A new species of Oligodon Fitzinger, 1826 from the Langbian Plateau, southern Vietnam, with additional information on Oligodon annamensis Leviton, 1953 (Squamata: Colubridae)

Nomenclatural acts

The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone (see Articles 8.5–8.6 of the Code). This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information can be viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is as follows: urn:lsid:zoobank.org:pub:51B851C2-5D34-4065-86EA-CF18DDD94419. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Sampling. The adult male specimen of Oligodon sp. was collected by Bang Van Tran and Linh Hoang Nguyen during the field trip in June 2017 in Bidoup–Nui Ba National Park (hereafter NP), Lam Dong Province, Vietnam (locality 1; Fig. 1). After euthanasia with 20% solution of benzocaine, the specimen was initially preserved in 95% alcohol for one day then subsequently stored in 70% alcohol. Additional specimens of Oligodon annamensis were collected in Chu Yang Sin NP, Dak Lak Province, southern Vietnam, by Nikolay A. Poyarkov (locality 3; Fig. 1); and in Phnom Samkos Wildlife Sanctuary (hereafter WS) of the Cardamom Mountains, Pursat Province, southwest Cambodia by Seiha Hun (locality 4; Fig. 1); both records made in April, 2012. Geographic position of the surveyed localities is shown in Fig. 1. Because of certain morphological similarity with Oligodon annamensis in coloration and scalation (Orlov et al., 2010), we also included sequences of O. lacroixi in our phylogenetic analysis based on a specimen from Phu Tho Province, northern Vietnam.

Specimen collection protocols and animal operations followed the Institutional Ethical Committee of Southern Institute of Ecology, Vietnamese Academy of Science and Technology (certificate number 114/QD-STHMN of November 8, 2016).

Field work, including collection of samples and animals in the field, was authorized the Bureau of Forestry, Ministry of Agriculture and Rural Development of Vietnam (permits Nos. 170/ TCLN–BTTN of 07/02/2013; 400/TCLN-BTTN of 26/03/2014; 831/TCLN–BTTN of 05/07/2013) and Forest Protection Department of the Peoples’ Committee of Dak Lak Province (permit No. 388/SNgV-LS of 24/04/2019); the fieldwork in Bidoup–Nui Ba NP was conducted under scope of the contract between Sustainable Nature Resource Management Project (SNRM) under Japan International Cooperation Agency and Southern Institute of Ecology to perform the “Biodiversity Baseline Survey” project of September 24, 2018.

Morphological analysis. Color characters and patterns were recorded during examination of specimens in life and taken from digital images of the living specimens. Morphological characters and morphometric ratios considered to be of taxonomic importance for Oligodon were used for species descriptions and followed a number of recent revisions of the genus (David, Das & Vogel, 2008; David et al., 2012; Leviton, 1953; Leviton, 1960; Neang & Hun, 2013; Nguyen et al., 2016; Nguyen et al., 2017; Vassilieva et al., 2013; Vassilieva, 2015). All body measurements, except body and tail lengths, were taken under a binocular microscope using digital slide-caliper to the nearest 0.1 mm. Body and tail lengths were measured to the nearest millimetre with a measuring tape. The right hemipenis was forcedly everted by using water injection prior the preservation of the specimen. Methodology of ventral and subcaudal scales counts followed Dowling (1951). Terminology for hemipenial structures generally followed Smith (1943) and Dowling & Savage (1960). Maxillary teeth of the specimens were counted by examining both maxillae, directly by pushing back the soft tissue with a needle under binocular microscope prior to preservation.

The following measurements (all in mm) and counts were taken: snout to vent length (SVL)—measured from the tip of the snout to the vent; tail length (TaL)—measured from the vent to the tip of the tail; total length (TL)—sum of SVL and TaL; relative tail length to total length (RTL) calculated as tail length to total length ratio (TaL/TL); head length (HL) from the tip of the snout to the posterior margin of the mandible; head width (HW) measured at the widest part of the head immediately posterior to the eye; head width to head length ratio (HW/HL); snout length (SnL)—distance between the tip of the snout and anterior edge of eye; eye diameter (EyeL)—maximal horizontal length of the eye; frontal scale length/width (FrL/FrW)—length and width of the frontal scale; interorbital distance (IOD)—the shortest distance between the eyes; internarial distance (IND)—distance between the nostrils; number of maxillary teeth (DEN); dorsal scale rows at neck (ASR)—number of scale rows at one head length behind the head; midbody scale rows (MSR)—number of scale rows at midbody; dorsal scale rows anterior to the vent (PSR)—number of dorsal scale rows at one head length prior to the vent; dorsal scale rows formula (DSR)—referred to as a general scale formula in the form “ASR-MSR-PSR” (for number of dorsal scale rows at neck, midbody and prior to vent, respectively); first dorsal scale reduction (RED1)—the first reduction of dorsal scale rows, corresponding to a ventral scale; ventral scales (VS)—number of scales from the second ventral scale posterior to gulars to the vent excluding cloacal plate; cloacal plate (AP)—number of terminal ventral scales immediately anterior to vent; subcaudal scales (SC)—number of paired subcaudal scales excluding the terminal scute; total belly scales (Total Sc.)—sum of ventral and subcaudal scales; supralabials (SL)—number of scales on upper lip; SL-Eye—number of SL entering orbit; infralabials (IL)—number of scales on lower lip; infralabials contacting each other (IL-contact)—number of pairs of infralabial scales in contact; infralabials contacting the anterior chin shields (IL-CS)—infralabial scales contacting the upper chin shields; number of preocular scales (PrO); number of presubocular scales (PrsO); number of postocular scales (PtO); number of anterior temporals (Ate)—temporal scales which contact the postocular scales; number of posterior temporals (Pte)—temporal scales immediately contacting the anterior temporal scales; condition of loreal scale (LOR)—1–present, 0–absent, *–vestigial; condition of nasal scale (NAS)—D–vertically divided, E–entire, PD–partially divided; hemipenis shape—(1) unforked, a single organ with no lobes at apex; (2) bilobed, organ contains two lobes at its apex; hemipenis ornamentation—notes on ornamentation of organ (i.e., spinules, calyces or flounces); presence of appendages seen in situ (papillae sensu Smith, 1943); hemipenis length—length of the everted hemipenis in mm and relative to number of subcaudal scales. Symmetric characters are given in left/right order. Other abbreviations: a.s.l.: above sea level; Div.: Division; Comm.: Commune; Dist.: District; Mt.: mountain; NP: National Park; NR: Nature Reserve; Prov.: Province; WS: Wildlife Sanctuary.

The type material was deposited in the herpetological collection of the Department of Zoology, Southern Institute of Ecology (SIEZC) in Ho Chi Minh City, Vietnam. Additional material used for comparisons is stored in the herpetological collections of Centre for Biodiversity Conservation of the Royal University of Phnom Penh, Phnom Penh, Cambodia (CBC RUPP); United States National Museum, Washington, D.C., USA (USNM); Museum National d’Histoire Naturelle, Paris, France (MNHN) and Zoological Museum of Lomonosov Moscow State University, Moscow, Russia (ZMMU).

Molecular analyses. Total genomic DNA was extracted from muscle tissue preserved in 95% ethanol using the Qiagen DNAeasy Blood & Tissue Kit following manufacturers’ protocol. We used the polymerase chain reaction (PCR) to amplify two fragments of mitochondrial DNA (hereafter mtDNA): the first fragment including partial sequences of 12S ribosomal RNA (rRNA), tRNA-Valine and 16 rRNA genes (total length up to 2035 bp) and a complete sequence of cytochrome b gene (1,096 bp). Primers used for both PCR and sequencing are summarized in Table 1.

PCR protocol for 12S–16S rRNA mtDNA fragment in general followed Green, Orlov & Murphy (2010). For both primer pairs of 12S and 16S rRNA, we used the following PCR protocol: (1) initial denaturation step at 94 °C for 5 min; (2) 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min and extension at 72 °C for 1 min; (3) final extension at 72 °C for 10 min; and (4) cooling step at 4 °C for storage.

For cytochrome b sequences (fragment up to 1,096 bp) we used a modified PCR protocol of Dahn et al. (2018) with touchdown: (1) initial denaturation step at 94 °C for 5 min; (2) 10 cycles of denaturation at 94 °C for 1 min, annealing for 1 min with temperature decreasing from 50 °C to 45 °C (with cool-down at 0.5 °C per each cycle) and extension at 72 °C for 1 min; (3) 24 cycles of denaturation at 94 °C for 1 min, annealing at 45 °C for 1 min and extension at 72 °C for 1 min; (4) final extension at 72 °C for 10 min; and (5) cooling step at 4 °C for storage.

All PCR products were sequenced in both directions by Genomics BioSci & Tech Corp. (Taipei, Taiwan). Sequences were assembled and checked using sequencher 4.9 (GeneCodes). The obtained sequences are deposited in GenBank under the accession numbers MN395601–MN395604 and MN396762; MN396765 (Table 2).

Phylogenetic analyses. The 12S–16S rRNA datasets of Green, Orlov & Murphy (2010), Pyron et al. (2013), our newly obtained sequences and other Oligodon sequences available in GenBank were used to examine the position of the Lam Dong Oligodon sp. in the matrilineal genealogy of the genus (summarized in Table 2). In total, we analysed mtDNA sequence data for 52 specimens, including 43 samples of ca. 24 species of Oligodon, and eight outgroup sequences of other Colubrinae representatives, and sequences of Hebius vibakari (Natricinae) which were used to root the tree.

Nucleotide sequences were initially aligned in MAFFT v.6 (Katoh et al., 2002) with default parameters, and subsequently checked by eye in BioEdit 7.0.5.2 (Hall, 1999) and slightly adjusted. MODELTEST v.3.6 (Posada & Crandall, 1998) was applied to estimate optimal evolutionary models for the data set analysis. Mean uncorrected genetic distances (p-distances) were calculated in MEGA 6.0 (Tamura et al., 2013).

The matrilineal genealogy was inferred using Bayesian inference (BI) and Maximum Likelihood (ML) approaches. The best-fitting model for both BI and ML analyses for 12S–16S rRNA fragments was the GTR+G+I model as of DNA evolution suggested by the Akaike Information Criterion (AIC); for cyt b gene AIC suggested GTR+G model for first and third codon partitions, and HKY+G+I for second codon partition. BI was conducted in MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003); Metropolis-coupled Markov chain Monte Carlo (MCMCMC) analyses were performed run with one cold chain and three heated chains for twenty million generations and sampled every 2000 generations. Five independent MCMCMC run iterations were performed and 1000 trees were discarded as burn-in. The convergence of the runs were checked by exploring examining the likelihood plots in TRACER v1.6 (Rambaut et al., 2014); the effective sample sizes (ESS) were all above 200. Nodal support was assessed by calculating posterior probabilities (BI PP).

ML was conducted using the RAxML web server (http://embnet.vital-it.ch/raxml-bb/; Kozlov et al., 2018). Confidence in nodal topology was estimated by non-parametric bootstrapping (ML BS) with 1000 pseudoreplicates (Felsenstein, 1985).

We a priori regarded tree nodes with BI PP values over 0.95 and ML BS values 75% or greater as sufficiently resolved; while BI PP values between 0.95 and 0.90 and ML BS values between 75% and 50% were regarded as tendencies. Lower values were regarded as indicating unresolved nodes (Huelsenbeck & Hillis, 1993).

Phylogenetic relationships of Oligodon

Sequence and statistics. The final concatenated alignment of the 12S rRNA –16S rRNA fragment and cyt b gene sequences contained 3131 base pairs, of which, 1959 sites were conserved and 1049 sites were variable, of which 713 were found to be parsimony-informative. The transition–transversion bias (R) was estimated as 1.89. Nucleotide frequencies were 38.0% (A), 22.0% (T), 24.5% (C), and 15.4% (G) (all data given for ingroup only).

MtDNA-based genealogy. Our mtDNA-based genealogy for the genus Oligodon (Fig. 2) inferred the following set of phylogenetic relationships, which is generally consistent with the results of Green, Orlov & Murphy (2010). Several well-supported clades were recovered within Oligodon (see Fig. 2):

1. Clade 1: Indian and Sri Lankan species (O. taeniolatus, O. calamarius, O. sublineatus; 1.0/100; hereafter node support values are given for BI PP/ML BS, respectively); O. arnensis from the same region tends to group with this clade, however with no node support (0.52/-).

2. Clade 2: Species from northern Vietnam (O. lacroixi and O. eberhardti) (1.0/100).

3. Clade 3: Joining the O. cinereus species group (Indochina and Myanmar), and some taxa from Myanmar (O. splendidus, O. theobaldi, O. cruentatus, O. torquatus, O. planiceps) and Philippines (O. maculatus) (1.0/100).

4. Clade 4: Joining other species of Oligodon from Indochina and southern China, clustered in the O. taeniatus species group (O. taeniatus and O. barroni; 1.0/98) and the O. cyclurus species group (O. cyclurus, O. formosanus, O. chinensis and O. ocellatus; 1.0/98).

5. The newly discovered Oligodon sp. from Bidoup–Nui Ba NP is reconstructed as a sister lineage with respect to two specimens of O. annamensis from Vietnam and Cambodia (1.0/100); O. octolineatus from Sundaland tends to group with this clade, however with no node support (0.62/-). All these species are clustered together with Clade 4 with strong support (1.0/100) (see Fig. 2).

Sequence divergence. The uncorrected p-distances for the 16S rRNA gene fragment among and within examined Oligodon species are presented in Table 3. Intraspecific distances varied significantly and ranged from p = 0% in a number of examined species to p = 2.3% in the O. cinereus complex and p = 2.8% in the O. cyclurus complex, what is most likely explained by incomplete taxonomy of these groups (Green, Orlov & Murphy, 2010; David, Das & Vogel, 2008; David et al., 2012); a more detailed study including topotype materials on these species complexes is required.

The interspecific genetic distances within examined Oligodon varied from p = 1.8% (between O. chinensis and O. formosanus) to p = 8.5% (between O. maculatus and O. octolineatus) (Table 3). The newly discovered Oligodon sp. lineage from Bidoup–Nui Ba NP is highly divergent from other congeners and is most closely related to O. annamensis with p = 3.3% of sequence divergence in 16S rRNA gene between these taxa. This divergence value is notably higher than the genetic differentiation between many other recognized Oligodon species (see Table 3), thus suggesting that the divergence between Oligodon sp. and O. annamensis likely reached species status. Genetic divergence between Vietnamese and Cambodian populations of O. annamensis is minimal and comprised p = 0.9% of substitutions (Table 3).

Systematics

Our mtDNA-genealogy of Oligodon demonstrated that Oligodon sp. from Bidoup–Nui Ba NP represents a new previously unknown lineage, sister to O. annamensis; both species are clustered with the O. taeniatus and O. cyclurus species groups with strong support. Though genetic divergence between Cambodian and Vietnamese populations of O. annamensis, separated from each other by over 600 km distance, is small (p = 0.9%); genetic differentiation between Oligodon sp. from Bidoup–Nui Ba NP and O. annamensis is much higher (p = 3.3%) and reaches species-level (see Table 3). We thus confirm the identification of the Cambodian population as O. annamensis (previously described by Neang & Hun, 2013), and also provide a morphological analysis of all presently known specimens of O. annamensis (see Table 4). Our results are further corroborated by our morphological analysis (see below), which uncovered significant differences between Oligodon sp. from Bidoup–Nui Ba NP, O. annamensis and other congeners. These results support our hypothesis that this recently discovered lineage of Oligodon represents an undescribed species, which we describe below:

Oligodon rostralis sp. nov.

(Figures 3–7; Tables 4–5)

Holotype. SIEZC 20201, adult male from Bidoup–Nui Ba National Park, ca. 6 km northwards from Da Nhim village, Da Chais Commune, Lac Duong District, Lam Dong Province, southern Vietnam (12.1518°N, 108.5279°E; elevation 1,622 m a.s.l.), collected on a steep slope near to mountain summit in montane evergreen pine forest by Bang Van Tran and Linh Hoang Nguyen at 23 h on June 13, 2017.

Diagnosis. The new species is assigned to the genus Oligodon Fitzinger, 1826 on the basis of its phylogenetic position and the following morphological attributes: posterior maxillary teeth enlarged and compressed; head short, barely distinct from neck; eye well-developed with round pupil; rostral enlarged; body cylindrical with smooth scales; ventrals rounded; subcaudals paired. Oligodon rostralis sp. nov. is distinguished from its congeners by a combination of the following morphological characters: medium size in adults (male TL = 582 mm); head small and broad with long largely protruding snout; 15 dorsal scale rows at neck and midbody and 13 rows before vent; ventrals 167, subcaudals 47 in male; single preocular, single postocular; loreal and presubocular absent; six supralabials, third and fourth entering orbit; six infralabials, anterior four contacting chin shields; internasals separate from prefrontals; nasal divided; single anterior and two posterior temporals; cloacal plate undivided; comparatively short hemipenis, forked in anterior one third of their length, extending to 8th subcaudal, lacking spines and papillae, bearing prominent transverse flounces and distal calyces; six maxillary teeth, the posterior three being enlarged; dark temporal streak present, edged with white; 14+4 large dark-brown dorsal blotches; bright-orange vertebral stripe on tail and dorsum; and ventral surfaces in life cream with quadrangular spots.

Description of holotype. Measurements and scale counts of the holotype are presented in Table 4. Adult male of medium size (TL 582 mm), body robust and cylindrical (Fig. 3); SVL 468 mm; head small, comparatively short and wide (HW/HL = 73.2%), ovoid in dorsal view, faintly distinct from the poorly defined neck; tail quite long (19.6% of total length), 114 mm in length; robust, abruptly tapering; eye small, comprising approximately 13.5% of the head length; snout long, protruding (SnL/HL ratio 43.6%); eye diameter much shorter than the distance between eye and nostril; pupil round;

Body scalation. Dorsal scales smooth, in 15-15-13 rows, scale row reduction from 15 to 13 at ventral 113; vertebral scales similar to other dorsal scales in size and shape; outermost dorsal scales slightly enlarged; 167 ventrals; cloacal plate entire; 47 subcaudals, all paired, terminal caudal scale in a shape of sharply pointed cap (Fig. 3B).

Head scalation. Details of head scalation are shown in Fig. 4. From dorsal view (Figs. 4A–4B), head scalation comprising single rostral, two internasals, two prefrontals, two supraoculars, single frontal, and two parietals. Rostral large, thick, wider than high, extending on to the dorsal surface of the snout, visible from above, pointed posteriorly and inserting deeply between internasals, with a deep crease ventrally, contacting nasals, internasals and first supralabial on both sides; the portion of rostral visible from above shorter than its distance from frontal; internasals sub-rectangular, in broad contact, shorter than prefrontals, each contacting rostral, prefrontal, internasal and paired nasals on both sides; prefrontals large, pentagonal, wider than long and larger than internasals, curving dorsolaterally into loreal region, each contacting internasal and posterior portion of nasal anteriorly, second supralabial laterally, and preocular, supraocular and frontal posteriorly; supraoculars subrectangular, elongated, widening posteriorly, approximately half as wide as long, contacting the orbit, preocular and postocular laterally, prefrontal, frontal and parietal medially; frontal large, pentagonal, longer than wide, narrowing posteriorly, posterior angle rather acute, contacting prefrontals, supraoculars and parietals on both sides; parietals irregularly trapeziform, about 1.5 time larger than frontal, anteriorly contacting frontal, supraoculars and postoculars on each side, bordered posteriorly by five small scales and laterally by the first and upper second temporals; no enlarged nuchal scales present.

In lateral view (Figs. 4C–4D), head scalation comprising a sub-rectangular nasal, vertically divided by prominent suture and pierced by large nostril, nasal on each side contacting rostral anteriorly, internasal and prefrontal dorsally, and first two supralabials ventrally; loreal and presubocular scales absent; 1/1 rectangular preocular, notably higher than wide, separated from nasal by the lateral part of the prefrontal, also contacting second and third supralabials ventrally and supraocular dorsally; 1/1 rectangular postocular, almost equal in size to preocular, contacting fourth and fifth supralabials ventrally, anterior temporal and parietal posteriorly and supraocular dorsally; six supralabials: I. the smallest, in contact with nasal, II. in contact with nasal, prefrontal and preocular, III. in contact with preocular and the orbit, IV. in contact with the orbit and postocular, V. in contact with postocular, anterior temporal and lower posterior temporal, VI. in contact with lower posterior temporal and scale dorsally, and with two smaller scales posteriorly, V. and VI. strongly enlarged; supralabial scale size formula: I<II<III=IV<V<VI; 1+2 temporals on each side, the upper ones pentagonal, elongated and narrow, upper posterior temporal slightly larger than the anterior, the lower posterior temporal rhomboid, ca. two times smaller than the upper ones, posteriorly contacting an enlarged scale of same size.

In ventral view (Figs. 4E–4F), 6/6 infralabials: I. in contact with mental anteriorly, in contact with each other medially; anterior three in contact with anterior chin shield; the fourth largest and touching posterior chin shield; mental small, triangular; 2/2 enlarged, elongated chin shields, anterior pair twice as long as the posterior pair; three small gular scales between posterior chin shields and first preventral.

Dentition. Maxillary teeth 6, curved posteriorly, smaller and shorter anteriorly; posterior three being notably enlarged, flattened and kukri-shaped.

Hemipenial morphology. Right hemipenis was everted prior to preservation and is shown in Fig. 5. Hemipenis rather short, the everted organ hardly reaching 8th subcaudal; bilobed, bifurcating at distal fifth of its length; organ semi-capitate and semi-calyculate; the sulcus spermaticus is bifurcated at around the proximal one-fifth of the hemipenial body and centrolineal along both lobes (Fig. 5A). The sulcal surface of hemipenis is mostly smooth (Fig. 5A), laterally and on asulcal surface hemipenis covered with several fleshy flounces, lacking spines or papillae-like structures (Fig. 5B); distal ends of hemipenial lobes with small indistinct calyces.

Colouration (in life). Dorsal ground color (Fig. 6A) dark brownish-gray with dense white reticulation between scales; dorsal pattern consisting of 18 large irregular blackish butterfly-shaped blotches, of which 14 are located on body and 4 on tail, the distances between two blotches comprises ca. 8–10 dorsal scale lengths; a bright orange vertebral stripe running from the base of the head to the tail tip; vertebral stripe width comprising from one to three dorsal scale rows; some dorsal scales edged with dark-brown forming an indistinct speckled or dashed pattern between blotches, lower rows of dorsal scales fringed with white. Dorsal ground color along head is grayish-brown (Fig. 6B), a butterfly-shaped marking with rusty tint with a rounded dark spot located on frontal, three separated dark-brown chevrons (one short between the eyes, forming two dark brown streaks running across the eye to the angle of the mouth; and two longer ones running from frontal postero-ventrally to neck and posteriorly to the base of the head, respectively); throat and ventral underside pale-cream laterally with alternating quadrangular black spots scattered from throat until tail (Fig. 7A); underside of tail orange-cream.

Colouration (in preservative). (Fig. 3), after two years in alcohol, coloration faded but pattern remained unchanged; body brown, vertebral stripe became somewhat dark-orange and less distinct (Fig. 3, A); dorsal blotches and head marking dark brown with blackish margins remained unchanged; throat, venter and tail underside cream-white, black quadrangular spots remained unchanged (Fig. 3B).

Etymology. The specific name “rostralis” is a Latin adjective in the nominative singular, masculine gender, derived from Latin words “rostrum” for “snout” or “beak” in reference to protruding snout distinctive for the new species. We suggest the following common names for the new species: “Long-snouted kukri snake” (English), “Răn khiêm mõm dài” (Vietnamese), and “Dlinnorylyi oligodon” (Russian).

Distribution. At present the new species is known only from the type locality in Bidoup–Nui Ba NP, in the eastern part of Langbian Plateau, southern Vietnam (see Fig. 1, locality 1). This montane area is characterized by high levels of local endemism (Nazarov et al., 2012; Poyarkov et al., 2014; Poyarkov et al., 2015a; Poyarkov et al., 2015b; Poyarkov et al., 2017; Poyarkov et al., 2019b; Stuart et al., 2011; Rowley et al., 2016); further research is needed to clarify the distribution of the new species.

Habitat and natural history. The type specimen was collected on the steep slope close to the mountain summit (Fig. 7), at late night (23 h). The animal was found on ground in leaf litter on the edge of the mixed-pine forest (dominated by Pinus keysia Royle ex Gordon) and evergreen montane broadleaf forest (dominated with trees of the families Fabaceae, Fagaceae, and few large pine trees of Pinus keysia, with understory consisting mostly of Poaceae –different species of bamboo) (Fig. 7B). In the pine forest, understory is dominated by Fagaceae family while ground is covered mostly by grasses and receives high grazing impact by livestock from the villages nearby. In the type locality the new species was recorded in sympatry with some other species of reptiles, including Cyrtodactylus bidoupimontis Nazarov, Poyarkov, Orlov, Phung, Nguyen, Hoang & Ziegler, Scincella rufocaudata (Darevsky & Nguyen), and Pareas hamptoni (Boulenger).

Phylogenetic position. Oligodon rostralis sp. nov. is suggested as a sister species of O. annamensis (Fig. 2), from which it is genetically divergent with p-distance 3.3% in 16S rRNA gene (Table 3). Both species are clustered together with the O. cyclurus and O. taeniatus species groups (Fig. 2).

Comparisons. Morphological diagnostics of species based exclusively on hemipenial morphology is often complicated due to insufficiency of data and certain controversy in describing hemipenis character states in Oligodon existing in literature (Smith, 1943; Wagner, 1975; Vassilieva, 2015); scalation and coloration features often might be more useful for species identification (Pauwels et al., 2002; David, Das & Vogel, 2008; David et al., 2012; Neang, Grismer & Daltry, 2012; Nguyen et al., 2016; Nguyen et al., 2017). By having 15-15-13 dorsal scale rows, Oligodon rostralis sp. nov. can be distinguished from other species inhabiting mainland Southeast Asia having greater number of MSR, namely all members of the O. cyclurus species group: O. cyclurus (Cantor) (19 or 21); O. formosanus (Günther) (19); O. ocellatus (Morice) (19); O. fasciolatus (Günther) (21 or 23); O. kheriensis Achraji & Ray (19); O. juglandifer (Wall) (19); O. chinensis (Günther) (17); O. saintgironsi David, Vogel & Pauwels (17 or 18); O. culaochamensis Nguyen, Nguyen, Nguyen, Phan, Jiang & Murphy (17); O. condaoensis Nguyen, Nguyen, Le & Murphy (17); O. macrurus (Angel) (17); O. arenarius Vassilieva (17) and O. cattienensis Vassilieva, Geissler, Galoyan, Poyarkov, Van Devender & Böhme (17); phylogenetic position of the latter two species is unclear.

Similarly, by having 15 MSR the new species can be diagnosed from the members of the O. taeniatus species group: O. taeniatus (Günther) (19); O. barroni (Smith) (17); O. mouhoti (Boulenger) (17); O. pseudotaeniatus David, Vogel & Van Rooijen (17); O. moricei David, Vogel & Van Rooijen (17) and O. deuvei David, Vogel & Van Rooijen (17).

Most members of the O. cinereus species group, which all are believed to have an unforked hemipenis (vs. bilobed hemipenis in the new species; see Green, Orlov & Murphy, 2010), can be also distinguished from Oligodon rostralis sp. nov. by larger MSR: O. cinereus (Günther) (17); O. nagao David, Nguyen, Nguyen, Jiang, Chen, Teynié & Ziegler (17); O. joynsoni (Smith) (17); O. saiyok Sumontha, Kunya, Dangsri & Pauwels (17); O. huahin Pauwels, Larsen, Suthanthangjai, David & Sumontha (17), and O. albocinctus (Cantor) (19 or 21); another member of the O. cinereus species group –O. inornatus (Boulenger) has 15 MSR and is compared with the new species below.

Diagnostics of Oligodon rostralis sp. nov. from other mainland Southeast Asian species of Oligodon with 15 or 13 dorsal scale rows appear to be the most pertinent (as the number of MSR may vary between these two values due to the position of the dorsal scale row reduction, see David et al., 2012); it is summarized in Table 5. From most species with 15 or 13 MSR, the new species can be distinguished by absence of loreal vs. loreal present in O. eberhardti Pellegrin; O. inornatus; O. kampucheaensis Neang, Grismer & Daltry; O. jintakunei Pauwels, Wallach, David, Chanhome (vestigial loreal); O. planiceps (Boulenger); O. torquatus (Boulenger); O. dorsalis (Gray) and O. melaneus Wall (vestigial loreal). By presence of an entire cloacal plate Oligodon rostralis sp. nov. can be diagnosed from those species who have the cloacal plate divided, namely from O. catenatus (Blyth), O. eberhardti, O. lacroixi Angel & Bourret, O. jintakunei, O. lungshenensis Zheng & Huang, O. ornatus Van Denburgh, O. hamptoni Boulenger, O. mcdougalli Wall, O. planiceps, O. torquatus, O. dorsalis, O. melaneus, and O. erythrorhachis Wall. By having internasals separate from prefrontals the new species can be readily diagnosed from those Oligodon species which have these scales fused, including O. catenatus, O. eberhardti, O. lacroixi, O. jintakunei, O. brevicauda and O. hamptoni. By having a single postocular scale Oligodon rostralis sp. nov. is distinguished from those species which have two postocular scales: O. catenatus, O. lacroixi, O. inornatus, O. kampucheaensis, O. lungshenensis, O. hamptoni, O. planiceps, O. torquatus, O. melaneus, O. brevicauda and O. erythrorhachis. The new species can be further distinguished from O. brevicauda by having a single preocular scale (vs. 2 preoculars). By having six supralabials the new species can be distinguished from Oligodon species with five (O. lacroixi, O. hamptoni, and O. planiceps), seven (O. jintakunei, O. mcdougalli, O. torquatus, O. dorsalis, O. melaneus, O. brevicauda and O. erythrorhachis), or eight (O. inornatus and O. kampucheaensis) supralabials.

Among all congeners Oligodon rostralis sp. nov. morphologically is most similar to O. annamensis, to which this species is also most closely related phylogenetically (see Results). However, the new species can be distinguished from males of O. annamensis by the following combination of morphological characters: (1) greater number of dorsal scale rows, DSR formula 15-15-13 (vs. DSR formula 13-13-13 in O. annamensis); (2) hemipenis bilobed, lobes bifurcating at distal third of body with flounces and lacking papillae (vs. hemipenis bilobed and elongate, lobes bifurcating proximally with papillae and transverse ridges in O. annamensis), (3) nasal vertically divided (vs. nasal entire in O. annamensis); (4) generally larger total length, 582 mm (vs. maximal total length 412 mm in O. annamensis); (5) generally wider head, HW/HL ratio 73.2% (vs. HW/HL ratio 53.6–56.3% in O. annamensis males, and 61.7% in female holotype; see Table 4); (6) generally higher number of subcaudals, 47 (vs. 30–46 in O. annamensis); (7) dorsal pattern consisting of large dark butterfly-shaped blotches and a light middorsal orange stripe (vs. white narrow crossbars edged with black and no middorsal stripe in O. annamensis); (8) ventral color in life cream-white with black quadrangular spots not forming transverse bars (vs. ventral surfaces in life bright coral-red to bright orange with black quadrangular spots forming transverse bars in O. annamensis) (see Tables 4 and 5). Finally, the new species is distinguished from O. annamensis by a significant divergence in mtDNA gene sequences (up to 3.3% of substitutions in 16S rRNA gene, see Table 3).

Additional information on Oligodon annamensis Leviton, 1953

Oligodon annamensis Leviton, 1953 Jour. Washington Acad. Sci., 43(12):422.

Figures 8–11; Table 4.

Holotype. USNM 90408, young female from “Blao, Haut Donai, Annam, French Indo-China” (today environs of Bao Loc, Lam Dong Prov., Vietnam), collected by E. Poilane on March 11, 1933 (Fig. 8).

Referred specimens. Three male specimens, including MNHN 8815, young male from Blao, Haut Donai, Station Agricole, collected by E. Poilane on March 11, 1933 (Fig. 9). MNHN 8815, young male from “Blao, Haut Donai, Station Agricole”, collected by E. Poilane on March 11, 1933 (Fig. 9); ZMMU R-14304, adult male from Chu Pan Phan Mt., Chu Yang Sin NP, Khue Ngoc Dien Comm., Krong Bong Dist., Dak Lak Prov., Vietnam (12.3950°N, 108.3503°E; 1050 m a.s.l.), collected by N.A. Poyarkov on April 14, 2012, described herein (Fig. 10); and CBC 01899, young male from Phnom Samkos WS, Pursat Prov., Cambodia (12.1690°N, 102.9721°E; 916 m a.s.l.), collected by Hun Seiha on April 26, 2012, 1933 (Fig. 11).

Revised diagnosis. An Oligodon with medium body size in adults (adult male TL up to 412 mm); head small, comparatively narrow, snout not protruding; 13 dorsal scale rows at neck, midbody and before vent; ventrals 146–157, subcaudals 43–46 in males; ventrals 170, subcaudal 30 in female; single preocular, single postocular; loreal and presubocular absent; generally six supralabials, third and fourth entering orbit; six infralabials, anterior three or four contacting the first chin shield; internasals separate from prefrontals; nasal entire; single anterior and one or two posterior temporals; cloacal plate entire; hemipenis deeply bilobed, bearing two long and thin papillae, reaching the 20th subcaudal; 7–8 maxillary teeth; broad dark temporal streak; ground color on dorsum dark brown, 9–10+2–5 light crossbars edged with black on dorsum and tail; vertebral stripe absent; and ventral surfaces in life coral-red to orange with black transverse bars or quadrangular spots.

Variation. Morphological data of all presently known specimens of O. annamensis are summarized in Table 4; color pattern of all O. annamensis specimens is remarkably similar (Figs. 8–11). The holotype of O. annamensis, USNM 90408, corresponds well to the original description by Leviton (1953) (Fig. 8), thus we do not provide its formal redescription. The type specimen is a female with several morphological characters different from the known male specimens (see Table 4): it has a relatively shorter tail, RTL 11.7% (vs. RTL 16.6–19.7% in three males), a greater number of ventrals, 170 (vs. 146–157 in males), and a lesser number of subcaudals, 30 (vs. 43–46 in males). The second already known specimen of O. annamensis, MNHN 8815, a subadult male, was described in detail by Leviton (1960) (Fig. 9). Though in general morphology of MNHN 8815 corresponds well to the description by Leviton (1960), we found several differences in scale counts: MNHN 8815 has 146 ventrals + 2 preventrals (vs. 159 ventrals, as stated by Leviton (1960) (courtesy of P. David). The reasons behind such significant differences in scale counts remain unclear; this result further underlines the importance of double-checking specimens preserved in historical collections in taxonomic practice.

The newly reported specimen of O. annamensis from Vietnam, ZMMU R-14304, was collected from Chu Yang Sin NP in Dak Lak Province at the northern edge of Langbian Plateau (see Fig. 1, locality 3). This specimen is an adult male and has the largest total length of all known O. annamensis specimens (412 mm); in scalation and coloration characters it agrees very well with the original description (Leviton, 1953) and the description of male specimen by Leviton (1960) (see Table 4). The tail of ZMMU R-14304 was dissected for examination of hemipenial structures; in full accordance with description by Leviton (1960) this specimen had deeply bilobed hemipenes each bearing two long and thin appendages seen in situ (papillae sensu Smith, 1943), reaching the 20th subcaudal. Coloration of ZMMU R-14304 in life is shown in Fig. 10; among other features, the characteristic coral-red background coloration of the ventral surfaces and black quadrangular spots forming complete transverse bars appear to be diagnostic from Oligodon rostralis sp. nov. (vs. in life ventral surfaces cream-white, black spots do not form transverse bars in the new species).

We present additional morphological information (see Table 4) and photos in life (Fig. 11) of the single known Cambodian specimen of O. annamensis CBC 01899 (see Fig. 1, locality 4) described by Neang & Hun (2013). Based on relative tail length (16.6%) this specimen is identified as male. In accordance with earlier results of Neang & Hun (2013) it shows certain morphological differences from the Vietnamese specimens, namely: having 6/5 supralabials of which 3–4/2–3 touching the orbit (vs. 6/6 and 3–4/3–4 in Vietnamese specimens); infralabials I–III contacting chin shields (vs. I–IV in Vietnamese specimens); posterior temporal single (vs. two posterior temporals in Vietnamese specimens); ventral coloration in life orange red with black markings not forming transverse bars, see Fig. 11B (vs. coral-red belly getting lighter anteriorly; black markings form numerous transverse bars in Vietnamese specimen, see Fig. 10B).

Distribution. To date O. annamensis is reliably known from two provinces of southern Vietnam (Lam Dong and Dak Lak), where it was recorded in montane forests of Langbian Plateau at elevations around 1,000 m a.s.l., and from similar elevations in montane forests of Phnom Samkos Mt. in the western part of the Cardamoms, Pursat, Cambodia. The record of O. annamensis from Dak Lak Province is a range extension and the first provincial record of this species.

Etymology. The specific name “annamensis” is a Latin adjective derived from “Annam”, the historical name of Truong Son Mountains and central Vietnam. Common name in English: “Leviton’s kukri snake” (English), “Răn khiêm trung bo” (Vietnamese), and “Annamskiy oligodon” (Russian).