Systematic position

Viperidae Gray, 1825

Gloydius Hoge and Romano-Hoge, 1981

Gloydius rubromaculatus sp. n. Shi, Li

and Liu, 2017

ZooBank accession: BF478EA7-C2D6-4F0E-B133-F4D624C3EA2A

Etymology The specific name of the new species is made up of the Latin word “rubro” (red) and “maculatus” (spot), indicating cardinal crossbands on the body. Its common name is suggested to read “Red-spotted alpine pit viper” or “Tongtianhe pit viper” in English and “Hóng Bān Gāo Shān Fù ( )” in Chinese.

Holotype and paratypes Holotype: IOZ032317, adult male, collected by Jingsong Shi (JS) and Xi’er Chen (XC) from the mid-upper reaches of the Tongtianhe River, Qumarleb, Qinghai Province, on 9 July 2016. Paratypes: NWIPB790058 (allotype, adult female), IOZ 032318 (adult male), JS1607Y3 (subadult female), NWIPB790056 (adult female), NWIPB 630064 (adult female), and NWIPB0512 (adult male). Referred specimens: NWIPB 17092:1, from Jyekundo, Qinghai Province; NWIPB 790 060-790 067, from Zhiduo, Qinghai Province. See table 2 for detailed information. Note: some of the specimens of the old species are labelled with “G. strauchi” which are identified as G. rubromaculatus sp. n. in this study.

Diagnosis The above mentioned specimens were identified as the members of Gloydius judging on their small body size, bilateral pits and divided subcaudal scales (Hoge and Romano-Hoge, 1981), while differ from other congeneric species in the following characteristics: 1. two rows of cardinal crossbands on the back, regularly spaced along the body; 2. glossy dorsal scales, compared to matte scales in other members of Gloydius; 3. colubrid-liked dome shaped head in lateral view and oval shaped in dorsal view, compared to flat-shaped head in lateral view and triangular in dorsal view in other Gloydius; 4. irregular small black spots dispersed on the head scales; 5. inconspicuous canthus rostralis; 6. dark brown eyes with black pupils (figs 1 and 2). Figure 1. Download Figure

Download figure as PowerPoint slide 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). Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134 Figure 2. Download Figure

Download figure as PowerPoint slide 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. Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134 The new species is distinct from species in the Gloydius blomhoffii complex by having three palatine teeth (versus four palatine teeth); from the species in the Gloydius halys-intermedius complex by having 21 rows of mid-body dorsal scales (versus 23-rows). Thus, the new species is suggested to belong to the Gloydius strauchi complex. Within the Gloydius strauchi complex, G. rubromaculatus sp. n. is distinct from G. monticola by its 21-rows mid-body scales (versus 19-rows mid-body scales and 6 supralabials); from G. himalayanus by its indistinct canthus rostralis (versus very distinct canthus rostralis); from G. qinlingensis and G. liupanensis by its oval head (versus triangular head), regular crossbands (versus irregular crossbands) and the lack of the white line on each side of body (versus obvious white line); from G. strauchi by its brownish black eyes (versus light brown eyes), and two rows of regular round crossbands (versus four irregular longitudinal strips). See table 3 and fig. S1 for the morphological comparisons within the Gloydius strauchi complex. The validity of this new species is also supported by molecular genetics. Three samples of G. rubromaculatus sp. n. (Y2, Y4 and Y5) form a strongly supported monophyletic group. In addition, the corrected ND4 p-distance between G. rubromaculatus sp. n. and other species is greater than the ones between most of the other species (8.6-12.4 percent, see online supplementary table S3).

Description of the holotype Adult male, a small slender pit viper with a total length of 554 mm (body length 473 mm and tail length 81 mm), preserved in 75% ethanol with its bilateral hemipenis extruded. The head is spoon-shaped in dorsal view and dome-shaped in lateral view, 24.6 mm in length, 15.4 mm in width and 7.4 mm in height. Rostral scale slightly turns up to the upper side of the head. Seven bilateral supralabials are presented: the second one smallest, not reaching the pit; the third and fourth largest, with the former extending to the bottom of orbit. Three preoculars, two postoculars, and two rows of temporals (2 + 4). Ten infralabials are on the left while nine on the right, of which the first pair contact behind the mental, the second and third ones meet the chin shield. Mental groove is made up of paired parallelogram chin shields, which extend to the mental. The canthus rostralis are not distinct. The head has a clear border with the neck. The eyes are brownish black, with vertical spindly, light brown margined black pupil. A thick, black bordered yellowish red cheek stripe extends from the posterior side of each eye (separated from the orbit by the inferior postocular) to the first pair of neck crossbands, throughout the inferior temporal and the last two supralabials. Lots of irregular, uneven-sized black spots dispersed on most of the head scales, except for the temporals (figs 1 and 2). Mouth lining is pink in life. The anterior part of the tongue is black while the base is pink. Table 3. Detailed comparison between G. rubromaculatus sp. n. and the remaining species of the Gloydius strauchi complex. The body colour is light greyish yellow, with a column of complete regular round cardinal crossbands on each side of the back, black bordered and shallow inside, in pairs or interfaces, about three to four scales in length, and four to six scale rows in width, separated by blank areas one or two scales in width, extending down to lateral one or two ventral scales. Crossbands range from the neck to the tip of the tail, 49/46 on body and 12/16 on the tail (left/right). A range of triangular or irregular black ventrolateral blotches (irregular one is made up of the adjacent two or three ones) range on the boundary between back scales and ventrals on each side of the body, divided by one or two ventral scales (fig. 1). Ventral scales are opalescent, with mottled irregular black blotches. The black blotches are concentrated on the middle of scales. The edge and the junction of bilateral subcaudals are trimmed in black thread. One semicircular black blotch is present on the lateral edge of each subcaudal scale. A black stripe is present on each side of the boundary of the two bilateral ventrals. The tip of the tail is black and bony. Dorsal scales are in 21-21-15 rows (reduce from 21 to 15 rows beginning with the ventral 92/93), keeled (excluding the ones bordering the ventral scales) and glossy. Ventrals 158 (excluding three preventral scales). Anal plate is complete. Subcaudals divided in pairs (43 pairs). See table 2 for the detailed measurements of the specimens examined.

Skull The frontal of G. rubromaculatus sp. n. is inverted triangular in dorsal view, contrast to round in G. strauchi, and “T”-shaped in G. shedaoensis. The lateral margin of nasal is rounded, without any process, while in G. strauchi the lateral process is distinct. The fang is quite short, approximately one third length of the ectoptery (versus approximately a half-length of the ectoptery in G. halys-intermedius complex and G. blomhoffii complex; Gloyd and Conant, 1990). Six replacement fangs behind each primary fang, three palatine teeth, 12 pterygoid teeth and 11 alvenlus teeth on each side. The upper edge of postfrontal is in contact with the posterior-lateral process of the frontal, versus separated in G. strauchi (Gloyd and Conant, 1990). The quadrate is quite slender, about 1.2 times as long as the squamosal, versus almost equal to the squamosal in length in G. strauchi (fig. 3). Figure 3. Download Figure

Download figure as PowerPoint slide 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. Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134

Hemipenes The hemipenes of G. rubromaculatus sp. n. are generally similar to those of G. strauchi but differ by the podgier spines. Eight subcaudals in length, forked for two subcaudals. Small stubby spines range from the basal to the distal side of the organ, without any enlarged spines (versus three to five enlarged spines on the base in Gloydius halys complex; Gloyd and Conant, 1990). The spines gradually increase in size distally. More spines occur on the asulcate side on than the sulcus side. Spines and sulcus merge with the calyces on the distal side. No giant spines present on the basal of the hemipenis as Gloydius halys-intermedius complex (fig. 4). Figure 4. Download Figure

Download figure as PowerPoint slide Asulcate side (A) and sulcus side (B) of the right hemipenis of G. rubromaculatus sp. n. (holotype, Y2). Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134

Infraspecific morphological variation Some morphological variations are present within the specimens for this study: some of the G. rubromaculatus sp. n. have large, complete scarlet crossbands, while other have small irregular ones (e.g., Y4), or large brown regular crossbands (e.g., Y7) (fig. 1). Body colour is mostly grayish white, sometimes brownish yellow (Y9). Spots are mostly present on the head scales, but some have none or only a few small ones. Most have seven supralabials (rarely eight) and 10 infralabials (rarely 9 or 11, table 2). Ventrals range from 146 to 158 in males (mean 152.3, n = 3 ) while 153 to 163 in females (mean 159.5, n = 4 ). Subcaudals range from 41 to 43 in males (mean 42.3, n = 3 ), and 35 to 43 in females (mean 40, n = 4 ). See table 2 for the detailed measurements of the specimens examined.

Phylogenetic and phylogeographic analysis The validity of the new species is supported by phylogenetic analyzis, the topological structure of the maximum likelihood (ML) and Bayesian inference (BI) trees are approximately identical except for the clade of G. stejnegeri, G. rickmersi and G. caraganus (fig. 5). All members of Gloydius perform as a monophyletic group. The clade of the new species from along the Tongtianhe river (Y2, Y4 and Y5) performs as a strongly supported monophyletic group (Clade A, in red), and constitutes sister groups with the clade of G. monticola from Yunnan (Clade B). Despite the geographic proximity with G. strauchi, G. rubromaculatus’s group does not perform as sister groups with G. strauchi (Clade C). Thus, the new species is more closely related to G. monticola than to G. strauchi. The samples of G. qinlingensis (Clade D) and G. liupanensis (Clade E) from near the Yellow River do not present as sister groups, moreover, the p-distance between them is relatively greater (7.2 percents for ND4) than the ones between other congeneric species, thus they should be regarded as distinct species. In general, the samples of the Gloydius strauchi complex, including qinlingensis (Shaanxi), liupanensis (Ningxia), monticola (Yunnan), strauchi (Sichuan) and rubromaculatus sp. n. (Qinghai) do not completely constitute a monophyletic group as the Gloydius halys-intermedius complex (Clade F) and Gloydius blomhoffii complex (Glade G). Figure 5. Download Figure

Download figure as PowerPoint slide 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 “∗”. Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134 The validity of G. rickmersi Wagner, Tiutenko, Mazepa, Simonov, Borkin, 2016 is reconfirmed in this study. On the other hand, the populations distributed in Jiaodong Peninsular (Shandong Province) had long been regard as a subspecies of G. intermedius (G. i. changdaoensis Li, 1999), however, the molecular phylogeny shows a significant distance between G. i. changdaoensis and G. intermedius (p-distance: 5.4 percent), the clade of changdaoensis (Z1 and C1) does not constitute as sister groups with G. intermedius (SX1, 22, Q4 and QS002), but firstly separates from the remaining taxa of the G. halys-intermedius complex. Therefore, G. i. changdaoensis should be regarded as a full species, but not a subspecies of G. intermedius. Additionally, the samples of the different subspecies of G. halys (e.g. caraganus, stejnegeri and cognatus) do not performed as a monophyletic group, and none of them performed as sister groups with G. h. halys, allowing for the high p-distance between the different subspecies, this study fully shares the taxonomy of Shi et al. (2016), suggesting that G. changdaoensis, G. caraganus, G. stejnegeri and G. cognatus should be elevated as full species. (See Orlov and Barabannov, 1999 and Shi et al., 2016 for the detailed comparisons between different subspecies of G. halys.)

Diet We checked the faeces samples of four snakes (Y1, Y2, Y5 and Y6). The faeces contain almost entirely the debris of moths, such as undigested bodies, siphoning mouthparts, wings, periopods and arms of the furcas (supplementary fig. S2). One of the moths could be identified as Sideridis sp. (female). No hairs, bones or feathers of birds or mammals can be found in the faeces yet. Two undigested neonate zokors (Eospalax fontanierii) are found in another specimen’s stomach (NWIPB 630064). Two juvenile pit vipers are observed to prey on moths and pink mice in captivity (Y1 and Y5). Two hypotheses can account for the moth debris: they may be from the moths preyed and excreted directly by snakes; or alternatively, these items were secondarily ingested along with the primary prey items, frogs and lizards, which are more routinely found to be insectivores compared to snakes. However, one of the snakes (Sample Y3) was observed to vomit a whole undigested moth, which could confirm the former hypothesis.

Distribution and habitat Gloydius rubromaculatus sp. n. is distributed mainly along the Tongtianhe River, in the Sanjiangyuan region of Qinghai Province. Additionally, G. rubromaculatus sp. n. is also found in Tibet (Tongpu village, Jiangda Country) and Sichuan (Shiqu Country) (table 1 and supplementary fig. S3). The distribute altitude ranges from 3300 to 4770 m. G. rubromaculatus sp. n. holds the highest snake distribution report within Chinese venomous snakes, and the second highest one all over the world, next to G. himalayanus, which can occur up to 4880 m (Sharma et al., 2013). Gloydius rubromaculatus sp. n. tends to stay in the mountain passes, sandy riversides, sunny slopes, bushes and shales (fig. 6). Figure 6. Download Figure

Download figure as PowerPoint slide Habitat of Gloydius rubromaculatus sp. n. (type locality), along the Tongtianhe River, Qumarleb Country, Qinghai Province, 4154 above sea level. Citation: Amphibia-Reptilia 38, 4 (2017) ; 10.1163/15685381-00003134