Abstract
This study presents a comprehensive morphological comparison along with molecular phylogeny of the genus Gloydius based on five mitochondrial genes (12S, 16S, COI, cytb, and ND4). The specimens collected from Jiuzhaigou National Nature Reserve are shown to be a new species, Gloydiuslateralissp. nov. Zhang, Shi, Jiang & Shi based on a combination of morphological and molecular accounts. G.lateralissp. nov. differs from other congeneric species by a series of diagnostic morphological characteristics and forms a strongly supported monophyletic group. The new species is phylogenetically closely related to G.swild, another recently described species from Heishui, Aba, Sichuan.
Keywords: Asian pitviper, Gloydiuslateralis , Jiuzhaigou National Nature Reserve, morphology, new species, phylogenetics
Introduction
Jiuzhaigou National Nature Reserve (JNNR; 32.900–33.266°N, 103.767–104.050°E, 1996–4764 m a.s.l.), a World Heritage Site, lies in the transition zone from the eastern edge of the Qinghai-Tibet Plateau to the Sichuan Basin (Sichuan Province, China), and occupies an area of 651 km2 (Li et al. 2004). The reserve is covered with well-preserved original forests, and numerous alpine lakes, inhabiting many world-famous rare animals, such as Giant Panda (Ailuropodamelanoleuca), and Golden Snub-nosed Monkey (Rhinopithecusroxellana). In contrast to the mammals, the herpetological diversity here is relatively low due to the harsh alpine environment (e.g., low temperatures, low oxygen levels, and intense solar radiation: Li et al. 2004; Shi et al. 2017). To further investigate the herpetological biodiversity and post-earthquake ecological system in this region, we conducted a series of investigations from April to September 2021, and collected several specimens of small-bodied pitvipers in the genus Gloydius Hoge & Romano-Hoge, 1981.
Pitvipers of the genus Gloydius, or Asian pitvipers, are small-bodied venomous snakes distributed in Asia. At present, at least 23 species of the genus belonging to three groups (i.e., the G.blomhoffii group, G.halys-intermedius group, and G.strauchi group) are recognized (Orlov and Barabanov 1999; Zhao 2006; Shi et al. 2017, 2018). From 2017 to 2021, five taxa initially thought to be G.strauchi (Bedriaga, 1912) had been shown to be new species based on morphological and genetic accounts: Gloydiusrubromaculatus (Shi et al. 2017), G.angusticeps (Shi et al. 2018), G.huangi (Wang et al. 2019), G.lipipengi (Shi et al. 2021), and G.swild (Shi et al. 2021). Previous studies have suggested that there still might be hidden species within the G.strauchi complex, given that the G.strauchi complex is widely distributed in western China (Zhao et al. 1998; Zhao 2006; Shi et al. 2021). Shi et al. (2018) indicated the underestimated diversity of alpine pitvipers, and emphasized the necessary of further elucidating of the biodiversity in southwest China.
During the herpetological surveys in JNNR up to August 2021, we collected nine specimens of Gloydius from Zharu Valley. Subsequent examination of these specimens, and assessment of their morphological and genetic data showed that these individuals differ from the topotypic G.angusticeps, G.strauchi, and G.swild from Sichuan Province, as well other congers of the genus. Herein, we report a new Gloydius species. The discovery of this new species once more highlights the species diversity of Gloydius in the Hengduan Mountains.
Materials and methods
Sampling
Nine specimens collected from Jiuzhaigou National Nature Reserve were fixed in 10% buffered formalin after removing the liver tissues for molecular analyses, and then transferred to 80% ethanol for permanent preservation (Fig. 1). The above-mentioned specimens were deposited in the Institute of Chengdu Institute of Biology (CIB), Chinese Academy of Sciences (CAS), Institute of Vertebrate Paleontology and Paleoanthropology (IVPP), Chinese Academy of Sciences (CAS) and Sichuan Academy of Forestry (SAFS).
Figure 1.
The type locality of Gloydiuslateralis sp. nov. in Jiuzhaigou National Nature Reserve, denoted by a black square, with the collection localities of some other congeneric species.
Morphology
A total of 15 morphological characters of the candidate new species were measured. Snout-vent length (SVL), tail length (TL), and total length (TTL) were measured with a measure to the nearest 1 mm. Other morphological measurements were taken with Vernier calipers to the nearest 0.1 mm: head length (HL, from the tip of snout to the line connecting left and right posterior margins of mandible), head width (HW, the widest part of the head in dorsal view), head depth (HD, the deepest part of the head in lateral view), snout length (SL, from the tip of snout to the anterior margin of the eye), eye diameter (ED, measured as a horizontal distance), interorbital space (IOS, the distance between the top margin of eyes), and internasal space (INS, the distance between nostrils). The numbers of supralabials (SPL), infralabials (IFL), dorsal scales (DS), ventral scales (V, excluding preventral scales), and subcaudal scales (SC) were counted. Dimensions and counts are listed in Table 1. Coloration is described according to the HTML color codes (https://html-color.codes). For morphological comparison, specimens of seven congers also from eastern part of Qinghai-Tibet Plateau were examined (Table 1).
Table 1.
Measurements of Gloydiuslateralis sp. nov. and other species of the genus from eastern part of Qinghai-Tibet Plateau.
| Taxa | Museum Vouchers | Preserve | Localities | Gender | SVL | TTL | TL | HL | HW | HH | SL | ED | IOS | INS | V | Sc | DS | SPL (L/R) | IFL (L/R) | ED/HL | Reference |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Gloydiuslateralis sp. nov. | CIB 119377 (JZ02)** | CIB | JNNR | F | 440.0 | 498.0 | 58.0 | 21.9 | 15.1 | 8.3 | 6.4 | 3.2 | 7.8 | 4.2 | 158 | 38 | 20-20-15 | 6/6 | 10/10 | 0.146 | This study |
| G.lateralis sp. nov. | IVPP OV 2727 (JZ01)* | IVPP | JNNR | F | 428.0 | 434.1 | 60.1 | 19.2 | 14.4 | 7.4 | 6.2 | 3.2 | 8.2 | 4.3 | 161 | 44 | 21-21-15 | 8/7 | 10/10 | 0.167 | This study |
| G.lateralis sp. nov. | CIB 119378 (JZ03)* | CIB | JNNR | F | 421.0 | 478.0 | 57.0 | 18.5 | 13.4 | 8.5 | 6.8 | 2.9 | 7.8 | 5.1 | 155 | 39 | 21-21-16 | 7/7 | 10/10 | 0.157 | This study |
| G.lateralis sp. nov. | CIB 119379 (JZ04)* | CIB | JNNR | F | 393.0 | 454.0 | 61.0 | 19.3 | 12.0 | 6.3 | 5.3 | 2.8 | 7.4 | 3.8 | 158 | 42 | 21-20-16 | 7/7 | 11/11 | 0.145 | This study |
| G.lateralis sp. nov. | CIB 87280* | CIB | JNNR | M | 394.0 | 464.7 | 70.7 | 21.0 | 15.2 | 8.0 | 6.4 | 3.6 | 8.5 | 4.3 | 151 | 49 | 21-21-15 | 7/7 | 9/10 | 0.171 | This study |
| G.lateralis sp. nov. | SAFS2021001* | SAFS | JNNR | F | 445.0 | 481.0 | 36+ | 23.0 | 1.6 | 8.2 | – | – | 9.1 | 4.0 | 155 | 22+ | 21-21-17 | 7/7 | 10/10 | – | This study |
| G.lateralis sp. nov. | SAFS2021002* | SAFS | JNNR | F | 489.0 | 533.0 | 44+ | 23.0 | 1.6 | 8.4 | – | – | 9.4 | 4.0 | 163 | 22+ | 21-21-17 | 7/7 | 10/10 | – | This study |
| G.lateralis sp. nov. | SAFS2021003* | SAFS | JNNR | F | 430.0 | 488.0 | 58.0 | 24.0 | 1.5 | 7.8 | – | – | 9.1 | 4.0 | 162 | 41 | 21-21-17 | 7/7 | 10/10 | – | This study |
| G.lateralis sp. nov. | SAFS2021004* | SAFS | JNNR | F | 376.0 | 424.0 | 48+ | 20.0 | 1.2 | 6.5 | – | – | 7.0 | 3.3 | 160 | 33+ | 21-21-17 | 7/7 | 10/10 | – | This study |
| G.angusticeps | IVPPOV 2634** | IVPP | Xiaman, Sichuan | M | 373.2 | 439.7 | 66.5 | 21.2 | 12.4 | 6.6 | 6.7 | 2.2 | 9.1 | 4.1 | 148 | 39 | 19-19-15 | 7/7 | 10/10 | 0.104 | Shi et al. (2018) |
| G.angusticeps | JS1507G5A* | SYNU | Xiaman, Sichuan | M | 283.4 | 331.6 | 42.2 | 16.9 | 9.8 | 6.3 | 4.5 | 2.0 | 7.5 | 3.3 | 151 | 39 | 19-20-15 | 6/6 | 9/10 | 0.118 | Shi et al. (2018) |
| G.angusticeps | JS1306G1A* | SYNU | Golog, Qinghai | F | 443.1 | 502.3 | 59.2 | 23.6 | 13.2 | 7.0 | 5.3 | 2.8 | 8.3 | 4.3 | 162 | 31 | 21-21-15 | 7/6 | 8/9 | 0.119 | Shi et al. (2018) |
| G.angusticeps | IOZ002317* | IOZ | Golog, Qinghai | F | 457.2 | 459.4 | 72.2 | 22.1 | 11.8 | 7.1 | – | – | 8.0 | 4.5 | 157 | 35 | 19-21-15 | 6/6 | 10/10 | – | Shi et al. (2018) |
| G.huangi | KIZ 027654** | KIZ | Chaya, Chamdo, Tibet | F | 532.0 | 455.0 | 67.0 | 23.2 | 14.6 | – | – | 3.1 | 8.4 | 4.3 | 174 | 43 | 21-21-15 | 7/7 | 10/10 | 0.134 | Wang et al. (2019) |
| G.lipipengi | IVPP OV 2720** | IVPP | Zawalong, Zayu, Tibet | M | 540.6 | 628.2 | 87.6 | 25.2 | 13.2 | 8.2 | 7.4 | 2.9 | 9.6 | 5.4 | 165 | 46 | 23-21-15 | 7/7 | 10/11 | 0.115 | Shi et al. (2021) |
| G.monticola | CIB 72553 | CIB | Zhongdian, Yunnan | F | 274.0 | 308.0 | 34.0 | 18.1 | 9.5 | 6.4 | – | 1.5 | 6.9 | 4.7 | 145 | 30 | 19-19-15 | 6/6 | 9/10 | 0.083 | Shi et al. (2017) |
| G.rubromaculatus | IOZ 032317** | IOZ | Yushu, Qinghai | M | 473.0 | 554.0 | 81.0 | 24.6 | 15.8 | 7.4 | 7.8 | 3.1 | 8.2 | 4.6 | 158 | 43 | 21-21-15 | 7/8 | 10/11 | 0.126 | Shi et al. (2017) |
| G.strauchi | SUNU1410G3△ | SYNU | Kangding, Sichuan | M | 407.3 | 482.7 | 75.4 | 21.5 | 13.4 | 7.8 | – | 2.8 | 9.3 | 4.4 | 144 | 45 | 21-21-15 | 7/7 | 10/10 | 0.130 | Shi et al. (2017) |
| G.strauchi | CIB 14356△ | CIB | Kangding, Sichuan | M | 338.5 | 405.0 | 66.3 | 19.4 | 11.8 | 6.2 | – | 2.1 | 7.7 | 4.2 | 151 | 38 | 21-21-16 | 7/7 | – | 0.108 | Shi et al. (2017) |
| G.strauchi | CIB 14357△ | CIB | Kangding, Sichuan | M | 347.2 | 412.4 | 65.2 | 19.9 | 12.1 | 8.7 | – | 2.2 | 7.8 | 3.7 | 146 | 41 | 21-21-15 | 7/7 | – | 0.111 | Shi et al. (2017) |
| G.strauchi | SYNU1508G4 | SYNU | Litang, Sichuan | M | 372.3 | 436.4 | 64.1 | 20.3 | 12.7 | 6.5 | 5.9 | 2.1 | 8 | 4.3 | 148 | 42 | 21-21-15 | 7/7 | 10/10 | 0.103 | Shi et al. (2017) |
| G.strauchi | CIB 78588 | CIB | Litang, Sichuan | M | 427.3 | 504.6 | 77.3 | 24.6 | 15.6 | 8.2 | – | 2.7 | 9.9 | 5.3 | 151 | 40 | 21-21-16 | 7/7 | 10/10 | 0.110 | Shi et al. (2017) |
| G.strauchi | CIB 14358△ | CIB | Kangding, Sichuan | F | 384.1 | 438.3 | 54.2 | 22.4 | 12.4 | 7.9 | – | 2.4 | 8.4 | 5.6 | 158 | 35 | 21-21-15 | 7/7 | – | 0.107 | Shi et al. (2017) |
| G.strauchi | CIB 14359△ | CIB | Kangding, Sichuan | F | 450.3 | 505.5 | 55.2 | 20.9 | 12.4 | 7.2 | – | 1.9 | 7.8 | 6 | 160 | 33 | 21-21-15 | 7/7 | – | 0.091 | Shi et al. (2017) |
| G.swild | IVPP OV 2725** | IVPP | Heishui, Aba, Sichuan | F | 462.0 | 529.5 | 67.5 | 20.8 | 12.2 | 6.6 | 5.8 | 2.4 | 7.6 | 4.1 | 170 | 46 | 21-21-15 | 7/7 | 10/10 | 0.115 | Shi et al. (2021) |
| G.swild | IVPP OV 2726* | IVPP | Heishui, Aba, Sichuan | F | 552.0 | 629.1 | 77.1 | 23.8 | 15.7 | 8.4 | 6.2 | 3.2 | 9.6 | 5.0 | 168 | 43 | 21-21-17 | 7/7 | 10/10 | 0.134 | Shi et al. (2021) |
Note: **, holotype; *, paratype; △, topotype. The missing data are marked as “–”.
Molecular analyses
Genomic DNA was extracted from four specimens collected in this study using the Qiaprep Spin Miniprep kit (QiaGen). Five mitochondrial genome fragments were specifically amplified for this study: an 859 bp fragment of 12S ribosomal RNA (12S) using primers 12SFPhe and 12SRVal (Knight and Mindell 1993); a 465 bp fragment of 16S ribosomal RNA (16S) using primers 16sFL and 16sRH (Palumbi et al. 1991); a 657 bp fragment of cytochrome c oxidase subunit I (COI) using primers L14919 and H16064 (Burbrink et al. 2000); a 1065 bp fragment of cytochrome b (cytb) using primers L14919 and H16064 (Burbrink et al. 2000); and a 666 bp fragment of NADH dehydrogenase subunit 4 (ND4) using the primers ND4 and Leu (Arevalo et al. 1994). The standard PCR protocol was performed in a 20 µl reaction with at least 20 ng of template DNA and 10 pmol of primers. PCR conditions consisted of an initial denaturation for 3 min at 94 °C, followed by 35 cycles: denaturation at 94 °C for 30 sec, annealing temperature 52 °C for 12S, 54 °C for 16S, 50 °C for COI, 48 °C for cytb, and 56 °C for ND4 for 30 sec, elongation at 72 °C for 60 sec, and then finalized with an extension step at 72 °C for 10 min. Sequencing was conducted by Beijing Tianyi Huiyuan Biotech Co., Ltd. New sequences are deposited in GenBank (Table 2).
Table 2.
Molecular samples included in this study.
| Taxa | Museum voucher | Code | Locality | locus | |||||
|---|---|---|---|---|---|---|---|---|---|
| 12S | 16S | cytb | COI | ND4 | Reference | ||||
| Gloydiuslateralis sp. nov. | CIB 119377 (JZ02)** | CIB | JNNR | ON362225 | ON362229 | ON423417 | ON399075 | ON423421 | This study |
| G.lateralis sp. nov. | IVPP OV 2727 (JZ01)* | IVPP | JNNR | ON362226 | ON362230 | ON423418 | ON399076 | ON423422 | This study |
| G.lateralis sp. nov. | CIB 119378 (JZ03)* | CIB | JNNR | ON362227 | ON362231 | ON423419 | ON399077 | ON423423 | This study |
| G.lateralis sp. nov. | CIB 119379 (JZ04)* | CIB | JNNR | ON362228 | ON362232 | ON423420 | ON399078 | ON423424 | This study |
| G.angusticeps. | IOZ 002317* | G1 | Golog, Qinghai | KY040541 | KY040572 | KY040627 | KY040604 | KY040647 | Shi et al. 2018 |
| G.angusticeps | IVPP OV 2634** | G5 | Zoige, Sichuan | KY040545 | KY040577 | KY040631 | KY040609 | KY040652 | Shi et al. 2018 |
| G.blomhoffii | B524 | B524 | Japan | AY352719 | AY352719 | AY352751 | – | AY352814 | Malhotra and Thorpe 2004 |
| G.brevicaudus | DL70 | B1 | Liaoning | KY040552 | KY040584 | HQ528467 | – | HQ528303 | Shi et al. 2017 |
| G.caraganus | CR1 | CR1 | Kazakhstan | – | – | MF490455 | – | MF490453 | Shi et al. 2017 |
| G.caraganus | RIZ 20426.1 | 426 | Kyzylorda, Kazakhstan | MZ958021 | MZ957012 | MZ959165 | – | MZ959158 | Shi et al. 2021 |
| G.caucasicus | RIZ 29913 | 913 | Mazandaran, Iran | MZ958022 | MZ957013 | MZ959166 | – | MZ959159 | Shi et al. 2021 |
| G.caucasicus | NEZMUT_61 | NE61 | Alborz, Iran | – | – | MH378692 | – | MH378729 | Asadi et al. 2019 |
| G.changdaoensis | SYNUSHF01△ | C1 | Changdao, Shandong | KY040522 | KY040554 | KX063823 | KY040586 | KX063796 | Shi et al. 2017 |
| G.cognatus | CIB-QY224 | QY224 | Zoige, Sichuan | KY040529 | KY040561 | KY040619 | KY040593 | KY040640 | Shi et al. 2017 |
| G.cognatus | SYNU 13109I3 | I3 | Saihan, Inner Mongolia | KY040531 | KY040563 | KY040621 | KY040595 | KY040642 | Shi et al. 2017 |
| G.halyshalys | SYNU 1510151 | H9 | Greater-Xing’an, Heilongjiang | KY040528 | KY040560 | KY040618 | – | KY040639 | Shi et al. 2017 |
| G.huangi | CIB 533422012 | MK | Mangkang, Tibet | – | MZ957017 | MZ355578 | – | MZ355578 | Shi et al. 2022 |
| G.huangi | KIZ 027654* | 027654 | Chaya, Chamdo, Tibet | MK227409 | MK227412 | MK227415 | – | MK227418 | Wang et al. 2019 |
| G.intermedius | SYNU 150622 | 22 | Zhuanghe, Liaoning | KY040524 | KY040556 | KY040617 | – | KY040638 | Shi et al. 2017 |
| G.liupanensis | GP198 | S083 | Ningxia | – | MK193903 | MK201255 | – | JQ687472 | Li et al. 2019; Xu et al. 2012 |
| G.lipipengi | IVPP OV 2720 | G2 | Zawalong, Zayu, Tibet | KY040542 | KY040574 | KY040628 | AY352751 | KY040649 | Shi et al. 2021 |
| G.liupanensis | LP1 | LP1 | Guyuan, Ningxia | MZ958024 | MZ957015 | MZ959168 | KY040599 | MZ959161 | Shi et al. 2021 |
| G.liupanensis | LP4 | LP4 | Guyuan, Ningxia | MZ958025 | MZ957016 | MZ959169 | ON399079 | MZ959162 | Shi et al. 2021 |
| G.liupanensis | TC1 | TC1 | Tanchang, Gansu | MZ958023 | MZ9570124 | MZ959167 | ON399080 | MZ959160 | Shi et al. 2021 |
| G.monticola | SYNU 1607DL1 | DL1 | Dali, Yunnan | KY040549 | KY040581 | KY040635 | – | MG025935 | Shi et al. 2017 |
| G.qinlingensis | SYNU QL1△ | QLS | Xunyangba, Shanxi | KY040534 | KY040566 | KY040623 | KY040598 | KY040644 | Shi et al. 2017 |
| G.rickmersi | MHNG 2752.69 | R1 | Kyrgyzstan | – | – | – | – | KM078592 | Wagner et al. 2016 |
| G.rubromaculatus | IOZ 032317** | Y2 | Qumarleb, Qinghai | KY040546 | KY040578 | KY040632 | KY040610 | KY040653 | Shi et al. 2017 |
| G.stejnegeri | SYNU 1508S4△ | S4 | Linfen, Shanxi | KY040537 | KY040569 | KX063818 | KY040601 | KX063791 | Shi et al. 2017 |
| G.shedaoensis | SYNU 110D2△ | D2 | Lvshun, Liaoning | KY040523 | KY040555 | KX063819 | KY040587 | KX063792 | Shi et al. 2017 |
| G.strauchi | SYNU 1501G3△ | G3 | Kangting, Sichuan | KY040543 | KY040575 | KY040629 | KY040607 | KY040650 | Shi et al. 2017 |
| G.strauchi | SYNU 1508G4 | G4 | Litang, Sichuan | KY040544 | KY040576 | KY040630 | KY040608 | KY040651 | Shi et al. 2017 |
| G.swild | IVPP OV 2725 | GR1 | Heishui, Aba, Sichuan | OK210582 | OK184551 | OK239647 | – | OK239652 | Shi et al. 2021 |
| G.swild | IVPP OV 2726 | GR2 | Heishui, Aba, Sichuan | OK210583 | OK184552 | OK239648 | – | OK239653 | Shi et al. 2021 |
| G.tsushimaensis | – | Ts1 | Japan | JN870203 | JN870196 | JN870203 | JN870203 | JN870211 | Fenwick 2011 |
| G.ussuriensis | U1 | U1 | Heilongjiang | KP262412 | KP262412 | KP262412 | KP262412 | KP262412 | Xu et al. 2012 |
| Deinagkistrodonacutus | – | A | Fujian | DQ343647 | DQ343647 | DQ343647 | DQ343647 | DQ343647 | Yan et al. 2008 |
Note: **, holotype; *, paratype; △, topotype. The missing data are marked as “–”.
For phylogenetic comparisons, corresponding sequences of 22 recognized species of the genus Gloydius, and one representative of the outgroup (Deinagkistrodonacutus) were obtained from GenBank (Table 2; Shi et al. 2021). Sequences were assembled, and aligned using MEGA 6 (Tamura et al. 2013) with default settings, and were further revised manually when necessary. With respect to the different evolutionary characters of each molecular marker, the dataset was initially split into eight partitions by gene and codon positions and then combined into nine partitions taking advantage of PartitionFinder 2.1.1 (Lanfear et al. 2012) to find similarly evolving partitions.
A Bayesian phylogenetic analysis was performed using MrBayes 3.1.2 (Ronquist et al. 2012). All searches consisted of three heated chains and a single cold chain. Three independent iterations each comprising two runs of 100 million generations were performed, sampling every 10,000 generations, and parameter estimates were plotted against generation. The first 25% of the samples were discarded as burn-in, resulting in a potential scale reduction factor (PSRF) of < 0.005. A maximum-likelihood analysis was run with the IQtree tool in the webserver CIPRES (https://www.phylo.org/index.php), with 1000 fast bootstrap repeats. The General time-reversible (GTR) model, the most probable substitution model for the corrected ND4p-distance matrix was calculated in PAUP 4.0 (https://people.sc.fsu.edu/~dswofford/paup_test/).
Results
Morphology
Comparative data of specimens examined are listed in Tables 1, 2, and 4, as well as Figs 2–6. The holotype and paratypes are illustrated in Figs 2–4. The Gloydius specimens from JNNR are different from recognized species of the genus by a combination of morphological characters including ventrals 151–163 (n = 9), subcaudals 38–49, laurel green dorsal body with deep-colored patches, and regular greyish brown ventrolateral stripes.
Table 4.
Corrected distance between Gloydiuslateralis and other Gloydius species based on ND4 and GTR model.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | ||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | G.intermedius (22) | - | ||||||||||||||||||||
| 2 | G.shedaoensis (D2) | 0.011 | - | |||||||||||||||||||
| 3 | G.halys (H9) | 0.041 | 0.042 | - | ||||||||||||||||||
| 4 | G.cognatus (I3) | 0.033 | 0.033 | 0.033 | - | |||||||||||||||||
| 5 | G.stejnegeri (S4) | 0.045 | 0.050 | 0.047 | 0.041 | - | ||||||||||||||||
| 6 | G.rickmersi (R1) | 0.052 | 0.051 | 0.054 | 0.049 | 0.065 | - | |||||||||||||||
| 7 | G.caraganus (CR1) | 0.038 | 0.046 | 0.049 | 0.042 | 0.059 | 0.050 | - | ||||||||||||||
| 8 | G.changdaoensis (C1) | 0.054 | 0.049 | 0.050 | 0.042 | 0.069 | 0.066 | 0.054 | - | |||||||||||||
| 9 | G.qinlingensis (QL1) | 0.110 | 0.122 | 0.106 | 0.104 | 0.113 | 0.113 | 0.114 | 0.113 | - | ||||||||||||
| 10 | G.liupanensis (LP1) | 0.090 | 0.099 | 0.088 | 0.086 | 0.097 | 0.102 | 0.097 | 0.095 | 0.039 | - | |||||||||||
| 11 | G.strauchi (G3A) | 0.098 | 0.110 | 0.102 | 0.097 | 0.111 | 0.116 | 0.107 | 0.105 | 0.074 | 0.059 | - | ||||||||||
| 12 | G.angusticeps (G5C) | 0.101 | 0.112 | 0.097 | 0.099 | 0.112 | 0.104 | 0.102 | 0.104 | 0.063 | 0.068 | 0.067 | - | |||||||||
| 13 | G.monticola (DL1) | 0.120 | 0.130 | 0.122 | 0.112 | 0.137 | 0.134 | 0.135 | 0.120 | 0.076 | 0.078 | 0.079 | 0.076 | - | ||||||||
| 14 | G.huangi (R86*) | 0.111 | 0.123 | 0.117 | 0.112 | 0.122 | 0.122 | 0.119 | 0.124 | 0.081 | 0.080 | 0.086 | 0.080 | 0.078 | - | |||||||
| 15 | G.rubromaculatus (Y2*) | 0.106 | 0.115 | 0.100 | 0.109 | 0.118 | 0.109 | 0.113 | 0.112 | 0.086 | 0.085 | 0.090 | 0.079 | 0.089 | 0.085 | - | ||||||
| 16 | G.lipipengi (G2) | 0.112 | 0.125 | 0.114 | 0.127 | 0.124 | 0.121 | 0.119 | 0.132 | 0.078 | 0.081 | 0.092 | 0.081 | 0.088 | 0.089 | 0.042 | - | |||||
| 17 | G.swild (GR1) | 0.116 | 0.125 | 0.110 | 0.108 | 0.102 | 0.123 | 0.114 | 0.119 | 0.099 | 0.085 | 0.089 | 0.086 | 0.089 | 0.103 | 0.085 | 0.097 | - | ||||
| 18 | G.brevicaudus (B1) | 0.143 | 0.152 | 0.135 | 0.145 | 0.155 | 0.150 | 0.145 | 0.156 | 0.117 | 0.113 | 0.124 | 0.121 | 0.122 | 0.138 | 0.124 | 0.126 | 0.138 | - | |||
| 19 | G.ussuriensis (U1) | 0.106 | 0.116 | 0.131 | 0.115 | 0.133 | 0.133 | 0.119 | 0.123 | 0.119 | 0.109 | 0.110 | 0.102 | 0.122 | 0.105 | 0.110 | 0.128 | 0.118 | 0.107 | - | ||
| 20 | G.blomhoffii (B524) | 0.132 | 0.144 | 0.148 | 0.142 | 0.157 | 0.147 | 0.135 | 0.144 | 0.119 | 0.110 | 0.117 | 0.110 | 0.112 | 0.119 | 0.116 | 0.125 | 0.125 | 0.117 | 0.068 | - | |
| 21 | G.sushimaensis_Ts1 | 0.121 | 0.135 | 0.145 | 0.133 | 0.152 | 0.142 | 0.139 | 0.146 | 0.126 | 0.113 | 0.118 | 0.108 | 0.110 | 0.121 | 0.130 | 0.138 | 0.133 | 0.122 | 0.054 | 0.053 | - |
| 22 | G.lateralis sp. nov. | 0.108 | 0.112 | 0.100 | 0.101 | 0.111 | 0.120 | 0.099 | 0.100 | 0.095 | 0.083 | 0.090 | 0.092 | 0.099 | 0.101 | 0.091 | 0.101 | 0.061 | 0.147 | 0.126 | 0.142 | 0.149 |
Figure 2.
Holotype of Gloydiuslateralis sp. nov. in life, adult female, CIB 119377 (JZ02) A dorsolateral view B ventral view.
Figure 6.
Lateral (dorsolateral) view of the alpine pitvipers (Gloydiusstrauchi complex) that distributed in Sichuan and Tibet (not to scales); the lateral stripes are pointed by arrows AG.lateralis sp. nov., CIB 119377 (JZ02), female, from Jiuzhaigou, Sichuan BG.swild, IVPP OV 2725, female, from Heishui, Sichuan CG.angusticeps, male, from Golog, Qinghai (not included in this study) DG.liupanensis, male, from Longnan, Gansu EG.qinlingensis, male, from Ningshaan, Shaanxi FG.strauchi, from Sichuan GG.lipipengi, IVPP OV 2720, male, from Zayu, Tibet HG.rubromaculatus, male from Yushu, Qinghai IG.huangi, CIB 533422012, male, from Hola, Mangkang. Copyright: Sheng-Chao Shi (A, I), Jing-Song Shi (B, E, G, H), Hong Zhao (C), Zu-Yao Xia (D) and Zhi-Yuan Tang (F).
Figure 4.
Paratypes of Gloydiuslateralis sp. nov. in life A, BCIB 119379 (JZ04), adult female in dorsolateral and ventral views C, D CIB119378 (JZ03), adult female in dorsolateral and ventral views.
Molecular phylogeny
The final dataset with 3,722 bp of 37 specimens was analysed in this study. The evolutionary models assigned to each of the nine partitions by PartitionFinder are shown in Table 3. In this study, the topological structures of the maximum likelihood (ML) and Bayesian inference (BI) trees are generally consistent (Fig. 7). The four specimens from JNNR formed a strongly supported monophyletic group. This lineage is sister to the clade of G.swild from Heishui, Sichuan. The clade including the new species and G.swild is sister to the clade formed by 11 species of the G.intermedius-halys complex. The result conforms to the earlier studies of the genus (Xu et al. 2012; Shi et al. 2017, 2018; Wang et al. 2019).
Table 3.
Partitions and their evolutionary models selected by PartitionFinder 2.1.1.
| Partitions | Locus | Length (bp) | Models |
|---|---|---|---|
| Partition 1 | 12S | 859 | GTR+I+G |
| Partition 2 | 16S | 475 | GTR+I+G |
| Partition 3 | cytb pos1, ND4 pos1 | 577 | TVM+I+G |
| Partition 4 | ND4 pos2 | 222 | TVM+I+G |
| Partition 5 | ND4 pos3 and cytb pos3 | 577 | GTR+G |
| Partition 6 | cytb pos2 | 355 | K81uf+I+G |
| Partition 7 | COI pos1 | 219 | GTR+G |
| Partition 8 | COI pos2 | 219 | K81UF+I |
| Partition 9 | COI pos3 | 219 | GTR+I+G |
GTR: General Time-Reversible model; K81uf: Kimura 1981 TVM: transversional substitution model; TIM: transitional substitution model; +G: rate heterogeneity; +I: proportion of invariable sites
Figure 7.
Maximum-likelihood tree of the genus Gloydius based on 12S, 16S, COI, ND4 and cytb sequences, with the maximum likelihood bootstrap supports (left, regular) and Bayesian posterior probabilities (right, italic) displayed on the nodes (those < 50% are displayed as “-”).
The corrected p-distance based on the ND4 gene between the new species and its closest related congeners, G.swild is 6.1%, higher than many pairs of substantial species, such as G.intermedius vs G.shedaoensis (1.1%), G.halys vs G.cognatus (3.3%), G.qinlingensis vs G.liupanensis (3.9%), and G.lipipengi vs G.rubromaculatus (4.2%; Table 4). Thus, the molecular phylogeny supports these new specimens from JNNR as phylogenetically independent species.
Both morphological and molecular analyses support that the specimens from JNNR represent a new species, and it is described herein.
Taxonomic account
. Viperidae
Oppel, 1811
646559CB-25AF-52DD-8FE1-3A68BD6DEC75
Gloydius Hoge & Romano-Hoge, 1981
. Gloydius lateralis
Zhang, Shi, Jiang & Shi sp. nov.
774B80B3-FCF2-5623-B361-E6D8488FC62C
http://zoobank.org/6553A5A1-6A2A-4605-B5F7-64724A2DAB7B
Figure 3.
Head squamation of holotype of Gloydiuslateralis sp. nov. CIB 119377 (JZ02) A lateral view B ventral view C dorsal view. Scale bar: 10 mm.
Figure 5.
Lateral (dorsolateral) heads of the alpine pitvipers (Gloydius) distributed in Sichuan and Tibet (not to scales) AG.lateralis sp. nov., CIB 119377 (JZ02), female, from Jiuzhaigou National Nature Reserve, Sichuan BG.swild, IVPP OV 2725, female, from Heishui, Sichuan CG.angusticeps, IVPP OV 2634, male, from Zoige, Sichuan DG.liupanensis, male, from Longnan, Gansu EG.qinlingensis, male, from Ningshaan, Shaanxi FG.strauchi, SYNU1508G4, male, from Litang, Sichuan GG.lipipengi, IVPP OV 2720, male, from Zayu, Tibet HG.rubromaculatus, male, from Yushu, Qinghai (not included in this study) IG.huangi, CIB 533422012, male, from Hola, Mangkang. Copyright: Sheng-Chao Shi (A, I), Jing-Song Shi (B, C, E, F, G, H), Zu-Yao Xia (D).
Chresonymy.
Gloydiusstrauchi – Li et al. 2004
Holotype.
CIB 119377 (collection number: JZ02, Figs 2–3), аdult female, collected from Zharu Valley, Jiuzhaigou National Nature Reserve, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China (33.26°N, 103.93°E, 2072 m a.s.l.), leg. Chun-Lin Zhao, Peng Yan, and Tao Yang, 8 Jun. 2021.
Paratypes.
Three adult females: IVPP OV 2727, CIB 119378, and CIB 119379 (JZ01, JZ03, and JZ04; Fig. 4), leg. Peng Yan and Mei-Hua Zhang 31 Aug. 2021. Four adult females (SAFS2021001–SAFS2021004), leg. Ping Wang, Jun. 2021; one adult male (CIB 87280), leg. Cheng Li, 23 May 2002. All paratypes were collected from the same locality of the holotype.
Diagnoses.
The above-mentioned specimens were identified as members of the genus Gloydius based on the small body size, bilateral pits, and divided subcaudal scales (Zhao 2006; Shi et al. 2016, 2017, 2018, 2021). G.lateralis sp. nov. differs from other congeneric species by a combination of the following characteristics: (1) relatively larger eyes (ED/HL: 0.145–0.171, n = 5); (2) three palatine teeth; (3) 20 or 21 rows of mid-body dorsal scales; (4) ventrals 151–163 (n = 9); (5) subcaudals 38–49 (n = 6); (6) dorsal body laurel green or light brown with four rows of zigzag, dark brown patches, the medial two rows separated from each other by alternate phyllotaxis pattern in background color; (7) continuous, regular greyish-brown ventrolateral stripe on each side of body and tail.
Comparisons.
Compared to other species in the genus Gloydius, G.lateralis sp. nov. has continuous, regular greyish-brown and pale, greyish-white ventrolateral stripes on both sides (vs disconnected white upper bordered ventrolateral stripes in G.qinlingensis and G.liupanensis; lacking the ventrolateral stripes in other congeneric species), and relatively larger eyes than the congeneric species (the ratio between the eye diameter and head length ranges from 0.145–0.171 in G.lateralis sp. nov. vs < 0.134 in others).
Gloydiuslateralis sp. nov. can be differentiated from the species in the G.blomhoffii group by having three palatine teeth (vs four), from the G.halys complex by having 20 or 21 rows of mid-body dorsal scales (vs 22 or 23).
For species in the G.strauchi group, Gloydiuslateralis sp. nov. can be differentiated from G.monticola by having 20 or 21 rows of mid-body dorsal scales (vs 19 in G.monticola). Given the similar to G.angusticeps, G.lateralis sp. nov. can be differentiated from the latter by the larger eyes (ED/HL 0.145–0.171 vs 0.104–0.119) and the ticker postorbital stripes. Additionally, the ventrolateral stripes sometimes appear in some other Gloydius species, such as G.qinlingensis and G.liupanensis, but G.lateralis sp. nov. differs from them by having the ventrolateral stripe lacking a white upper edge. Gloydiuslateralis sp. nov. differs from G.strauchi, G.rubromaculatus, G.lipipengi, and G.huangi by the triangular head in dorsal view (vs spoon-shaped head: Figs 5, 6).
Gloydiusswild is another species from Heishui, Aba, Sichuan (female holotype IVPP OV 2725 and paratype IVPP OV 2726) that is phylogenetically most closely related to G.lateralis sp. nov., but G.swild can be separated from the latter by significant branch lengths and p-distance (6.1%). G.lateralis sp. nov. differs from G.swild by having fewer ventrals 151–163 (n = 9) (vs 168–170, n = 2), laurel-green dorsal body with deep-colored patches (vs dark, greyish-brown background dorsal color), relatively larger eyes (ED/HL 14.5–17.1%, n = 5 vs 11.5–13.4%, n = 2), the thicker postorbital stripes (2/3 the width of the anterior temporal vs half the width of the anterior temporal), and the regular greyish-brown ventrolateral stripes (vs irregular ventrolateral stripes).
Description of the holotype.
CIB 119377 (JZ02), adult female, body slender, medium-sized, tail short (SVL 440.0 mm, TL 58.0 mm, TL/TTL 0.116). Head triangular in dorsal view, 1.45× the length of the width, distinct from the neck (HW 15.1 mm, HL 21.9 mm, HH 8.3 mm); snout bluntly protruding (SL 6.4 mm) from dorsal view; upper jaw slightly protruding beyond lower jaw; rostral scales barely seem from dorsal view; canthus rostralis blunt; eyes relatively large (ED 3.2 mm), pupil vertical, ED/HL 0. 146. Pupil vertical. Fang not exceeding third infralabial (Fig. 2).
Scalation. Internals wider than long, near right triangular (IN 4.4 mm); prefrontals larger, pentagonal; frontal shield-like; the curve edges of two parietals contacting 13 small scales posterior to frontal and supraoculars; supraocular large, slightly smaller than frontal (IOS 7.8 mm); remaining dorsal head scales smaller posteriorly, first few rows irregular and smooth, gradually rhomboidal and keeled posteriorly. Nasals partially divided into two parts by two disconnected vertical sutures touching rear edge of nostril; two loreals, upper loreal forms part of canthus rostralis, lower loreals distinctly smaller and join pit; preoculars 3/2 (left/right), upmost forms part of canthus rostralis, lower join the pit; postoculars 2/2, upper pair small, lower pair larger, and crescent-shaped, surrounding about one-third of eye, touching third supralabial; temporals 2+3/2+2. Supralabials 6/6: first supralabial in contact with both parts of nasals; second supralabial smallest, fourth and fifth supralabials longest; third supralabial reaching the bottom of orbit; fourth supralabial slightly larger than the following. Infralabials 10/10; first pair extends behind mental, first four pairs narrow and touching chin shields, fifth and sixth infralabials largest, similar in size; one pair of chin shields enlarged, forming a distinct mental groove. Dorsal body scales rhombic with matte surface, keeled except the rows bordering ventrals, increasing in size from medial to lateral; dorsal scales rows 20-20-15; ventral scales 158; anal undivided; subcaudal scales 45 pairs (Fig. 3).
Coloration in life (Figs 2, 4–6). Description based on observation immediately after shedding. Dorsal head gray with distinct smoky-black markings resembling a human in half squat; one gray patch present on middle of frontal; one gray U-shaped marking present on parietals. Lateral head light gray; postorbital stripe otter brown, wider than half of the largest anterior temporal, extending to lateral neck, without white margins; supralabials and infralabials light gray without conspicuous spots; iris bicolored, upper one-third gold, lower part marbled with smoky black; edges of pupil gold. Ventral head white; one faint yellow-orange stripe present on inner edges of infralabials and adjacent edges of contacting scales on both lateral sides of the lower lip. Tone uniformly purple-taupe.
Dorsal body laurel green; two rows of pine-needle colored irregular patches present on dorsolateral body behind head markings, each patch involving several scales (mostly 4–8) on seventh to higher dorsal scale rows, and partially connected or separated by one laurel-green scale; vertebral scales mostly laurel green, forming an alternate phyllotaxis pattern on the body after neck; a row of copper patches present on both sides of lateral body behind postorbital stripe, involving several scales (5–7) on dorsal scale rows 3–6, also partially connected or separated by one laurel-green scale. Ventral body white right behind head, mottled with sparse smoky-black spots, gradually dense to posterior; a distinct, continuous, regular, greyish-brown ventrolateral stripe present on each side of body, behind faint yellow-orange stripe, lie on junction of ventrals and lower edge of first dorsal scales. Dorsal tail smoky black, covered with a dozen of small, laurel-green patches or transverse bands. Ventral tail laurel green with dense, smoky-black spots, continuous, regular, greyish brown, extending from body to middle of ventrolateral tail. Skin between all dorsal scales black. Front edge of most dorsal scales dyed black.
Variations.
Measurements and body scalation variations are listed in Table 1. One of the paratypes, CIB 119378 (JZ03) has a more deeply brown background color than other specimens, dorsal scales on body ginger, and lateral patches on body chestnut; ventrolateral stripes saddle brown. A small, dark-brown spot presents on the middle of the posterior head of one of the paratypes CIB 119378 (JZ03), IVPP OV 2727 (JZ01). Postoculars 3/3 in CIB 87280, IVPP OV 2727 (JZ01); 2/3 in CIB 119379 (JZ04).
Etymology.
The specific epithet lateralis refers to the unique continuous, regular, greyish-brown ventrolateral stripes at the junction of ventrals and the first row of dorsal scales. The common name is suggested as “Jiuzhai pitviper” in English, “Jiǔ Zhài Fù” (九寨蝮) in Chinese, refer to its type locality, JNNR.
Distribution and ecology.
At present, G.lateralis sp. nov. is only known from JNNR, Sichuan, China. The type specimens were collected from the middle of June to the end of August. Gloydiuslateralis sp. nov. is active on sunny days by the roadside in a hot, dry valley (Fig. 8). This species is sympatric with Protobothropsjerdonii, Rhabdophisnuchalis, and Scincellatsinlingensis. The food spectrum of the new species includes small mammals based on a small patch of fur observed in feces. They fed on suckling mice in captivity.
Figure 8.
A, C Macrohabitats and D microhabitats of Gloydiuslateralis sp. nov. at the Jiuzhaigou National Nature Reserve. Note that another Protobothropsjerdonii sympatric to G.lateralis sp. nov. is shown in B.
Discussion
Recent studies continue to improve our understandings of the taxonomy and phylogeny of Asian pitvipers (Xu et al. 2012; Shi et al. 2016, 2017, 2018, 2021; Wang et al. 2019). However, the recent molecular phylogenic trees of Asian pitvipers did not resolve the phylogenic relationship between Gloydiusqinlingensis and G.liupanensis due to the inconsistencies between ML and BI trees. In this study conversely, the topological structures of the ML and BI trees based on five mitochondrial genome fragments are generally consistent. The clades of G.qinlingensis and G.liupanensis form a strongly supported monophyletic group. Furthermore, the molecular phylogeny in this study reveals the sister relationship between Gloydiuslateralis sp. nov. and G.swild, another recently described species from Heishui, Aba, Sichuan (6.1% corrected p-distance for ND4; Shi et al. 2021). The linear distance between the type localities of G.lateralis sp. nov. and G.swild is only 148 km (Fig. 1).
The discovery of G.lateralis sp. nov. provides new insights into the diversity and the distribution patterns of Asian pitvipers. The genetic differentiation from its closest congener, G.swild, might suggest that the formation of the Qinghai-Tibet Plateau might be one of the key factors to the geographical isolation of the alpine pitvipers in southwest China. As discussed by Shi et al. (2021) and authors of many similar studies (Shi et al. 2017, 2018; Wang et al. 2019), the wide-ranging G.strauchi complex spans several biogeographic barriers and distinct environments across poorly investigated regions. The discovery of G.lateralis sp. nov. verifies the hypothesis that there might be additional hidden species within the G.strauchi complex.
The type locality of G.lateralis is located in Jiuzhaigou National Nature Reserve, a world-famous heritage site that receives millions of tourists every year. The only known habitat of the new species is Zharu Valley, and it is now under touristic development. Walkways for tourists have been built in the region, but some people are still venturing off of the walkways. Thus, warning signs are still needed to remind visitors to watch out for the venomous pitviper, since this species and the sympatric Protobothropsjerdonii are often found in grass or bushes on both sides of roads. On the other hand, reptiles are one of the vertebrate groups most affected by roads through vehicle collisions, both because they are intentionally killed by drivers, and due to their biological needs, such as thermoregulation, making them more prone to collisions (Gonçalves et al. 2018). The observation of the dead bodies of G.laterialis shows the necessity to remind the drivers to slow down, and avoid road killings.
Supplementary Material
Acknowledgements
This work was funded by the National Key Research and Development Program of China (2020YFE0203200), the Second Tibetan Plateau Scientific Expedition and Research Program (2019QZKK0705, Tao Deng). Construction of Basic Conditions Platform of Sichuan Science and Technology Department (2019JDPT0020), Jiuzhaigou Post-earthquake Recovery Project: Vegetation Succession and Landscape, Ecology, Environment Protection for Jiuzhaigou World Natural Heritage (5132202020000046), China Biodiversity Observation Networks (Sino BON), and Jiuzhaigou Nature Reserve Comprehensive scientific investigation (Purchase No:5132202019000439). We thank Chun-Lin Zhao, Tao Yang, and Tian Zhao for their help in field surveys; Zhong-Yi Yao, Si-Meng Du, Sheng-Bo Zhou, and Peng Guo for their help with the lab work and corrections of the manuscript. We thank Jia-Tang Li, Ke Jiang, Dong-Sheng Li, and Ke Lv for helping with museum specimen preserving and examining.
Citation
Zhang M-H, Shi S-C, Li C, Yan P, Wang P, Ding L, Du J, Plenković-Moraj A, Jiang J-P, Shi J-S (2022) Exploring cryptic biodiversity in a world heritage site: a new pitviper (Squamata, Viperidae, Crotalinae) from Jiuzhaigou, Aba, Sichuan, China. ZooKeys 1114: 59–76. https://doi.org/10.3897/zookeys.1114.79709
Contributor Information
Jian-Ping Jiang, Email: jiangjp@cib.ac.cn.
Jing-Song Shi, Email: shijingsong@ivpp.ac.cn.
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