the ectoptery (versus approximately a half- imens examined. length of the ectoptery in G. halys-intermedius Phylogenetic and phylogeographic analysis. complex and G. blomhoffii complex; Gloyd The validity of the new species is supported by and Conant, 1990). Six replacement fangs be- phylogenetic analyzis, the topological structure hind each primary fang, three palatine teeth, 12 of the maximum likelihood (ML) and Bayesian pterygoid teeth and 11 alvenlus teeth on each inference (BI) trees are approximately identical side. The upper edge of postfrontal is in contact except for the clade of G. stejnegeri, G. rick- with the posterior-lateral process of the frontal, mersi and G. caraganus (fig. 5). All members versus separated in G. strauchi (Gloyd and Co- of Gloydius perform as a monophyletic group. nant, 1990). The quadrate is quite slender, about The clade of the new species from along the 1.2 times as long as the squamosal, versus al- Tongtianhe river (Y2, Y4 and Y5) performs as a most equal to the squamosal in length in G. strongly supported monophyletic group (Clade strauchi (fig. 3). A, in red), and constitutes sister groups with Hemipenes. The hemipenes of G. rubromacu- the clade of G. monticola from Yunnan (Clade latus sp. n. are generally similar to those of G. B). Despite the geographic proximity with G. strauchi but differ by the podgier spines. Eight strauchi, G. rubromaculatus’s group does not subcaudals in length, forked for two subcaudals. perform as sister groups with G. strauchi (Clade Small stubby spines range from the basal to the C). Thus, the new species is more closely re- distal side of the organ, without any enlarged lated to G. monticola than to G. strauchi.The spines (versus three to five enlarged spines on samples of G. qinlingensis (Clade D) and G. Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 527

Figure 3. CT scanned skull imagine of (A) G. rubromaculatus sp. n. (Y2, holotype) and (B) G. strauchi (G4). 1: ventral view; 2: dorsal view; 3: lateral view. liupanensis (Clade E) from near the Yellow plex (Clade F) and Gloydius blomhoffii complex River do not present as sister groups, more- (Glade G). over, the p-distance between them is relatively The validity of G. rickmersi Wagner, Tiutenko, greater (7.2 percents for ND4) than the ones Mazepa, Simonov, Borkin, 2016 is reconfirmed between other congeneric species, thus they in this study. On the other hand, the populations should be regarded as distinct species. In gen- distributed in Jiaodong Peninsular (Shandong eral, the samples of the Gloydius strauchi com- Province) had long been regard as a subspecies plex, including qinlingensis (Shaanxi), liupa- of G. intermedius (G. i. changdaoensis Li, nensis (Ningxia), monticola (Yunnan), strauchi 1999), however, the molecular phylogeny shows (Sichuan) and rubromaculatus sp. n. (Qinghai) a significant distance between G. i. chang- do not completely constitute a monophyletic daoensis and G. intermedius (p-distance: 5.4 group as the Gloydius halys-intermedius com- percent), the clade of changdaoensis (Z1 and Downloaded from Brill.com10/07/2021 08:28:56AM via free access 528 J. Shi et al.

Figure 4. Asulcate side (A) and sulcus side (B) of the right hemipenis of G. rubromaculatus sp. n. (holotype, Y2).

C1) does not constitute as sister groups with such as undigested bodies, siphoning mouth- G. intermedius (SX1, 22, Q4 and QS002), but parts, wings, periopods and arms of the furcas firstly separates from the remaining taxa of the (supplementary fig. S2). One of the moths could G. halys-intermedius complex. Therefore, G. be identified as Sideridis sp. (female). No hairs, i. changdaoensis should be regarded as a full bones or feathers of or mammals can be species, but not a subspecies of G. intermedius. found in the faeces yet. Two undigested neonate Additionally, the samples of the different sub- zokors (Eospalax fontanierii) are found in an- species of G. halys (e.g. caraganus, stejnegeri other specimen’s stomach (NWIPB 630064). and cognatus) do not performed as a mono- Two juvenile pit vipers are observed to prey phyletic group, and none of them performed on moths and pink mice in captivity (Y1 and as sister groups with G. h. halys, allowing for Y5). the high p-distance between the different sub- Two hypotheses can account for the moth de- bris: they may be from the moths preyed and ex- species, this study fully shares the taxonomy creted directly by snakes; or alternatively, these of Shi et al. (2016), suggesting that G. chang- items were secondarily ingested along with the daoensis, G. caraganus, G. stejnegeri and G. primary prey items, and lizards, which are cognatus should be elevated as full species. (See more routinely found to be insectivores com- Orlov and Barabannov, 1999 and Shi et al., 2016 pared to snakes. However, one of the snakes for the detailed comparisons between different (Sample Y3) was observed to vomit a whole subspecies of G. halys.) undigested moth, which could confirm the for- mer hypothesis. Diet. We checked the faeces samples of four snakes (Y1, Y2, Y5 and Y6). The faeces Distribution and habitat. Gloydius rubromac- contain almost entirely the debris of moths, ulatus sp. n. is distributed mainly along the Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 529

Figure 5. Bayesian phylogenetic tree of the Gloydius species (Asian pit vipers) based on concatenated 12S, 16S, ND4 and cytb gene sequences, 3129 bp, with the Bayesian posterior supports (left, italic) and ML bootstrap supports (right) showed on the nodes (the ones which are lower than 50 percent are noted as “-”). The holotypes are marked with “∗∗”, the topotypes are marked with “∗”.

Tongtianhe River, in the Sanjiangyuan region of stay in the mountain passes, sandy riversides, Qinghai Province. Additionally, G. rubromac- sunny slopes, bushes and shales (fig. 6). ulatus sp. n. is also found in Tibet (Tongpu village, Jiangda Country) and Sichuan (Shiqu Conservation. Gloydius rubromaculatus sp. n. is protected under the conservation regu- Country) (table 1 and supplementary fig. S3). lations of the Sanjiangyuan National Reserve. The distribute altitude ranges from 3300 to Traditional Tibetan culture also offers alterna- 4770 m. G. rubromaculatus sp. n. holds the tive knowledge and perspectives that facilitate highest snake distribution report within Chi- the environmental conservation throughout the nese venomous snakes, and the second highest region (Shen and Tan, 2012). Due to the faith one all over the world, next to G. himalayanus, of native Tibetans, the there, includ- which can occur up to 4880 m (Sharma et al., ing snakes, are fully respected and well pro- 2013). Gloydius rubromaculatus sp. n. tends to tected. Downloaded from Brill.com10/07/2021 08:28:56AM via free access 530 J. Shi et al.

Figure 6. Habitat of Gloydius rubromaculatus sp. n. ( locality), along the Tongtianhe River, Qumarleb Country, Qinghai Province, 4154 above sea level.

Discussion diet, which may be attributed to the lack of prey in the alpine habitat. It is still unknown whether In the past decades, the pit vipers with 21-rows the moths-preying behaviour is seasonal. More mid-back scales from Qinghai-Tibet Plateau data remain to be obtained over a longer dura- have been identified as G. strauchi (Zhao and tion of time (i.e., a proper diet study over an en- Yang, 1997; Zhao, 2006), while according to tire year), including adults, juveniles, and prey Pope’s study (1935), the type locality of G. availability for the habitat. The reason why the strauchi is restricted to Tungngolo (between pit vipers have a special preference for moths is Litang and Kangting, near Xindu, Sichuan) but unknown. A possible assumption would be that redefined as Da-Tsian-lu (Kangding, Sichuan, they are attracted by the smell of pheromone locality of the lectotype ZISP 8534) by Orlov given off by the moths for sex attraction (Groot and Barabanov (2000). Thus, we recognize et al., 2006). These questions need further in- sample G3 as topotype of G. strauchi. However, vestigation. there are both morphological and genetic dif- ferences between G. rubromaculatus sp.n.and G. strauchi (p-distance: 9.0 percent). The two Author contributions species did not recover as sister groups in the phylogenetic trees. Thus, G. rubromaculatus sp. Body article and figures: Jingsong Shi. Field n. is recognized as a new taxon in this study. work: Jingsong Shi and Xi’er Chen. Molecular This study confirmed that G. rubromacula- experiments: Jingsong Shi, Gang Wang and Yi- tus sp. n. is able to prey on moths in the wild, hao Fang. Specimen’s measurement: Jingsong and has the potential to prey on small mammals. Shi, Li Ding and Gang Wang. Data analysis: More attention should be paid to its unusual Jingsong Shi, Mian Hou, Li Ding and Song Downloaded from Brill.com10/07/2021 08:28:56AM via free access A new alpine pit viper species 531

Huang. Naming: Jingsong Shi, Pipeng Li and interspecific directional selection on moth pheromone Jun Liu. All authors gave the final approval for communication. PNAS 103 (15): 5858-5863. Hoge, A., Romano-Hoge, S. (1981): Poisonous snakes of publication. the world. Part 1: checklist of the pit vipers, Viperoidea, Viperidae, Crotalinae. Mem. Inst. Butantan 42 (43): 179- 309. Ethics statement Lanfear, R., Calcott, B., Ho, S.Y.W., et al. (2012): Partition- Finder: combined selection of partitioning schemes and This study is conducted with appropriate per- substitution models for phylogenetic analyzes. MBE 29 missions (letter voucher: IHSYNU [2016] 08) (6): 1695-1701. Li, D., Wang, Z., Wu, C. (1989): Economical Animals of for field survey and specimens’ collecting, and Qinghai Province. Qinghai People’s Press, Xining. guidelines from the responsible authority, the Li, J. 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Tingting Zhang, Bin Wang (CIB), Yemao Hou and Yong sourceforge.net/mb3.2_manual.pdf. Xu (IVPP) for the help with picture drawings and software Sharma, S., Pandey, D., Shah, K., Tillack, F., Chappuis, operations; to Ding Ding (IOZ), Xiaoping Wang (Liaoning F., Thapa, C., Alirol, E., Kuch, U. (2013): Island National Nature Reserve), Jianfang Gao, Xi- Snakes of . A Photographic Guide, 1st Edition, aoyu Zhu, Wei Xue, Jincheng Liu and Xinlei Huang for Feb. important samples; to Chaodong Zhu and Qingyan Dai for Shen, X., Tan, J. (2012): Ecological conservation, cultural moth identification; to Vivek Sharma, Upadhyay, Deepak preservation, and a bridge between: the journey of Shan- CK (Indiansnakes. org), Jiansheng Peng and Zhiyuan Tang shui Conservation Center in the Sanjiangyuan region, for nice photographs. Qinghai-Tibetan Plateau, China. Eco. Soc. 17 (4): 38. Shi, J., Yang, D., Zhang, W., Ding, L. 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