Abstract

We describe a new species of pitvipers from Trang Province of Thailand, near the Thailand–Malaysian border, based on morphological and molecular (2427 bp from cyt b, ND4, and 16S rRNA mitochondrial DNA genes) lines of evidence. Morphologically, Trimeresurus ciliaris sp. nov. is distinguished from its congeners by the following combination of morphological characters: a long papillose hemipenis; first supralabial and nasal scale fused; three to four small supraocular scales; internasals not in contact; small scale between nasal and the scale formed by the fused second supralabial and loreal present; dorsal scales in 17–17–15 rows across the body; ventral scales 172–175 in males, 171 in female; subcaudal scales 59–63 in males, 61 in female, all paired; in life an emerald-green dorsum with reddish-brown bands; creamy-white venter lacking dark dots or stripes on the lateral sides of the ventrals; white vertebral spots present in both sexes on every two or three dorsal scales; dark brown spots forming discontinuous pattern present on 1–3 lateral dorsal scale rows; males with reddish-brown postocular stripe. The new species forms a distinct clade on the phylogenetic tree of the genus Trimeresurus and differs from the morphologically similar species T. venustus by a significant divergence in cytochrome b mitochondrial DNA gene sequences (p = 12.5%). The new species is currently known from a small karstic area in the Nakawan Range spanning the border of Thailand and Malaysia, in particular in limestone forests in Trang and Satun provinces (Thailand); it likely also occurs in the adjacent parts of Perlis State (Malaysia). Our study also suggests that the taxonomy of T. kanburiensis species complex requires further studies; in particular our study suggests that the status of populations from Chumphon Province of Thailand and Pulau Langkawi Island of Malaysia should be re-assessed.

Biodiversity, Malaysia, morphology, phylogenetics, Reptilia, taxonomy, Thai-Malay Peninsula, Trimeresurus ciliaris sp. nov., Trimeresurus kanburiensis species complex

The Asian pitviper genus Trimeresurus Lacépède, 1804 is a complex and species-rich group of venomous snakes that currently comprises 46 nominal species of terrestrial to arboreal species distributed across South and Southeast Asia (Mirza et al. 2023; Uetz et al. 2023). The genus is known to contain numerous cryptic species which are very similar morphologically and are often hardly distinguishable in the wild. Recently, molecular methods have helped to resolve some particularly problematic taxonomic issues among such complexes of cryptic pitviper species, with new taxa being described every year such as in T. albolabris complex (e.g., Chen et al. 2020, 2021; Mirza et al. 2020), T. kanburiensis complex (e.g., Malhotra et al. 2011; Sumontha et al. 2021), T. popeiorum complex (e.g., Mirza et al. 2023), and T. stejnegeri complex (e.g., Rathee et al. 2022).

Subgeneric taxonomy of the genus Trimeresurus is a subject of a long-lasting debate. Following a pioneering attempt to reconstruct a molecular phylogeny of the snakes of the Trimeresurus radiation, Malhotra and Thorpe (2004a) split the genus Trimeresurus into eight full genera: Trimeresurus Lacépède, 1804, Cryptelytrops Cope, 1860, Himalayophis Malhotra & Thorpe, 2004, Parias Gray, 1849, Peltopelor Günther, 1864, Popeia Malhotra & Thorpe, 2004, Sinovipera Guo & Wang 2011, and Viridovipera Malhotra & Thorpe, 2004. Subsequently, David et al. (2011) demonstrated that the type-species of Trimeresurus Lacépède, 1804 is a member of the genus Cryptelytrops. Consequently, Cryptelytrops is a junior synonym of Trimeresurus sensu stricto, which thus includes only species related to Trimeresurus albolabris Gray, 1842. David et al. (2011) referred species placed by Malhotra and Thorpe (2004a) in their genus Trimeresurus, namely a group of Indian and Indo-Malayan species, to the next available generic nomen, Craspedocephalus Kuhl & van Hasselt, 1822. Lastly, David et al. (2011) recognized at subgeneric level the genera erected or recognized by Malhotra and Thorpe (2004a).

Recently, the peninsular Indian and Sri-Lankan taxon Peltopelor and the Southeast-Asian taxon Craspedocephalus were regarded as a distinct genus Craspedocephalus, following indirectly the conclusions of Malhotra and Thorpe (2004a), with two respective subgenera (e.g., Mallik et al. 2021). However, more recently Mirza et al. (2023), based on an extensive sampling and mitochondrial DNA (hereafter mtDNA)-based genealogy, suggested that these two taxa should be better treated as separate genera due to their deep divergence, along with biogeographic and morphological distinctiveness; this taxonomy was not followed by the Reptile Database (Uetz et al. 2023). Recognizing the limitations of the mtDNA-markers in reconstruction of phylogenetic relationships (see, e.g., Hillis et al. 2021; Gallego-Garcia et al. 2023), we herein treat Peltopelor and Craspedocephalus as subgenera of the genus Craspedocephalus, and recognize the genus Trimeresurus sensu lato with six subgenera pending subsequent multilocus and genome-level studies on evolutionary relationships of this group.

The subgenus Trimeresurus is diagnosed from other members of the genus Trimeresurus by the combination of a long papillose or calyculate hemipenis and a (partially) fused first supralabial and nasal scales (Malhotra and Thorpe 2004a; Sumontha et al. 2021). Currently, this subgenus includes 21 species namely: Trimeresurus albolabris, T. andersonii Theobald, 1868, T. cantori (Blyth, 1846), T. cardamomensis (Malhotra, Thorpe, Mrinalini & Stuart, 2011), T. caudornatus Chen, Ding, Vogel & Shi, 2020, T. davidi Chandramouli, Campbell & Vogel, 2020, T. erythrurus (Cantor, 1839), T. fasciatus (Boulenger, 1896), T. guoi Chen, Shi, Vogel & Ding, 2021, T. honsonensis (Grismer, Ngo & Grismer, 2008), T. insularis Kramer, 1977, T. kanburiensis Smith, 1943, T. kuiburi Sumontha, Suntrarachun, Pauwels, Pawangkhanant, Chomngam, Iamwiriyakul & Chanhome, 2021, T. labialis Fitzinger in Steindachner, 1867, T. macrops Kramer, 1977, T. mutabilis Stoliczka, 1870, T. purpureomaculatus (Gray, 1832), T. rubeus (Malhotra, Thorpe, Mrinalini & Stuart, 2011), T. salazar Mirza, Bhosale, Phansalkar, Sawant, Gowande & Patel, 2020, T. septentrionalis Kramer, 1977, and T. venustus Vogel, 1991 (David et al. 2004; Malhotra and Thorpe 2004a, 2004b; Malhotra et al. 2011; Mirza et al. 2020, 2023; Sumontha et al. 2021; Poyarkov et al. 2023; Uetz et al. 2023).

The Trimeresurus kanburiensis species complex (previously also often referred to as ‘T. venustus species complex’, see David et al. 2004; Sumontha et al. 2021; however as T. kanburiensis represents the earliest described taxon of this group, the name ‘T. kanburiensis species complex’ is more appropriate) encompasses karst-dwelling pitvipers distributed across the Thai-Malay Peninsula and currently includes three nominal species: T. kanburiensis (northern Tenasserim mountains in Kanchanaburi Province of Thailand), T. kuiburi (central Tenasserim mountains in Prachuap Khiri Khan Province of Thailand), and T. venustus (currently known to occupy the southern part of Thai-Malay Penisula from the Isthmus of Kra in Chumphon Province of Thailand in the north to northernmost Peninsular Malaysia in the south, Sumontha et al. 2021) (see Fig. 1). For a long time T. kanburiensis and T. venustus were considered to be synonyms (Viravan et al. 1992; Warrell et al. 1992), until 2004, when both were recognized as distinct species (David et al. 2004; Malhotra and Thorpe 2004b).

Among other members of the T. kanburiensis species complex, the Beautiful Pitviper, T. venustus apparently has the widest distribution and is reported from Thailand (Surat Thani, Phang-Nga, Trang, Nakhon Si Thammarat, Krabi, and Chumphon provinces), and northern Peninsular Malaysia (Kedah State) (Grismer et al. 2006; Chanhome et al. 2011; Chan-ard et al. 2015; Sumontha et al. 2021; see Fig. 1 for details). However, recently Sumontha et al. (2021) demonstrated some distinctive features of the population from Chumphon and reported that this population differs in coloration and pattern from typical T. venustus s. str., and its taxonomic status requires further investigations.

Figure 1. 

Distribution of the Trimeresurus kanburiensis species complex in Thai-Malay Peninsula: Localities. T. kanburiensis (yellow): 1 Kanchanaburi Prov., Sai Yok Dist., Wat Tham, Phom Lo Khao Yai; T. kuiburi (blue): 2 Prachuap Khiri Khan Prov., Kuiburi Dist., Wat Khao Daeng; 3 Prachuap Khiri Khan Prov., Kuiburi Dist., Khao Daeng Beach; 4 Prachuap Khiri Khan Prov., Kuiburi Dist., Khao Daeng, near Ban Thung Noi; T. cf. venustus (1) (green): 5 Chumphon Prov., Wat Tham Sanook; T. venustus s. str. (pink): 6 Krabi Prov., Mueang Krabi Dist., Tiger Cave viewpoint; 7 Thailand, Nakhon Si Thammarat Prov., Khao Luang; 8 Nakhon Si Thammarat Prov., Thung Song; 9 Surat Thani Prov.; Trimeresurus ciliaris sp. nov. (red): 10 Trang Prov., Palian Dist., Thum Khao Ting; 11 Tha Le Ban NP., Khuan Don Dist., Satun Prov.; 12 Perlis State National Park, Perlis State; T. cf. venustus (2) (purple): 13 Pulau Langkawi Island, Kedah State. Remark: stars denote type localities (the type locality is not shown for T. kanburiensis as it was not sampled). Abbreviations: MY – Myanmar, MA – Malaysia, Prov. – Province, Dist. – District.

During our recent field surveys in Trang Province of Thailand, we encountered several unusual pitviper specimens that were superficially similar to T. venustus in overall morphological habitus and body coloration. Closer morphological examination of scalation, pattern, color and morphometric characteristics of this population demonstrated clear morphological differences from T. venustus s. str. In the present paper we demonstrate that these differences are concordant with a significant divergence between mtDNA gene sequences among the two populations. Consequent phylogenetic analyses of three mtDNA genes (cyt b, ND4 and 16S) confirmed the placement of the Trang population within Trimeresurus s. str. where it forms a deeply divergent lineage, with sister relationships to a clade encompassing all other members of T. kanburiensis and T. macrops species complexes with exception of T. rubeus. Therefore, in the present paper we describe the Trimeresurus population from Trang Province as a new species and discuss the taxonomy of T. kanburiensis species complex.

Our molecular results are further corroborated by the morphological analysis, which recovered a number of important diagnostic characters which allow distinguishing the population of Trimeresurus sp. from Trang from T. venustus s. str. and all other congeners (summarized below). These results support our hypothesis that this recently discovered lineage of Trimeresurus sp. from Trang Province represents a previously unknown species, which we formally describe below.

Trimeresurus ciliaris sp. nov.

https://zoobank.org/898F9CF2-0457-4F77-B53A-BB8D1E8AB3F2

Figures 3, 4, 5 (A), 6 (A, B), 7 (B–E), Table 4

Chresonymy

Cryptelytrops cf. venustus (partim): Chan et al. (2011: 258).

Holotype

ZMMU Re-17661, adult male collected by S. Idiiatullina, P. Pawangkhanant and T. Woranuch on 23 January 2023 near Thum Khao Ting, Palian District, Trang Province, Thailand (07°09.943N, 99°48.142E; elevation 28 m a.s.l).

Paratypes (n = 4)

QSMI 1538, adult female collected by P. Pawangkhanant on 18 June 2016 from the same locality as the holotype; AUP-02011, ZMMU Re-17662, ZMMU Re-17663, three adult males collected on 23 January 2023 from the same locality and by the same collectors as for the holotype.

Diagnosis

A species of the genus Trimeresurus which is assigned to the subgenus Trimeresurus based on the following morphological attributes: a long papillose or calyculate hemipenis and partially fused first supralabial and nasal scales (Malhotra and Thorpe 2004a; David et al. 2011). The new species Trimeresurus ciliaris sp. nov. can be distinguished from all other congeneric species by the following combination of morphological characters: three or four small convex supraocular scales; internasals not in contact; small scale between nasal and the scale formed by the fused second supralabial and loreal present; dorsal scales in 17–17–15 rows; ventral scales 172–175 in males, 171 in a single female; subcaudal scales 59–63 in males, 52 in a single female, all paired; in life an emerald-green dorsum with reddish-brown cross-bands; creamy-white venter lacking dark dots or stripes on the lateral sides of the ventrals; iris olive-green with faded-brown horizontal stripe; tail dark brown mottled with rusty spots; white vertebral spots present in both sexes located approximately every two or three dorsal scales; dark brown spots forming discontinuous pattern present on 1–3 lateral dorsal scale rows; males having a reddish-brown postocular stripe.

Description of the holotype

Adult male (Fig. 3), specimen in a good state of preservation. Body cylindrical, long and thin (SVL 345 mm, TaL 72 mm, TL 417 mm, TaL/TL 0.17) (Fig. 3A,B). Head triangular in dorsal view (Fig. 3C), elongate, clearly distinct from the neck (HL 16.5 mm, HW 11.4 mm, HW/HL 0.69). Snout elongate, flattened and rounded in dorsal view (Fig. 3C), rather rectangular in lateral view (Fig. 3E,F), with a very distinct and sharp canthus rostralis (SnL/HL 0.30, SnL/ED 1.92). Distance between eye and nostril 3.9 mm on both sides. Rostral slightly visible in dorsal aspect, triangular (Fig. 3C). Pupil vertically elliptical, loreal pit present, triangular in shape (Fig. 3E,F). Nostril completely enclosed in nasal scale; nasal scale partially fused with first supralabial (Fig. 3E). Shield bordering the anterior edge of loreal pit fused with second supralabial. Anterior subocular long, thin, and crescent-like, separated from the 4^th and 5^th supralabials by one row of scales; posterior subocular ovoid, separated from the 6^th supralabial by 2/1 scales (Fig. 3E, F). Three preoculars on each side of the head; two upper preoculars located above the loreal pit, elongated, in contact with the single loreal which separates them from the nasal; lower preocular forming the lower margin of the loreal pit, lower preocular in contact with third supralabial (Fig. 3E, F). A small scale between the nasal and the scale formed by the fused second supralabial and loreal; 2/2 postoculars; 9/9 supralabials, third the largest (Fig. 3E,F); 11/10 infralabials, those of the first pair in contact with each other behind the mental; the first three pairs of infralabials in contact with the single pair of chin shields (Fig. 3D). Six pairs of gulars aligned between the chin shields and the first preventral (Fig. 3D). One large pair of enlarged internasals, separated by three small scales. Three small slightly convex supraoculars, same size as or smaller than preoculars and postoculars (Fig. 3C). Scales on snout and in the interorbital region smooth, irregular, subimbricate; temporal and occipital scales moderately keeled (Fig. 3C,E,F). Dorsal scales in 17–17–15 rows. Dorsal scales all strongly keeled, except the first row, which is smooth (Fig. 3H). One preventral + 172 ventrals. Cloacal plate single; 61 subcaudals, all divided. Hemipenes long, papillose, and deeply forked with small, soft, basal spines (Fig. 3G). Each maxilla bearing a single large venom tooth; three teeth on palatine, 11 on pterygoid, and 12 on dentary.

Figure 3. 

The holotype of Trimeresurus ciliaris sp. nov. in life (ZMMU Re-17661, adult male) from Thum Khao Ting, Palian District, Trang Province, Thailand. A dorsolateral and B ventral view of body; C head in dorsal, D in ventral, E in lateral right, and F in lateral left aspects; G right hemipenis, partially everted; H close-up of dorsal scales. Photographs by P. Pawangkhanant.

Coloration in life (Figs 3–5)

Dorsal surface of the head emerald-green with many scales partly or entirely dark red, especially on the snout, and on the interorbital, temporal and occipital regions (Fig. 3C). A reddish-brown postocular stripe extends from postocular scales to the neck (Fig. 3E,F). Black vertical pupil; iris olive-green with faded brown horizontal stripe (Fig. 3E,F). The background color of the dorsum emerald-green, similar to that of the head, with about 75 irregular, dark red cross-bands (Fig. 3A). These red bands are about two dorsal scales long mediodorsally, but getting narrower on the lower flanks where they are about 1–2 dorsal scale in length. At the level of the vertebral row, the red bands are generally separated by one dorsal scale. White vertebral spots present along the vertebral scale row, approximately every two or three scales (Fig. 3A). Ventrolateral stripe absent. Dark brown spots forming discontinuous pattern present on first to third lateral dorsal scale rows (Fig. 3H). Tail dark brown mottled with rusty spots. Ventral surface of the tail dark brown with white irregular bands, tail tip completely black. Infralabials greenish-white with faint brown spots (Fig. 3E,F); the ventral surfaces of the head creamy white with bluish tint (Fig. 3D); the ventral surfaces of body creamy white (Fig. 3B).

In preservative the background dorsal color faded to greyish-brown, with less contrasting dark marks on the head and bands on the dorsum; the ventral color became whitish grey.

Variation

The main meristic and morphometric characters of the type series of Trimeresurus ciliaris sp. nov. are summarized in Table 4; color variation of the type series is presented in Figures 4, 5A, 6A–B, 7B–E. All members of the type series, similarly to the holotype, show a nasal scale partially fused with the first supralabial. Head scalation of the paratypes generally agrees with that of the holotype, however male ZMMU Re-17662 has a suboculars fused in a single long crescent-shaped scale (Fig. 4B), while the two males AUP-02011 (Fig. 4C) and ZMMU Re-17663 (Fig. 4D) have four small supraoculars on each side of the head instead of three as in the holotype. Coloration of paratypes is very similar to that of the holotype; all specimens have a creamy-white belly and lack ventrolateral stripe. Female specimen QSMI 1538 has slightly lower number of subcaudals than in males (SC 52 vs. 59–63). The white vertebral spots are present both in males and in females; they are regularly spaced by two or three vertebral scales. The number of dark bands on dorsum varies from 70 to 81 among the members of the type series, without apparent sexual dimorphism. Coloration of female specimen QSMI 1538 largely faded due to preservation in formalin for seven years.

Figure 4. 

Variation in head scalation and coloration in male paratypes of Trimeresurus ciliaris sp. nov.: A ZMMU Re-17661; B ZMMU Re-17662; C AUP-02011; and D ZMMU Re-17663. See Table 4 for details. Photographs by P. Pawangkhanant.

Figure 5. 

Comparison of body coloration between males of A Trimeresurus ciliaris sp. nov.; B T. kanburiensis; C T. kuiburi; and D T. venustus s. str. Photographs by P. Pawangkhanant (A, B, D) and T. Woranuch (C).

Figure 6. 

Comparison of head coloration (left profile and dorsal view of the head) between males of A–B Trimeresurus ciliaris sp. nov.; C–D T. kanburiensis; E–F T. kuiburi; and G–H T. venustus s. str. Photographs by P. Pawangkhanant (A–D), A. Kaosung (E–F, H) and M. Naiduangchan (G).

Figure 7. 

Habitat of Trimeresurus ciliaris sp. nov. A microhabitat of the new species near the Thum Khao Ting Cave, Palian District, Trang Province, Thailand; B holotype ZMMU Re-17661, adult male, in life in situ; C paratype QSMI 1538, adult female, in life in situ; D–E male specimen from Wang Kelian, Perlis State, Malaysia (not collected). Photographs by P. Pawangkhanant (A–C) and Azrul Azrizal Azmy (D–E).

Table 4.

Download as

CSV

XLSX

Morphological data on the type series of Trimeresurus ciliaris sp. nov. Notes: For abbreviations see Materials and methods section.

No.	ZMMU Re-17661	AUP-02011	ZMMU Re-17662	ZMMU Re-17663	QSMI 1538
Type status	Holotype	Paratype	Paratype	Paratype	Paratype
Sex	Male	Male	Male	Male	Female
SVL (mm)	345	357	360	352	333
TaL (mm)	72	73	72	71	51
TL (mm)	417	430	432	423	384
HL (mm)	16.5	16.9	17.1	16.8	16
HW (mm)	11.4	12.2	12.7	11.3	11.3
HD (mm)	5	5.7	6.1	5.6	5.7
SnL (mm)	5	5.1	5.3	4.9	5.6
ED (mm)	2.6	2.8	3	2.5	2.7
VED (mm)	2.3	2.1	2.5	2.1	2.2
SOL (mm)	2.8	3	2.8	2.7	3.4
SOW (mm)	0.7	0.6	0.7	0.7	0.8
Internasals in contact	no	no	no	no	no
Contact nasal-1st SL	partially fused	partially fused	partially fused	partially fused	partially fused
SupOc	3	3	4	4	3
PosOc	2/2	2/2	2/2	2/2	2/2
IOS	11	14	11	11	12
SL	9/9	9/8	9/9	9/9	9/9
IL	11/10	10/10	10/10	11/10	10/11
ASR	17	17	17	17	17
MSR	17	17	17	17	17
PSR	15	15	15	15	15
SRR 17 to 15 (V)	120–121	123–124	123–124	125–126	123
PreVEN	2	2	2	2	2
VEN	172	173	175	172	171
CP	1	1	1	1	1
SC	61	59	63	61	52
Postocular stripe	reddish-brown	reddish-brown	reddish-brown	reddish-brown	Faint, reddish-brown
Number of red bands on dorsum	74	81	73	70	80
White vertebral dots	every 2–3 scales	every 2–3 scales	every 2–3 scales	every 2–3 scales	every 2–3 scales

Distribution and natural history

Currently, Trimeresurus ciliaris sp. nov. is known only from a narrow limestone area in the Nakawan Range spanning the border of Thailand and Malaysia, in particular in limestone forests in Trang (Palian District) and Satun (Tha Le Ban National Park) provinces, Thailand (Fig. 1). It is highly likely that the new species also inhabits the northernmost part of Perlis State of Peninsular Malaysia (Fig. 1, see Discussion for details). The new species occurs in lowland dipterocarp forest on limestone rocks and appears to be strongly associated with a karst landscape. The new species was recorded for the first time in limestone formations within the Tha Le Ban National Park, Satun Province of Thailand, in June 2016 (P. Pawangkhanant pers. obser.), where it was observed in a lowland dipterocarp forest with numerous large karst rocks. Almost all specimens of Trimeresurus ciliaris sp. nov. were collected during daytime (except ZMMU Re-17663 that was collected at night around 20h50). All specimens were found near large boulders of karst rocks covered with dense vegetation and leaf litter (Fig. 7A); one specimen was found after heavy rain around 18h00 while perching vertically on a limestone rock surface ca. 5 m above the ground.

In captivity, due to its small size, this species of pitviper mostly feeds on small geckos and microhylid frogs [Microhyla butleri Boulenger, M. cf. heymonsi Vogt, and Micryletta cf. lineata (Taylor)]. Nothing is known about the diet of the new species in the wild, but in one specimen from Mueang District, Satun Province, caudal luring behavior was observed: the male specimen was wiggling its tail tip when an adult gecko Cnemaspis biocellata Grismer, Chan, Nasir & Sumontha approached its shelter. Other amphibian and reptile species recorded in syntopy with the new species include: Ingerophrynus parvus (Boulenger), Micryletta cf. lineata, Microhyla butleri, Kaloula latidisca (Chan, Grismer & Brown), Cnemaspis niyomwanae Grismer, Sumontha, Cota, Grismer, Wood, Pauwels & Kunya, C. biocellata, Gekko gecko (Linnaeus), Cyrtodactylus astrum Grismer, Wood, Quah, Anuar, Muin, Sumontha, Ahmad, Bauer, Wangkulangkul, Grismer & Pauwels, C. quadrivirgatus Taylor, Cyrtodactylus sp., Elaphe taeniura ridleyi (Cope), and Tropidolaemus wagleri (Boie).

Etymology

The species name “ciliaris” is a Latin adjective in the nominative singular, masculine gender, derived from Latin word “cilium” meaning “an eyelash”, and is given in reference to the characteristic small and distinct supraocular scales in the new species, which resemble eyelashes or eyebrows in lateral view. We suggest the following common names for the new species: “Ngu Hang Mhai Khao Hin Poon” (งูหางไหม้เขาหินปูน) (in Thai), “Limestone Eyelash Pitviper” (in English), and “Resni­tchataya ukrashennaya kufiya” (in Russian).

Comparisons

The new species is morphologically and phylogenetically placed within the subgenus Trimeresurus (Malhotra and Thorpe 2004a; David et al. 2011) and morphologically is overall most similar to other limestone-dwelling pitviper species, including T. kanburiensis, T. kuiburi, and T. venustus s. str., so the comparisons with these three species appear to be the most pertinent. Trimeresurus ciliaris sp. nov. can be easily distinguished from other congeners by having: 17 midbody scale rows, three or four small supraocular scales, and a small scale between the nasal and the scale formed by the fused second supralabial and loreal. The main diagnostic characters separating Trimeresurus ciliaris sp. nov. from these three species are summarized in Table 5. The comparison of body coloration and head scalation of these species is presented in Figures 5 and 6, respectively. From the superficially similar Trimeresurus truongsonensis Orlov, Ryabov, Vu & Ho, a member of the subgenus Viridovipera, the new species can be easily distinguished by having a long papillose and deeply forked hemipenis (vs. short spinose hemipenis) and partially fused first supralabial and nasal scales (vs. separate).

Table 5.

Download as

CSV

XLSX

Comparison of morphological characters of Trimeresurus ciliaris sp. nov. with T. kanburiensis, T. kuiburi, and T. venustus s. str. Diagnostic differences from the new species are marked in bold.

Character	T. ciliaris sp. nov.	T. kanburiensis	T. kuiburi	T. venustus s. str.
Max SVL (males, mm)	360	412	376	490
Max SVL (females, mm)	333	572	451	456
IOS	11–14	7–9	9–11	8–12
Internasals in contact	0%	0%	100%	45%
ASR	17	23 (21 or 22)	21 (23)	23 (21 or 25)
MSR	17	19	19	21 (19)
PSR	15	15 (16)	15	15
VEN males	172–175	172	164–166	166–177
VEN females	171	170–178	164–171	172–181
SC males	59–63	59	63–65	63–72
SC females	52	41–51	51–53	50–66
Small scale between nasal and the scale formed by the fused second supralabial and loreal	present	present/absent	absent	absent
Dorsum color in life	Reddish-brown bands on emerald-green background	Dark olive-brown bands on olive-grayish background	Red/purple bands on bottle green background	Red/purple bands on dark/bottle green background
White vertebral dots (in males)	Present, every 2–3 scales	Present, every 3–5 scales	Present, every 5–6 scales	Absent
White vertebral dots (in females)	Present, every 2–3 scales	Absent	Absent	Absent
Belly background color in life	creamy white	creamy white	pale green	pale green
Lateral dark stripe on ventrals	Absent	Always present, discontinuous, olive-brown	Absent	Always present, continuous or discontinuous, red
Supraocular​ scale	Three or four, small	One, large	One, large	One, large
Iris color	Olive-green with faded brown horizontal stripe	brown, slightly golden	copper	yellowish-brown/gold
Tail color	dark brown mottled with rusty	brownish-gray with olive-brown blotches	red with some thin lighter bands	brown with dark purplish-brown crossbars
Distribution	Thailand (Trang, Satun), Malaysia (Perlis)	Thailand (Kanchanaburi)	Thailand (Prachuap Khiri Khan)	Thailand (Surat Thani, Phang-Nga, Trang, Nakhon Si Thammarat, Surat Thani, Krabi, Chumphon?), Malaysia (Kedah?)
Sources	our data	Smith (1943); David et al. (2004); our data	Sumontha et al. (2021); our data	Vogel (1991); David et al. (2004); Malhotra and Thorpe (2004b); our data

In particular, Trimeresurus ciliaris sp. nov. differs from T. kanburiensis by having: smaller maximal SVL (360 mm in male, 333 mm in female vs. 412 mm in male, 572 mm in female); lower anterior number of dorsal scale rows (17 vs. 23, rarely 21 or 22); lower number of midbody scale rows (17 vs. 19); absence of dark lateral stripe on ventrals, Fig. 5A (vs. dark lateral stripe on ventrals always present, discontinuous, olive-brown, Fig. 5B); three or four small supraocular scales, Fig. 6B (vs. single large supraocular​ scale, Fig. 6D); body color in life (reddish-brown bands on an emerald-green background [Fig. 5A] vs. dark olive-brown bands on olive-greyish background [Fig. 5B]); white vertebral spots present in both sexes separated by 2–3 dorsal scales vs. white vertebral spots present only in males and are separated by 3–5 dorsal scales; iris color (olive-green with faded brown horizontal stripe [Fig. 6A] vs. brown, slightly golden [Fig. 6C]); and tail color (dark brown mottled with rusty spots vs. brownish-gray with olive-brown blotches).

Trimeresurus ciliaris sp. nov. further differs from T. kuiburi by having: smaller maximal SVL in female (333 mm vs. 451 mm); internasals separated by two or three scales (vs. always in contact); lower anterior number of dorsal scale rows (17 vs. 21 rarely 23); lower number midbody scale rows (17 vs. 19); higher number of ventrals in females (172–175 vs. 164–166); ventral surfaces of body creamy-white (vs. pale-green); three or four small supraocular​ scales, Fig. 6A (vs. one large supraocular scale, Fig. 6F); small scale between nasal and second supralabial present vs. absent; body color in life (reddish-brown bands on emerald-green background [Fig. 5A] vs. red to purple bands on bottle-green background [Fig. 5C]); white vertebral spots present in both sexes separated by 2–3 dorsal scales vs. white vertebral dots present only in males and are separated by 5–6 dorsal scales; iris color (olive-green with faded brown horizontal stripe [Fig. 6A] vs. copper [Fig. 6E]); and tail color (dark brown mottled with rusty spots vs. red with some thin lighter bands).

Trimeresurus ciliaris sp. nov. can be further differentiated from T. venustus s. str. by having: smaller maximal SVL (360 mm in male, 333 mm in female vs. 490 mm in male, 456 mm in female); lower anterior number of dorsal scale rows (17 vs. 23, rarely 21 or 25); lower number midbody scale rows (17 vs. 21, rarely 19); white vertebral spots present in both sexes separated by 2–3 dorsal scales (vs. absent); ventral surfaces creamy-white (vs. pale-green); lateral dark stripe on ventrals absent (vs. lateral dark stripe on ventrals always present, discontinuous, red); three or four small supraocular scales, Fig. 6A (vs. one large supraocular scale, Fig. 6H); small scale between nasal and second supralabial present vs. absent; body color in life (reddish-brown bands on emerald-green background [Fig. 5A] vs. red to purple bands on dark bottle-green background [Fig. 5D]); white vertebral spots present in both sexes separated by 2–3 dorsal scales vs. white vertebral dots absent in both sexes; iris color (olive-green with faded brown horizontal stripe [Fig. 6A] vs. yellowish-brown to gold [Fig. 6G]); and tail coloration (dark brown mottled with rusty spots vs. brown with dark purplish-brown crossbars).

Among the other species in the subgenus Trimeresurus, the new species can be readily distinguished from T. albolabris, T. andersonii, T. cantori, T. cardamomensis, T. caudornatus, T. davidi, T. erythrurus, T. fasciatus, T. guoi, T. honsonensis, T. insularis, T. labialis, T. macrops, T. mutabilis, T. purpureomaculatus, T. rubeus, T. salazar, and T. septentrionalis by body coloration and pattern (reddish-brown bands on emerald-green background in the new species vs. uniform green or green coloration with no pattern, or pattern consisting of small brownish spots or speckles in other species, or straw-yellow background with irregular, dark-brown transverse body bands in T. honsonensis). Moreover, by having 17 MSR, the new species can be further separated from T. albolabris (21 MSR), T. andersonii (21 MSR), T. cantori (27, 29 or 31 MSR), T. cardamomensis (21 MSR), T. caudornatus (21 MSR), T. davidi (21 or 23 MSR), T. erythrurus (23 rarely 21, 25 MSR), T. fasciatus (21 MSR), T. guoi (21 MSR), T. honsonensis (21 MSR), T. insularis (21 MSR), T. labialis (21 or 23 MSR), T. macrops (21 MSR), T. mutabilis (21 MSR), T. purpureomaculatus (25 rarely 27, 29 MSR), T. rubeus (21 MSR), T. salazar (21 MSR), and T. septentrionalis (21 MSR) (see Gumprecht et al. 2004; Grismer et al. 2008; Malhotra et al. 2011; Chandramouli et al. 2020; Chen et al. 2020, 2021; Mirza et al. 2020; our data).

In the present study we report on a previously unknown lineage of limestone-associated pitvipers of the subgenus Trimeresurus. According to our mtDNA-based genealogy, the newly discovered species Trimeresurus ciliaris sp. nov. forms a highly divergent lineage of pitvipers, which is suggested as the sister lineage to all other members of T. kanburiensis and T. macrops species complexes (see Fig. 2). The new species is also separated from its congeners by a significant divergence in cyt b gene sequences (with p = 7.7–17.3%), which is higher than the genetic distances between many other recognized species of the group (see Table 3). The mtDNA-based genealogy presented herein is insufficiently resolved in several deeper nodes; however in general it agrees well with the earlier genome-wide analysis by Mrinalini et al. (2015) which demonstrated the non-monophyly of T. kanburiensis and T. macrops species complexes suggesting complicated evolutionary history of this group. Though the phylogenetic position of the new species is not completely resolved in our phylogenetic tree, it is obvious that Trimeresurus ciliaris sp. nov. represents a deeply-divergent, previously unknown taxon which warrants taxonomic recognition.

Our study also provides some insights on phylogenetic relationships within the subgenus Trimeresurus. This subgenus was originally proposed by Malhotra and Thorpe (2004a) as Cryptelytrops Cope, 1860 (type species: Trimeresurus carinatus Gray, 1842, subjective junior synonym of Trigonocephalus purpureomaculatus Gray, 1832 based on David et al. 2011) based on subtle morphological similarities (form of hemipenial structures and the fusion of the first supralabial and nasal scales). However, the monophyly of this group was not supported in the phylogenetic analysis of Malhotra and Thorpe (2004a). Later, David et al. (2011) demonstrated that the name Trimeresurus Lacépède, 1804 should be applied to this group (type species: Trimeresurus viridis Lacépède, 1804, nomen oblitum, a senior synonym of Trimeresurus albolabris insularis Kramer, 1977), however, these authors only discussed the nomenclature of the subgenus Trimeresurus and did not address its systematics. Our mtDNA-based genealogy suggested the significant differences between the members of T. albolabris and T. kanburiensis + T. macrops species groups included in this taxon. Though only based on limited data from three mtDNA genes, our study strongly suggests that the subgenus Trimeresurus sensu lato David et al. (2011) is likely non-monophyletic (Fig. 2). At the same time, the study by Malhotra et al. (2010) which included nuclear intron sequence data recovered the subgenus Trimeresurus as monophyletic. In summary, it is highly likely that the clade encompassing the T. kanburiensis + T. macrops species groups might warrant a taxonomic recognition as a separate subgenus of the genus Trimeresurus. Further comprehensive phylogenetic studies, including the analyses of multiple nuclear DNA genetic markers and additional morphological characters, are required to achieve a better resolution of evolutionary relationships within the subgenus Trimeresurus.

Our updated mtDNA-based genealogy also provides new data on phylogenetic relationships within the T. kanburiensis + T. macrops clade. Two morphologically defined species complexes were recognized within this clade: limestone-associated T. kanburiensis species group joining species with blotched coloration pattern on the dorsum (including T. kanburiensis, T. venustus, T. kuiburi, and also including Trimeresurus ciliaris sp. nov.) and lowland forest-associated T. macrops species group, which includes species with a uniform green coloration of dorsum (T. macrops, T. cardamomensis, and T. rubeus). Trimeresurus honsonensis has also a blotched dorsum coloration pattern and was reported to superficially resemble T. venustus, though this species is not associated with karst landscapes and occurs only in secondary forests with large granite boulders on Hon Son Island of southern Vietnam (Grismer et al. 2008). In our mtDNA-based genealogy the members of T. kanburiensis and T. macrops species complexes did not form reciprocally monophyletic clades, but were mixed with each other: T. cardamomensis is suggested as a sister species of T. venustus s. str., the phylogenetic position of T. rubeus remains unresolved, while T. macrops, T. honsonensis, T. kuiburi and T. cf. venustus 1 form a well-supported group (Fig. 2). Apparently, this result suggests that the transition between forest-dwelling and karst-dwelling lifestyle likely occurred multiple times independently within the subgenus Trimeresurus, often resulting in strikingly similar morphologies. It is noteworthy, that among other Trimeresurus subgenera, limestone-associated species often show coloration similar to that in T. kanburiensis group; for example, the karst-dwelling species Trimeresurus (Viridovipera) truongsonensis from central Vietnam and Laos also has a greenish dorsum with red blotches and superficially resembles T. venustus (Orlov et al. 2004). Further research is needed to investigate the consequences of lifestyle and habitat shifts in the evolution of ­Trimeresurus pitvipers (see discussion in Sanders et al. 2006).

Our work also provides new data on the diversity of limestone-associated Trimeresurus pitvipers in Thailand. In addition to the description of Trimeresurus ciliaris sp. nov., our study further corroborates the results of Sumontha et al. (2021), who suggested that the taxonomic status of T. venustus from Chumphon Province of Thailand required further study. Our molecular analyses demonstrated that despite its morphological similarity to T. venustus, the Chumphon population represents a distinct lineage of Trimeresurus, phylogenetically more closely related to T. kuiburi, T. macrops and T. honsonensis than to T. venustus sensu stricto. In the present paper, we tentatively refer to this population as T. cf. venustus 1, though we recognize this lineage as a distinct new yet undescribed species, the formal description of which is currently in progress and will be published elsewhere.

Further research is needed to clarify the taxonomic status of the T. kanburiensis species complex members occurring in Malaysia. A strange population of pitvipers superficially resembling T. venustus was recorded on Pulau Langkawi Island, Kedah State, Malaysia (Fig. 1, locality 13). This record published by Grismer et al. (2006: 65, fig. 18) is based on two specimens (LSUDP 974–75) originally identified as T. venustus. However, the coloration pattern of these specimens seem to be quite distinct as compared to other congeners, in particularly to Trimeresurus ciliaris sp. nov. and T. venustus s. str. As the genetic information for these specimens is still lacking, we tentatively refer to the Langkawi population as T. cf. venustus 2. Further integrative taxonomic analysis of additional materials from Malaysia, including morphological and molecular data, is required to clarify the status of this population which well may also represent a previously undescribed taxon.

Based on our observations, the newly described species Trimeresurus ciliaris sp. nov. inhabits not only Trang Province, but is also distributed in Satun Province of Thailand (recorded in Thale Ban National Park, Wang Prachan, Khuan Don District based on P. Pawangkhanant pers. obser.). Moreover, the new species likely also inhabits Perlis State of Malaysia (Fig. 1). Chan et al. (2011) reported on a strange population of T. cf. venustus from Perlis State Park, Northern Malaysia. This record was originally published under the name Cryptelytrops cf. venustus; based on examination of the available photo material it is conspecific with Trimeresurus ciliaris sp. nov. (Universiti Kebangsaan Malaysia Digital Photograph Collection: UKMDPC 1.0144). Additional records of a similarly-looking pitviper have been recently provided for Wang Kelian View Point, Kaki Bukit, Perlis State (Azrul Azrizal Azmy pers. obser.; see Fig. 6D,E). Morphologically these records are similar to Trimeresurus ciliaris sp. nov. in body habitus and coloration pattern; moreover the localities belong to the same limestone massif (Nakawan Range) continuing from Perlis State of Malaysia to Trang Province of Thailand and are located within approximately 90 km from the type locality of the new species. Located on the Malaysia-Thailand border, the Nakawan Range represents Malaysia’s largest continuous limestone area and is known as an area with high herpetofaunal diversity (Chan et al. 2009; Grismer et al. 2014). Additional sampling efforts and along with molecular and morphological data on the Perlis population of Trimeresurus ciliaris sp. nov. are required; we suspect that this locality represents the southern limit of the new species distribution. Toxicological status of the new species remains unknown. In summary, further investigations are required to elucidate the distribution, diet, reproductive biology, population size, population trends and conservation status of the new species.

Our work raises the total number of species of the genus Trimeresurus to 47; Trimeresurus ciliaris sp. nov. represents the fifteenth species of Trimeresurus known for Thailand. Despite the recent significant progress, our knowledge of molecular phylogeny, classification and distribution of Asian pitvipers remains incomplete. Last but not least, the medical relevance of Trimeresurus pitvipers further underlines the necessity for future studies on the taxonomy of this genus.

We would like to thank the Laboratory Animal Research Center, University of Phayao and The Institute of Animal for Scientific Purposes Development (IAD), Thailand for permission to do field work. We are deeply grateful to Thiti Ruangsuwan, Mali Naiduangchan, Kanokwan Yimyoo, and Priwan Srisom (Thailand) for help during the field surveys. We give thanks to Azrul Azrizal Azmy (Malaysia) for sharing information and providing us photographs of Trimeresurus ciliaris sp. nov. from Perlis State, Malaysia. The study was completed within the frameworks of the research project E-1.2 of the Joint Vietnam-Russia Tropical Science and Technology Research Centre for 2023. We are especially thankful to Gernot Vogel (Germany) for sharing information related to Trimeresurus spp. N.A. Poyarkov gives thanks to the members of MSU HerpLab including Vladislav A. Gorin, Evgeniya N. Solovyeva, Andrei M. Bragin, Alexey V. Trofimets, Nikita S. Klyukin, and Dmitriy V. Akhipov for support and assistance. S. Idiiatullina is thankful to Sunutcha Suntrarachun, Nararat Laopichienpong, and Apinya Longya (QSMI) for providing some important DNA samples and for helping in the lab. We thank Christopher Joldnall for proofreading and linguistic help. We are very grateful to Patrick David (MNHN, France), Anita Malhotra (Bangor University, UK), Zeeshan Mirza (Max Planck Institute for Biology, Germany) and Uwe Fritz (Museum of Zoology, Senckenberg Dresden, Germany) for helpful comments which allowed us the oppurtunity to improve the previous version of the manuscript.

This work was supported by the Russian Science Foundation to N.A. Poyarkov (Grant No. 22-14-00037, specimen collection and preservation, molecular and morphological analyses, data analysis), the Unit of Excellence 2023 on Biodiversity and Natural Resources Management, University of Phayao (UoE66001; data analysis), and in part by the Rufford Foundation (Grant No. 39897-1; data analysis). Specimen collection and animal use protocols were approved by the Institutional Ethical Committee of Animal Experimentation of the University of Phayao, Phayao, Thailand (certificate number UP-AE64-02-04-005, issued to C. Suwannapoom) and were strictly compliant with the ethical conditions of the Thailand Animal Welfare Act. Field work, including collection of animals in the field, was authorized by the Institute of Animals for Scientific Purpose Development (IAD), Bangkok, Thailand (permit numbers U1-01205-2558 and UP-AE59-01-04-0022, issued to C. Suwannapoom).