A new moth-preying alpine pit viper species from Qinghai-Tibetan Plateau (Viperidae, Crotalinae) - Brill
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Amphibia-Reptilia 38 (2017): 517-532
A new moth-preying alpine pit viper species from Qinghai-Tibetan
Plateau (Viperidae, Crotalinae)
Jingsong Shi1,2,∗ , Gang Wang3 , Xi’er Chen4 , Yihao Fang5 , Li Ding6 , Song Huang7 , Mian Hou8,9 ,
Jun Liu1,2 , Pipeng Li9
Abstract. The Sanjiangyuan region of Qinghai-Tibetan Plateau is recognized as a biodiversity hotspot of alpine mammals
but a barren area in terms of amphibians and reptiles. Here, we describe a new pit viper species, Gloydius rubromaculatus
sp. n. Shi, Li and Liu, 2017 that was discovered in this region, with a brief taxonomic revision of the genus 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 snake
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, China
Asian pit vipers are small venomous snakes
2 - University of Chinese Academy of Sciences, 100044
Beijing, China distributed mainly in Asia. 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 Herpetology, 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: shijingsong@ivpp.ac.cn himalayanus, above 4880 m; Sharma et al.,
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© 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 taxonomy (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
of Zoology; NWIPB: Northwest Institute of Plateau Biol-
1. Gloydius halys-intermedius complex (G. 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).
Morphology
2. Gloydius blomhoffii complex (G. blomhof-
fii, G. brevicaudus, G. tsushimaensis and Measurements were taken with vernier calliper (Guanglu,
0-200 mm, Made in China). Snout-vent length (SVL), tail
G. ussuriensis).
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
(CAS)) at the Key Laboratory of Vertebrate Evolution and
that G. monticola should be attributed to one Human Origins, CAS. Specimens were scanned at 140 kV
of the subspecies of G. strauchi and deny the with a flux of 100 μA at a resolution of 42.3 μm per pixel
validity of qinlingensis and liupanensis (Zhao, using a 360° rotation with a step size of 0.5° and an unfil-
tered aluminium reflection target. A total of 720 transmis-
1998; Zhao, 2006). Xu et al. (2012) conducted
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.
Laboratory protocols
However, the topological structures of the max-
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.
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Table 1. Details of the molecular samples for this study.
Museum voucher Code Taxa Locality GenBank Accession Numbers Reference
12s 16s Cytb ND4
JSSD1408Z1 Z1 G. changdaoensis Lianyungang, Jiangsu KY040521 KY040553 KX063821 KX063794 this study
JSSD1510C1 C1 G. changdaoensis Changdao, Shandong KY040522 KY040554 KX063823 KX063796 this study
JSSD11110D2 D2 G. shedaoensis Lvshun, Liaoning KY040523 KY040555 KX063819 KX063792 this study
JS150622 22 G. intermedius Zhuanghe, Liaoning KY040524 KY040556 KY040617 KY040638 this study
A new alpine pit viper species
JSSD1110Q4 Q4 G. intermedius Wafangdian, Liaoning KY040525 KY040557 KX063793 KX063820 this study
QS002 QS002 G. intermedius Anshan, Liaoning JX661216 / JX661205 JX661228 Wu et al. (2015)
– SX1 G. intermedius Heilongjiang KM434236 KM434236 KM434236 KM434236 Xu et al. (2012)
SYNU1301908 46 G. h. halys Lingyuan, Liaoning KY040526 KY040558 KX063802 KX063775 this study
JSSD1508X3 X3 G. h. halys Xilinhot, Inner KY040527 KY040559 KX063803 KX063776 this study
Mongolia
JS1407H9 H9 G. h. halys Greater Hinggan Mts., KY040528 KY040560 KY040618 KY040639 this study
Heilongjiang
CIBQY224 QY224 G. cognatus Zoige, Sichuan KY040529 KY040561 KY040619 KY040640 this study
JSSD13109I3 I3 G. cognatus Sonit Right Banner, KY040531 KY040563 KY040621 KY040642 this study
Inner Mongolia
JS131147 47 G. cognatus Yinchuan, Ningxia KY040532 KY040564 KY040622 KY040643 this study
JSSD1504N6 N6 G. cognatus Wuzhong, Ningxia KY040533 KY040565 KX063809 KX063782 this study
JS1505QL1 QLS G. qinlingensis Xunyangba, Shanxi KY040534 KY040566 KY040623 KY040644 this study
HS34 HS34 G. qinlingensis Taibai, Shaanxi / / KF997922 KF997981 this study
GP197 GP197 G. qinlingensis Zhouzhi, Shaanxi / / JQ687490 JQ687471 Xu et al. (2012)
GP198 GP198 G. liupanensis Ningxia / / JQ687491 JQ687472 Xu et al. (2012)
GP206 GP206 G. liupanensis Ningxia / / JQ687492 JQ687473 Xu et al. (2012)
GP215 GP215 G. liupanensis Ningxia / / JQ687493 JQ687474 Xu et al. (2012)
NNU95043 95043 G. liupanensis Liupanshan, Ningxia EF012814 / / EF012795 Zhou et al. (2006)
JSSD1409S3 S3 G. stejnegeri Tongchuan, Shaanxi KY040536 KY040568 KX063817 KX063790 this study
JSSD1508S4 S4 G. stejnegeri Linfen, Shanxi KY040537 KY040569 KX063818 KX063791 this study
SYNU1510145 53 G. stejnegeri Mentougou, Beijing KY040538 KY040570 KX063815 KX063788 this study
JSSD151054 54 G. stejnegeri Mentougou, Beijing KY040539 KY040571 KY040625 KY040646 this study
JS1501G3 G3 G. strauchi Kangting, Sichuan KY040543 KY040575 KY040629 KY040650 this study
JS1508G4 G4 G. strauchi Litang, Sichuan KY040544 KY040576 KY040630 KY040651 this study
IOZ032317∗∗ Y2 G. rubromaculatus sp. n. Yushu, Qinghai KY040546 KY040578 KY040632 KY040653 this study
IOZ032318∗ Y4 G. rubromaculatus sp. n. Yushu, Qinghai KY040547 KY040579 KY040633 KY040654 this study
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Table 1. (Continued.)
JS1607Y5 Y5 G. rubromaculatus sp. n. Yushu, Qinghai KY040548 KY040580 KY040634 KY040655 this study
JS1607DL1 DL1 G. monticola Dali, Yunnan KY040549 KY040581 KY040635 MG025935 this study
JS1607DL2 DL2 G. monticola Dali, Yunnan KY040550 KY040582 KY040636 MG025936 this study
GY001 GY001 G. monticola Lijiang, Yunnan JX661213 / JX661200 JX661243 Wu et al. (2012)
DL70 B1 G. brevicaudus Liaoning KY040552 KY040584 HQ528467 HQ528303 this study
U1 U1 G. ussuriensis Heilongjiang KP262412 KP262412 KP262412 KP262412 Xu et al. (2012)
B524 B524 G. blomhoffii Japan AY352780 AY352719 AY352751 AY352814 Malhotra et al. (2003)
Ts1 Ts1 G. tsushimaensis Japan JN870186 JN870196 JN870203 JN870211 Fenwick et al. (2011)
MHNG 2752.69 R1 G. rickmersi Kyrgyzstan / / / KM078592 Wagner et al. (2016)
MHNG 2752.70 R2 G. rickmersi Kyrgyzstan / / / KM096379
ISEA R290 R290 G. caraganus / / / / KM078594
CR1 CR1 G. caraganus / / / MF490455 MF490453 this study
CR2 CR2 G. caraganus / / / MF490456 MF490454 this study
KT2668 K T. sichuanensis Sichuan KT2668 KT2668 KT2668 KT2668 Zhu et al. (2015)
Acutus A D. acutus Fujian DQ343647 DQ343647 DQ343647 DQ343647 Yan et al. (2008)
Note: ∗∗ , holotype; ∗ , paratype; , topotype. Mts: Mountains.
J. Shi et al.
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Downloaded from Brill.com07/31/2021 02:12:20AMTable 2. Dimensions (mm) and scale data of the specimens of Gloydius snakes for this study.
Preserve Museum Taxon Locality Sex SVL TTL TL HL HW HH SL ED IOS INS V Sc DS SPL (L/R) AFL (L/R)
voucher
IOZ IOZ002317∗∗ G. rubromaculatus Qumarleb, Qinghai 473 554 81 24.6 15.8 7.4 7.8 3.1 8.2 4.6 158 43 21-21-15 7/8 10/11
A new alpine pit viper species
sp. n.
IOZ IOZ002318∗ G. rubromaculatus Jyekundo, Qinghai 452 529 77 24.8 15.4 7.8 7.6 3 9.2 5.4 146 41 21-21-15 7/8 11/9
sp. n.
SYNU JS1607Y3∗ G. rubromaculatus Qumarleb, Qinghai 354 414 60 19.4 11.6 6 5.3 2.6 6 3.7 153 43 21-21-15 8/8 10/11
sp. n.
SYNU JS1410G3 G. strauchi Kangding, Sichuan 407 482 75 21.5 13.4 7.8 / 2.8 9.3 4.4 144 45 21-21-15 7/7 10/10
CIB CIB14356 G. strauchi Kangding, Sichuan 338 404 66 19.4 11.8 6.2 / 2.1 7.7 4.2 151 38 21-21-16 7/7 /
CIB CIB14357 G. strauchi Kangding, Sichuan 347 412 65 19.9 12.1 8.7 / 2.2 7.8 3.7 146 41 21-21-15 7/7 /
CIB CIB14358 G. strauchi Kangding, Sichuan 384 438 54 22.4 12.4 7.9 / 2.4 8.4 5.6 158 35 21-21-15 7/7 /
CIB CIB14359 G. strauchi Kangding, Sichuan 450 505 55 20.9 12.4 7.2 / 1.9 7.8 6 160 33 21-21-15 7/7 /
SYNU JS1508G4 G. strauchi Litang, Sichuan 372 436 64 20.3 12.7 6.5 5.9 2.1 8 4.3 148 42 21-21-15 7/7 10/10
CIB CIB78588 G. strauchi Litang, Sichuan 427 504 77 24.6 15.6 8.2 / 2.7 9.9 5.3 151 40 21-21-16 7/7 10/10
NWIPB NWIPB 630064 G. rubromaculatus Jyekundo, Qinghai 436 498 62 19.8 15.2 7.2 / / / / 163 42 21-21-16 7/7 10/10
sp. n.
NWIPB NWIPB 0512 G. rubromaculatus Jiangda, Tibet 352 408 56 21.2 14.6 7 / / / / 154 43 21-21-22 7/7 10/11
sp. n.
NWIPB NWIPB 790056 G. rubromaculatus Chengduo, Qinghai 452 521 69 22.2 14.2 7.2 / / / / 160 40 21-21-15 7/7 /
sp. n.
NWIPB NWIPB 790058∗ G. rubromaculatus Chengduo, Qinghai 446 509 63 23.1 15.2 7.4 / / / / 162 35 21-21-15 7/7 9/10
sp. n.
CIB CIB12948 G. monticola Zhongdian, Yunnan 436 492 56 21.7 13.3 7.9 / 1.9 8.2 5.8 151 28 21-21-15 / /
CIB CIB72551 G. monticola Zhongdian, Yunnan 422 493 71 25.3 15.2 9.8 / 2.4 9.6 6.7 140 34 21-21-15 6/6 /
CIB CIB72553 G. monticola Zhongdian, Yunnan 274 308 34 18.1 9.5 6.4 / 1.5 6.9 4.7 145 30 21-21-15 6/6 9/10
Note: ∗∗ , holotype; ∗ , paratype; , topotype. SVL: snout-vent length, TL tail length, TTL: total length (SVL + TL), HL: head length, HW: head wide, HH: head height, SL: snout length,
ED: eye diametre (horizontal distance), IOS: interorbital space, INS: internasal space, SPL: supralabials, IFL: infralabials, DS: dorsal scales, V: ventral scales, Sc: subcaudal scales.
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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- Bān Gāo Shān 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
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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, Y4 and Y5)
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,
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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-
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via free accessTable 3. Detailed comparison between G. rubromaculatus sp. n. and the remaining species of the Gloydius strauchi complex.
Species Dorsal Lateral Snout Internasal Prefrontal Supralabials spot Mid-body Canthus Body colouration Body pattern
A new alpine pit viper species
head heal dorsal scale rostralis
G. qinlingensis triangular flat sharp flat flat none 21 obvious yellowish brown or two columns of irregular dark
dark brown brown crossbands, in contact
on the axis of the back
G. liupanensis triangular flat sharp flat flat none 21 obvious yellowish brown (male) similar with qinlingensis
or dark grey (female),
white stripe on the
body side
G. monticola rounded flat sharp flat convex whitish borders of 19 not dark grey or dark blackwish markings on a dark
the labials along obvious brown ground colour
the mouth line
G. himalayanus triangular flat sharp flat flat usually a 21 very brown, reddish brown variable dark markings with
triangular spot sharp or dark grey dark edges
between the third
and forth
G. strauchi rounded dome rounded convex convex large brown 21 Not greenish brown, four longitudinal zigzag
between the obvious yellowish brown or strips, interrupted at intervals,
second, third and nut-brown sometimes curving and
forth (G4) or none coalescing
(G3)
G. rubromaculatus rounded dome rounded convex convex irregular small 21 Not khaki of yellowish two columns of irregular dark
sp. n. black spots obvious brown brown crossbands
525
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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.
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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
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via free access528 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 birds 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, frogs 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
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via free accessA 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 animals 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.
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via free access530 J. Shi et al.
Figure 6. Habitat of Gloydius rubromaculatus sp. n. (type 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.com07/31/2021 02:12:20AM
via free accessA 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
(6): 1695-1701.
missions (letter voucher: IHSYNU [2016] 08)
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. (1999): Infraspecific classification of two species of
Forest Department, Ministry of Forest and En- Gloydius (Serpentes: Crotalinae). Acta Zootaxon. Sin.
4.
vironment, the People’s Republic of China. All Li, J., Sun, L., Wang, X., et al. (2007): Influence of popu-
tissues for DNA extracting are from ventral lation distribution pattern of Gloydius shedaoensis Zhao
scales, shed skins or road-killed dead bodies in on predatory rate. Journal of Snake 19 (1): 12-16.
Orlov, N.L., Barabanov, A.V. (1999): Analysis of nomencla-
this study; no snakes were killed or vivisected.
ture, classification, and distribution of the Agkistrodon
halys-Agkistrodon intermedius complexes: a critical re-
view. Russ. J. Herpetol. 6 (3): 167-192.
Acknowledgements. This study is supported by Ministry Orlov, N.L., Barabanov, A.V. (2000): About type locali-
of Science and Technology of China (2014FY210200), and ties for some species of the genus Gloydius Hoge et
National Natural Scientific Foundation of China (130204, Romano-Hoge, 1981 (Crotalinae: Viperidae: Serpentes).
130201). We are grateful to Songchang Guo, Wenjing Li, Russ. J. Herpetol. 7 (2): 159-160.
Xiaocheng Chen (NWIPB), Dajie Gong, Gang Liang, Xiang Pope, C.H. (1935): The Reptiles of China. Natural History
Zhao and Thupten Gyaltsen for the assistances in specimen of Central Asia, vol. 6. Am. Mus. Nat. Hist., New York,
collection and examination; to Kevin Messenger, Yulong Li, America.
Jinzhong Fu, David Cundall, Yunke Wu, Jiasheng Hao, Lip- Ronquist, F., Huelsenbeck, J., Teslenko, M. (2011): Draft
ing Dong, Zhiyong Yuan, Xuankun Li and Liqun Hao for MrBayes version 3.2 manual: tutorials and model sum-
the kindly language revisions and professional advices; to maries. Distributed with the software from mrbayes.
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): Venomous
Snake Island National Nature Reserve), Jianfang Gao, Xi- Snakes of Nepal. 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. (2016): Distribution
and infraspecies taxonomy of Gloydius halys-Gloydius
intermedius complex in China (Serpentes: Crotalinae).
References
C. J. Z. 51 (5).
Cai, B., Wang, Y., Chen, Y., Li, J. (2015): A revised taxon- Stamatakis, A. (2006): RAxML-VI-HPC: maximum
omy for Chinese reptiles. Biod. Sci. 23: 365-382. likelihood-based phylogenetic analyses with thousands
Daltry, J.C., Wüster, W., Thorpe, R.S. (1996): Diet and of taxa and mixed models. Bioinf. 22 (21): 2688-2690.
snake venom evolution. Nat. 379 (6565): 537-540. Swofford, D.L. (2003): PAUP. Phylogenetic Analysis Using
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. Parsimony (and Other Methods). Version 4. Sinauer
(1994): DNA primers for amplification of mitochondrial Associates, Sunderland, MA, USA.
cytochromec oxidase subunit I from diverse metazoan Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar,
invertebrates. Mol. Mar. Biol. Biotechnol. 3 (5): 294. S. (2013): MEGA6: molecular evolutionary genetics
Gloyd, H.K., Conant, R. (1990): Snakes of the Agkistrodon analysis version 6.0. M. B. E. 30 (12): 2725-2729.
Complex. A Morphologic Review. Contributions to Her- Wagner, P., Tiutenko, A., Mazepa, G., Simonov, E., Borkin,
petology, No. 6. Society for the study of Amphibians and L.J. (2016): Alai! Alai! – a new species of the Gloydius
Reptiles, Oxford, OH, vi + 614 pp.; 52 pl. halys (Pallas, 1776) complex (Viperidae, Crotalinae),
Groot, A.T., Horovitz, J.L., Hamilton, J., Santangelo, R.G., including a brief review of the complex. Amp. Rep. 37
Schal, C., Gould, F. (2006): Experimental evidence for (1): 15-31.
Downloaded from Brill.com07/31/2021 02:12:20AM
via free access532 J. Shi et al.
Xu, Y., Liu, Q., Myers, E.A., Wang, L., Huang, S., He, Y., Scientific Expedition to the Qinghai-Xizang Plateau.
Peng, P., Guo, P. (2012): Molecular phylogeny of the Chinese Academy of Science, Beijing.
genus Gloydius (Serpentes: Crotalinae). A. H. R. 3 (2):
127-132.
Zhao, E. (2006): Snakes of China. Anhui Science and Submitted: March 28, 2017. Final revision received: Octo-
Technology Publishing House, China. ber 1, 2017. Accepted: October 21, 2017.
Zhao, E., Huang, M., Zong, Y., Zheng, J., Huang, Z., Yang, Associate Editor: Sylvain Ursenbacher.
D., Li, D. (1998): Fauna sinica. Reptilia 3: 1-522.
Zhao, E., Yang, D. (1997): Amphibians and Reptiles of
the Hengduan Mountains Region. The Comprehensive
Downloaded from Brill.com07/31/2021 02:12:20AM
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