Another new species of karst-associated pitviper (Serpentes, Viperidae: Trimeresurus ) from the Isthmus of Kra, Peninsular Thailand

. We describe a new species of karst-dwelling pitviper from Chumphon Province of Peninsular Thailand, in the Isthmus of Kra, based on morphological and molecular data (2427 bp from cyt b , ND4 and 16S rRNA mitochondrial DNA genes). Morphologically, Trimeresurus kraensis sp. nov. is distinguished from other congeners by the following combination of morphological characters: a dark/bottle-green dorsum with reddish-brown or purple crossbands; pale green venter lacking dark dots; stripes present on the lateral sides of the ventrals; internasals generally in contact; one large supraocular scale on each side of the head; iris pale copper; tail brown with dark purplish-brown crossbars; dorsal scales in 21–21–15 rows; ventral scales 167 in a single male, 169–171 in females; subcaudal scales 62 in a single male, 52–54 in females, all paired. White vertebral spots present in males, located on approximately every two or four dorsal scales; dark brown spots forming discontinuous pattern present on 1–3 lateral dorsal scale rows; males with reddish-brown postocular stripe with jagged edges. The new species differs from the morphologically similar species Trimeresurus venustus s. str. by a notable divergence in cytochrome b mitochondrial DNA gene sequences ( p = 5.9%).

The subgenus Trimeresurus is distinguished 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 & Thorpe 2004a;Sumontha et al. 2021).Molecular data suggests the presence of numerous cryptic species which are very similar morphologically and are often hard to distinguish in the wild (Mrinalini et al. 2015;Vogel et al. 2023).Consequently, 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 the T. albolabris complex (e.g., Mirza et al. 2020;Chen et al. 2020Chen et al. , 2021)), T. kanburiensis complex (e.g., Sumontha et al. 2021;Idiiatullina et al. 2023), T. popeiorum complex (e.g., Mirza et al. 2023), and T. stejnegeri complex (e.g., Rathee et al. 2022).
In the frame of the revision of several complexes of species and the identification of cryptic species in the subgenus Trimeresurus, in the present study, we focus on the Trimeresurus kanburiensis species complex.This group comprises relatively small, brightly coloured and patterned, karst-dwelling pitvipers distributed across the limestone karst formations of western and southern Thailand, and northwestern Peninsular Malaysia.Following Idiiatullina et al. (2023), this complex currently includes the four species listed above, the ranges of which are as follows, from north to south (see Fig. 1): • T. kanburiensis: western Thailand, endemic to Kanchanaburi Province; • T. kuiburi: northern Peninsular Thailand, endemic to Prachuap Khiri Khan Province; • T. venustus: southern part of Peninsular Thailand, namely in the Thai-Malay Peninsula, southern Thailand, in the provinces of Krabi, Nakhon Si Thammarat, Phang-Nga, Surat Thani, and Trang.This species was also reported from the Langkawi Island off extreme northwestern Peninsular Malaysia, in Kedah State, but its status is not resolved (see below); • T. ciliaris: extreme southern part of the Thai-Malay Peninsula, from Trang and Satun provinces to the extreme northwestern Peninsular Malaysia, in Perlis State.
However, our investigation of the Trimeresurus kanburiensis species complex suggests that the status of two populations is problematical and has to be re-assessed.The first one inhabits the island of Langkawi, off the west coast of West Malaysia.This population was first reported by Grismer et al. (2006) and identified in Idiiatullina et al. (2023) as Trimeresurus cf.venustus 2; its status will not be addressed here.
The second problematical population was initially recorded as Trimeresurus venustus from Chumphon Province, in the central part of Peninsular Thailand, by Pauwels et al. (2013).It was further discussed by Sumontha et al. (2021), and identified in Idiiatullina et al. (2023) as Trimeresurus cf.venustus 1.These latter authors demonstrated that specimens of the population from Chumphon differ in colouration and pattern from the typical specimens of the south Thailand, namely T. venustus s. str., and pointed out that its taxonomic status required further investigation.During our recent field surveys in Chumphon Province, we collected four specimens of this population.Although they are superficially similar to T. venustus s. str. in overall morphological habitus and body colouration, they differ in a number of other taxonomically significant morphological characters.Furthermore, we demonstrate that they also distinctly differ by a notable divergence in mitochondrial DNA (hereafter mtDNA) gene sequences.Subsequent phylogenetic analyses of three mtDNA genes (cyt b, ND4, and 16S) by Idiiatullina et al. (2023), and the present paper confirm the placement of the population of Trimeresurus sp. from Chumphon within the T. kanburiensis species complex, but in which it forms a divergent lineage, sister to T. kuiburi and distantly related to T. venustus s. str., T. ciliaris, and T. kanburiensis.Consequently, based on our new data, we herein describe the Trimeresurus cf.venustus 1 population from Chumphon Province as a new species, and discuss the taxonomy and distribution of the T. kanburiensis species complex.

Sampling
Fieldwork was carried out in the area of Sanook Cave, Muang District, Chumphon Province, Peninsular Thailand, by P. Pawangkhanant, C. Suwannapoom, and N.A. Poyarkov in January 2022, and by P. Pawangkhanant and S. Idiiatullina in July 2023 (Fig. 1, locality 5).Geographic coordinates and altitude were obtained using a Garmin GPSMAP 60CSx GPS receiver (USA) and recorded in datum WGS 84.Specimens were collected in the field by means of snake hooks, photographed in life, and euthanized using a benzocaine solution within 24 h of capture.Specimens were fixated in 4% buffered formalin for 24 h

DNA isolation and sequencing
We extracted the total genomic DNA from ethanol-preserved muscle or liver tissues using standard phenol-chloroform extraction procedures (Sambrook et al. 1989), followed by isopropanol precipitation as described in Idiiatullina et al. (2023).We used the polymerase chain reaction (PCR) to amplify three mtDNA fragments: complete sequences of cytochrome b (cyt b) and NADH dehydrogenase subunit 4 (ND4) genes and a fragment of 16S rRNA gene.PCR conditions and primers used for both PCR and sequencing are described in detail in Idiiatullina et al. (2023).
All amplifications were run using a T100 Thermal Cycler (Bio-Rad).PCR products were loaded onto 1% agarose gels in the presence of ethidium bromide and visualized in electrophoresis.The successfully targeted PCR products were purified by Diatom DNA PCR Clean-Up kit and outsourced to Evrogen ® (Moscow, Russia) for sequencing; sequence data collection and visualization was performed on an ABI 3730xl Automated Sequencer (Applied Biosystems).

Molecular phylogeny
The matrilineal genealogy of the genus Trimeresurus was assessed using the Bayesian Inference (BI) and Maximum Likelihood (ML) approaches.The newly obtained cyt b, ND4, and 16S rRNA sequences were aligned together with all available sequences of T. ciliaris, T. kanburiensis, T. kuiburi, and T. venustus obtained from GenBank, as well as the sequences of 29 species of Trimeresurus representing all major lineages within the genus, and five species of the genus Craspedocephalus Kuhl & Hasselt, 1822.The sequence of Azemiops feae Boulenger, 1888 was used as an outgroup to root the tree.The data on voucher and locality information, and GenBank accession numbers is summarized in Table 1.
The nucleotide sequences were initially aligned using MAFFT ver.6 online webserver (Katoh et al. 2019) with default parameters, and subsequently checked by eye in BioEdit ver.7.0.5.2 (Hall 1999).The genetic divergence among the members of the subgenus Trimeresurus was assessed by calculating the mean uncorrected genetic p-distances between the cyt b sequences using MEGA ver.6.0.(Tamura et al. 2013).The best-fit DNA evolution models for the data set were estimated for genes and codon positions in Partitionfinder ver.2.1.1 (Lanfear et al. 2012)  Phylogenetic trees were estimated for the concatenated alignment including fragments of cyt b, ND4, and 16S rRNA gene sequences.The ML-tree was generated using IQ-TREE webserver (Nguyen et al. 2015); the confidence in tree topology was assessed by 1000 ultrafast-bootstrap replications (ML UFBS).
The BI analysis was conducted in MrBayes ver.chains for one million generations and sampled every 1000 generations; the run was checked to ensure the effective sample sizes (ESS) were all above 200 by exploring the likelihood plots using TRACER ver.1.7 (Rambaut et al. 2018).The initial 1000 trees were discarded as burn-in.The confidence in tree topology was assessed by calculating the posterior probability (BI PP) (Huelsenbeck & Ronquist 2001).We a priori regarded the tree nodes with ML UFBS values of 95% or higher and PP values over 0.95 as strongly supported; ML UFBS values between 95% and 90% and PP values between 0.95 and 0.90 were regarded as moderate support, while lower values were regarded as a lack of node support (Huelsenbeck & Hillis 1993).

Morphological differentiation
Morphological examination, including measurements and meristic counts, followed Idiiatullina et al. (2023).Paired meristic characters were given in the left/right order.The following measurements were taken with a Mitutoyo digital caliper to the nearest 0. We also counted the number of maxillary teeth for each specimen.The sex was determined by examination of the hemipenes that were forcedly everted by water injection in the tail base prior to the preservation of the specimen.Morphological data for the additional examined specimens are presented in Appendix 1.

Sequence characteristics
The final alignment of the three examined mtDNA fragments comprised 2427 base pairs, including: 1104 bp of cyt b, 803 bp of ND4, and 520 bp of 16S rRNA genes.Protein-coding sequences were translated into amino acids in order to confirm that no pseudogenes have been amplified.The GenBank accession numbers of the obtained sequences and specimen information are summarized in Table 1.

Phylogenetic relationships
The ML and BI phylogenetic analyses recovered trees with essentially identical topologies; the only minor topological differences were associated with several nodes with low values of nodal support, which are not relevant to the analysis of the focal group (Fig. 2).Monophyly of the genus Craspedocephalus is not supported in our tree: it consists of two deeply-divergent mtDNA clades corresponding to the Southeast-Asian subgenus Craspedocephalus (95/1.0,hereafter node support values are given for ML UFBS/ BI PP, respectively), and to the Indian and Sri-Lankan subgenus Peltopelor Günther, 1864 (100/1.0);however, this topology lacks significant nodal support (56/0.74;Fig. 2).The matrilineal genealogy strongly supported the monophyly of the genus Trimeresurus (100/1.0)with respect to its sister genus Craspedocephalus; however, the phylogenetic relationships among the main groups of Trimeresurus remained unresolved.The subgenus Trimeresurus was recovered as paraphyletic in our analysis with the T. albolabris species group (100/1.0;which included: T. albolabris Gray, 1842, T. guoi Chen, Shi, Vogel & Ding, 2021, T. septentrionalis Kramer, 1977, T. cantori (Blyth, 1846), T. erythrurus (Cantor, 1839), and T. purpureomaculatus (Gray, 1832)) which was suggested as a sister group to all other members of the genus, though with ambiguous topological support values (93/0.98;Fig. 2).
The remaining members of the subgenus Trimeresurus included the T. macrops species complex and the T. kanburiensis species complex, which formed a strongly supported monophyletic group (100/1.0)(Fig. 2).The genealogical relationships within this clade were generally sufficiently resolved and strongly suggested that Trimeresurus sp. from Chumphon Province belongs to one clade with T. honsonensis, T. macrops, and T. kuiburi (100/1.0;Clade 2 in Fig. 2).The matrilineal genealogy recovered Trimeresurus sp. from Chumphon as a monophyletic group (1.0/100), and suggested that it represents a sister species of T. kuiburi, though this topology was not supported in ML ananlysis and received only moderate support in BI analysis (66/0.95;Fig. 2).The phylogenetic position of T. ciliaris from southernmost Thailand and T. rubeus from Vietnam remained unresolved in our tree.Trimeresurus kanburiensis was strongly suggested as a sister species to the remaining members of the species complex with exception of T. ciliaris and T. rubeus (99/1.0;Fig. 2), while T. cardamomensis was recovered as a sister species of T. venustus s. str.with ambiguous support (92/1.0;Fig. 2)

Genetic distances
The uncorrected p-distances for the cyt b gene fragment among examined members of the subgenus Trimeresurus are presented in Table 2. Interspecific distances among the members of the subgenus Trimeresurus varied from p = 3.0% (between T. venustus s. str.and T. honsonensis) to p = 15.1% (between T. kuiburi, T. erythrurus, and T. albolabris).The Trimeresurus sp.lineage from Chumphon Province is highly divergent from other congeners in cyt b mtDNA gene sequences with genetic distance varying from p = 4.1% (with T. honsonensis) to p = 14.0%(with T. erythrurus).
In summary, our phylogenetic analyses demonstrated that Trimeresurus sp. from Chumphon Province clearly represents a highly divergent lineage of pitvipers, which is most closely related to T. honsonensis, T. macrops, and T. kuriburi, with more distant genealogical relationships to T. venustus s. str.and T. cardamomensis (Fig. 2).Sumontha et al. (2021) and Idiiatullina et al. (2023) suggested that the population of Trimeresurus sp.occurring in Chumphon Province, in the Isthmus of Kra, was distinct from Trimeresurus venustus s. str.Molecular analyses clearly demonstrate that this population forms a distinct clade in the phylogeny of the Trimeresurus kanburiensis species complex (Fig. 2), which is also significantly divergent from other members of the complex in cyt b mtDNA gene sequences (Table 2).Based on examination of our new material, morphological analyses also recovered a number of important diagnostic characters allowing us to distinguish the Chumphon population of Trimeresurus sp. from T. venustus s. str., with which it was previously confused, as well as from all other members of the genus Trimeresurus (summarized below in Table 3).Based on these results, we regard the population from Chumphon Province as a distinct species, which we formally describe below.T. cardamomensis 7.7 9.3 6.0 5.6 4.8 -4.9 10.8 13.5 13.2 12.9 12.4 14 T. guoi 14.1 10.5 10.8 12.5 8.5 12.2 9.9 3.5 3.4 4.1 3.1 3.5 8.1

Etymology
The species name is the modern Latin adjective ʻkraensisʼ in the nominative singular, masculine gender, combining the noun ʻKraʼ, from the name of the Kra Isthmus in Peninsular Thailand where the type locality of the species is located, and the Latin suffix ʻ-ensisʼ (-is, -e), meaning ʻfromʼ.The species name therefore means, ʻfrom Kraʼ.We suggest the following common names for the new species: งู หางแฮ่ มชุ มพร (Ngu Hang Ham Chumphorn) (in Thai), and Kra Isthmus Pitviper (English).
Colouration of holotype in life (Fig. 3) The background dorsal colour is bright olive-green above, slightly paler bluish-green on the lower part of each side; many dorsal scales heavily stippled with minute reddish-brown dots, especially on the lower part of the sides; a dorsal series of about 60 dorsal blotches, dark reddish-brown, transversally elongate, rather short, wider than long, not extending downwards on the flanks beyond the 5 th dorsal scale row and not forming true crossbands; these dorsal blotches are much irregular in shape, often constricted in their middle or partially divided into two lateral blotches, united or alternating with one another along the vertebral row; white, elongate narrow spots or dashes aligned on the vertebral row, located approximately every two or four dorsal scales; one or two series of dark brown spots, about one-scale long, scattered on the 1 st to 3 rd dorsal scale rows, irregularly spaced, usually forming pair obliquely arranged; some scales of the 1 st dorsal row cream as the tips of dorsals.The tail is coloured and patterned like the dorsum, with about 15 dorsal blotches, turning to dark brown on a pink background on the posterior quarter of the tail.The head is bright olive-green like the body above, largely covered with dark reddish-brown areas, of the same colour as the dorsal blotches, so extensively that the head appears reddish-brown above with narrow olive-green lines; rostral olive-green on its lower part, dark reddish-brown on its upper part; internasals and the canthal scales more extensively green than reddish-brown; side of the head pale green; paler than the upper head surface; nasal, loreal and preoculars with dark reddish-brown spots or blotches; supralabials dotted with pale blue; two broad vertical streaks, dark reddish brown covering the parts of the supralabials located below the loreal pit and the eye, respectively; a broad, dark reddish-brown postocular streak, edged above with a narrow area of bright olive-green, extends from the eye to the corner of the mouth.The infralabials, chin and throat are cream with dark brown spots, more numerous on the mental scale and the supralabials.The venter is pale creamish-green, mostly uniform but with some scattered dark brown spots; tips and outer parts of most ventral plates both cream and dark brown, forming a discontinuous, pale and dark, ventrolateral stripe extending from the neck to the vent.The ventral surface of the tail is cream heavily blotched with dark brown, becoming entirely dark brown posteriorly.

Colouration of holotype in preservative
In preservative, the background dorsal colour fades to dark grey, but the pattern remains generally unchanged.Idiiatullina et al., 2023, T. kanburiensis Smith, 1943, T. kuiburi Sumontha et al., 2021, and T. venustus Vogel, 1991.Diagnostic differences from the new species are marked in bold.For abbreviations see Material and methods section.n = number of specimens.

Head scalation
The internasals in contact in all examined specimens but in the male paratype ZMMU Re-17664 they are separated by a small scale; SL: 9-12; IL: 10-13.

Main characters of colouration pattern
Reddish-brown dorsal blotches not forming true crossbands, not extending downwards below the 5 th dorsal scale rows; postocular streak always present, dark reddish-brown, broad and conspicuous in both males and females; white ventrolateral stripe present in both sexes.

Distribution and natural history
Currently, Trimeresurus kraensis sp.nov. is known only from two small karst formations, namely the Sanook Cave (environs of Wat Tham Sanook Monastery; Fig. 4A) and Chang Phueak Cave (environs of Wat Tham Chang Phueak Monastery), both in Banna Subdistrict, Muang District, Chumphon Province, Thailand (approximate coordinates: 10.446216° N, 99.035114° E; 87 m a.s.l.; Sumontha et al. 2021;our data).The two localities are separated by a distance of 5.8 km from each other and are located on the opposite sides of a low limestone hilly area spanning from north-east to south-west for ca 22 km in the central part of the Chumphon Province of Thailand.It is highly likely that Trimeresurus kraensis sp.nov.can be found at additional localities along this karst area; nevertheless, the estimated distribution range of the new species seems to be restricted to this limestone landscape.
The new species appears to be a locally abundant species of snake.Adult individuals were recorded after dusk (18:30-22:00) while foraging on large karst rocks and walls (Fig. 4A).Young snakes were generally found while perching on small bushes growing among the limestone rocks; individuals in pre-shedding phase were also recorded while perching on bushes.Trimeresurus kraensis sp.nov. is quite aggressive when handled.Toxicological status of the new species remains unknown.Nothing is known about the diet of the new species in the wild either, but it is most likely a generalist predator, as captive individuals ate geckos, small frogs and mice, similar to T. kuiburi as described by Sumontha et al. (2021).Reproduction of the new species was not observed.

Comparisons
The new species is morphologically and phylogenetically placed within the subgenus Trimeresurus (Malhotra & Thorpe 2004a;David et al. 2011) and is morphologically overall most similar to other limestone-dwelling species of pitvipers belonging to the T. kanburiensis complex, including: T. ciliaris, T. kanburiensis, T. kuiburi, and T. venustus s. str.; therefore, the comparisons of the new species with these four congeners appear to be the most pertinent.The main diagnostic characters separating Trimeresurus kraensis sp.nov.from these four species are summarized in Table 3, additional morphological data are presented in Appendix 1.The comparison of body colouration and head scalation of these species is presented in Figs 5 and 6, respectively.
Trimeresurus kraensis sp.nov.differs from T. ciliaris by having lower total length in males (max TL 397 mm vs 432 mm); lower number of ventrals in males ); slightly lower number of ventrals plus subcaudals in males ); higher number of anterior dorsal scale rows (ASR 21 vs 17); higher number of midbody scale rows (MSR 21 vs 17); postocular stripe straight (vs concave); small scale between nasal and 2 nd supralabial absent (vs present); single large supraocular scale (vs three or four small supraocular scales); white vertebral dots absent in females (vs present, every 2-3 scales); ventral surface pale creamish-green (vs creamy white); pale and dark ventrolateral stripe always present, discontinuous, reddish-brown (vs absent); and iris pale copper (vs olive-green with faded brown horizontal stripe).
The new species differs from T. kanburiensis by having lower total length in both sexes (max TL 397 mm in male, 542 mm in female vs 500 mm in male, 667 mm in female, respectively); slightly lower number of ventrals in males (VEN 167 vs 172); slightly higher number of subcaudals in females ); slightly lower anterior number of dorsal scale rows (ASR 21 vs 21-23 [avg.22.2]); higher number of midbody scale rows (MSR 21 vs 19); higher number of interorbital scales (IOS 11-13 vs 7-9); internasals generally in contact (vs always separated); ventral surface pale creamish-green (vs creamy white).
Trimeresurus kraensis sp.nov.further differs from T. kuiburi by having lower total length in males (max TL 397 mm vs 465 mm); higher number of midbody scale rows (MSR 21 vs 19); postocular streak straight (vs concave); white vertebral dots present on every 2-4 scales in males (vs every 5-6 scales); and pale and dark lateral stripe on ventrals always present, discontinuous, cream, and reddish-brown (vs absent).

Discussion
Our phylogenetic data are congruent with the results of Idiiatullina et al. (2023) and suggest a similar set of genealogical relationships among the members of the T. kanburiensis species complex.Following the description of Trimeresurus kraensis sp.nov., the subgenus Trimeresurus, as defined by Mirza et al. (2023) and Idiiatullina et al. (2023), currently includes 23 species namely, Trimeresurus albolabris, T. andersonii, T. cantori, T. cardamomensis, T. ciliaris, T. caudornatus, T. davidi, T. erythrurus, T. fasciatus, T. guoi, T. honsonensis, T. insularis, T. kanburiensis, T. kraensis sp.nov., T. kuiburi, T. labialis, T. macrops, T. mutabilis, T. purpureomaculatus, T. rubeus, T. salazar, T. septentrionalis, and T. venustus (Sumontha et al. 2021;Mirza et al. 2023;Poyarkov et al. 2023).Within this group, the members of the T. kanburiensis and T. macrops species complexes do not form reciprocally monophyletic clades but form a strongly supported monophylum with poorly resolved phylogenetic relationships within it.Comparable results were reported by an earlier genome-wide analysis by Mrinalini et al. (2015) and mtDNA-based genealogy by Idiiatullina et al. (2023).Overall, these results suggest a complicated evolutionary history in this group, with the shifts between the limestone-associated and the forest-associated lifestyles likely happened several times during the evolution of Trimeresururs pitvipers (see discussion in Sanders et al. 2006 andIdiiatullina et al. 2023).
In the present study, we report on a previously undescribed lineage of limestone-associated pitvipers of the subgenus Trimeresurus.According to our mtDNA-based phylogeny, the newly discovered species Trimeresurus kraensis sp.nov.forms a highly divergent lineage of pitvipers, which is suggested as a sister species of T. kuiburi in our phylogenetic tree (Fig. 2).Both species occur in the Thai-Malay Peninsula which suggests that the area around the Isthmus of Kra likely played an important role in the differentiation of the T. kanburiensis and T. macrops species complexes.The importance of the Isthmus of Kra as an area of herpetofaunal turnover was demonstrated in numerous recent publications; this narrow zone likely shaped radiation in many groups of reptiles (e.g., Chomdej et al. 2021;Grismer et al. 2020aGrismer et al. , 2020bGrismer et al. , 2022;;Poyarkov et al. 2019Poyarkov et al. , 2022Poyarkov et al. , 2023) ) and amphibians (e.g., Chen et al. 2018;Gorin et al. 2020;Matsui et al. 2005;Pawangkhanant et al. 2018;Poyarkov et al. 2020Poyarkov et al. , 2021;;Suwannapoom et al. 2018Suwannapoom et al. , 2020Suwannapoom et al. , 2021Suwannapoom et al. , 2022) ) inhabiting Southeast Asia.Furthermore, the new species is also separated from its congeners by a significant divergence in cyt b gene sequences (with p = 4.1-14.0%),which is slightly higher than the genetic distances between many other recognized species of the subgenus Trimeresurus (Table 2).Therefore, the molecular and morphological evidence presented here confirm the status of Trimeresurus kraensis sp.nov.as a previously undescribed new species of pitvipers which warrants taxonomic recognition.
The Beautiful pitviper Trimeresurus venustus was described from southern Thailand in 1991.Subsequently, for a long time its relationships with the Kanburi pitviper T. kanburiensis remained controversial because very few specimens of T. kanburiensis were known at the time of its description (Vogel 1991).However, based on molecular and morphological evidence, Malhotra & Thorpe (2004b) and David et al. (2004) recognized T. venustus as a valid species, and formerly reported this species to be distributed in Southern Thailand and the Langkawi Island in northern West Malaysia (see Zimmerer 2004;Grismer et al. 2006;Chan-ard et al. 2015;Poyarkov et al. 2023).In the recent study of the T. venustus complex by Idiiatullina et al. (2023), a new species T. ciliaris was described from Trang and Satun provinces in southwestern part of Thailand, and from Perlis State of northern Peninsular Malaysia.At the same time, this work along with Sumontha et al. (2021) also underlined the need to study populations of T. cf.venustus from Chumphon Province, Thailand, which we herein describe as Trimeresurus kraensis sp.nov.The taxonomic status of the population of T. cf.venustus from Langkawi Island, Kedah State, West Malaysia, originally reported by Grismer et al. (2006), still remains controversial.In colouration, this population appears to be different from all presently recognized members of the T. kanburiensis species complex (see Fig. 5D).Therefore, based on the results of Vogel (1991), Malhotra & Thorpe (2004b), David et al. (2004), Idiiatullina et al. (2023), and our data, herein we propose to restrict the distribution of T. venustus, as now defined, to the central part of southern peninsular Thailand, in the provinces of Surat Thani, Phang-Nga, Trang, Nakhon Si Thammarat, Surat Thani, and Krabi.Meanwhile, the status of the insular population of T. cf.venustus from Pulau Langkawi, Malaysia, remains unclear and will be addressed elsewhere.
Trimeresurus kraensis sp.nov. is to date reliably recorded only from two small limestone caves in Chumphon Province, Thailand.This is a highly secretive snake which appears to be a strict forest-dweller, associated with limestone karst forests.Unfortunately, karst formations of Southeast Asia are being destroyed at an unprecedented rate by limestone quarrying, which constitutes the primary threat (see Grismer et al. 2021;Suwannapoom et al. 2018).These quarrying activities heavily affect the lowland evergreen or semi-evergreen tropical forests growing at the foot of karst formations.Given the available information, we suggest Trimeresurus kraensis sp.nov. to be considered Near Threatened (NT), following the IUCN's Red List categories (IUCN Standards and Petitions Committee 2019).David, Vidal & Pauwels, 2001(Poyarkov et al. 2023;this study).Despite the recent significant progress, our knowledge on molecular phylogeny, classification, distribution, and toxicology of pitvipers of the genus Trimeresurus in Thailand as well as Asia, is still far from being complete.Therefore, we call for more research, especially for integrative studies of taxonomically controversial species groups in Thailand, such as the members of the T. albolabris, T. macrops, T. purpureomaculatus, and T. popeiorum-sabahi species complexes.Further multilocus studies are also required to achieve a better phylogenetic resolution of evolutionary relationships within the T. kanburiensis and T. macrops complexes of the genus Trimeresurus, this would likely yield a better understanding of shifts between different morphotypes and habitat preferences during the evolution of this radiation of pitvipers.Finally, the medical importance of Trimeresurus pitvipers further underscores the need for future studies on the diversity and evolutionary relationships of this genus of snakes.et al., 2023, T. kanburiensis Smith, 1943, T. kuiburi Sumontha et al., 2021, and T. venustus Vogel, 1991 s. str.Remark: data for T. kuiburi from Sumontha et al. (2021).N/a = data not available.For abbreviations see Material and methods section.et al., 2023, T. kanburiensis Smith, 1943, T. kuiburi Sumontha et al., 2021, and T. venustus Vogel, 1991 s. str.Remark: data for T. kuiburi from Sumontha et al. (2021).N/a = data not available.For abbreviations see Material and methods section.
using Akaike information criterion (AIC).The optimal estimated models of DNA evolution included: GTR+I+G model for 16S rRNA and the second codon position of ND4, GTR+G model for the first codon position of cyt b, HKY+I+G model for the second codon position of cyt b and the first and the third codon positions of ND4, and HKY+I model for the third codon position of cyt b.When the same model was suggested for different codon partitions of a given gene, they were treated as a single partition, what resulted in four partitions in total.
1 mm.Abbreviations used were as follows: ED = horizontal eye diameter HD = maximum head depth HL = head length (from the tip of rostral to the posterior end of the jaw) HW = maximum head width SnL = snout length (from the tip of rostral to the anterior eye margin) SOL = supraocular length SOW = supraocular width SVL = snout-vent length TaL = tail length TL = total length VED = vertical eye diameter Meristic characters examined include: ASR = anterior number of dorsal scale rows (at one HL behind the head) CP = cloacal plate(s) DSR = dorsal scale rows IL = number of infralabial scales InN sep = internasal scales in contact (1) or not (0) IOS = interorbital scales, counted along a row between the middle of supraocular scales MSR = number of dorsal scale rows at midbody (at the level of the ventral plate corresponding to the half of the total number of ventrals) PosOc = number of postocular scale(s) PreV = number of preventrals (scales directly preceding the ventrals, unpaired, wider than long but not in contact on each side with the 1 st dorsal scale row) PSR = posterior number of dorsal scale rows (at one HL before anal plate) SC = number of subcaudal scales, not including the terminal pointed scute SL = number of supralabial scales SRR = dorsal scale rows reduction (counted following Dowling 1951a) VEN = number of ventral scales (counted following Dowling 1951b) : AUP = Agriculture University of Phayao, Phayao, Thailand NHMUK = The Natural History Museum, London, UK (formely BMNH) PSUZC = Prince of Songkhla University Zoological Collection, Songkhla, Thailand QSMI = Queen Saovabha Memorial Institute, Bangkok, Thailand ZMB = Zoologisches Museum für Naturkunde der Humbolt-Universität zu Berlin, Berlin, Germany ZMMU = Zoological Museum of Moscow State University, Moscow, Russia

Fig. 2 .
Fig. 2. Maximum Likelihood (ML) tree of the genus Trimeresurus Lacépède, 1804 derived from the analysis of 2427 bp of cyt b, ND4, and 16S rRNA mitochondrial DNA gene sequences.For voucher specimen information and GenBank accession numbers see Table 1.Numbers at tree nodes correspond to ML UFBS/BI PP support values, respectively.Colours of clades and locality numbers correspond to those on the map in Fig. 1.Photograph showing the new species Trimeresurus kraensis sp.nov.by P. Pawangkhanant.

Fig. 3 .
Fig. 3.The holotype of Trimeresurus kraensis sp.nov. in life (AUP-02036, adult female) from Wat Tham Sanook, Chumphon Province, Thailand.A. Dorsolateral view.B. Ventrolateral view.C. Close-up of dorsal scales.D. Left side of the head.E. Dorsal view of the head.F. Ventral view of the head.Photographs by P. Pawangkhanant.
1 (continued on next page).Main measurements and meristic characters of the examined specimens of Trimeresurus ciliaris Idiiatullina 2 (continued on next page).Main measurements and meristic characters of the examined specimens of Trimeresurus ciliaris Idiiatullina and later stored in 70% ethanol.Specimens were subsequently deposited in the herpetological collection of the School of Agriculture and Natural Resources, University of Phayao (AUP, Phayao, Thailand), and the Zoological Museum of Moscow State University (ZMMU, Moscow, Russia).Tissues for genetic analyses were taken from liver or heart prior to preservation of specimens, and stored in 96% ethanol.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 Chatmongkon Suwannapoom) and were strictly complacent 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 Chatmongkon Suwannapoom).

Table 1
(continued on next two pages).Sequences and voucher specimens of the genus Trimeresurus Lacépède, 1804 and outgroup taxa used in this study.

Table 1 (
continued).Sequences and voucher specimens of the genus Trimeresurus Lacépède, 1804 and outgroup taxa used in this study.

Table 1 .
(Malhotra & Thorpe 2004a;Idiiatullina et al. 2023)support values, respectively.Colours of clades and locality numbers correspond to those on the map in Fig. 1.Photograph showing the new species Trimeresurus kraensis sp.nov.byP.Pawangkhanant.TrimeresurusDiagnosisThe new species is assigned to the subgenus Trimeresurus based on the following morphological attributes: a long papillose hemipenis and partially fused first supralabial and nasal scales(Malhotra & Thorpe 2004a;Idiiatullina et al. 2023).The new species Trimeresurus kraensis sp.nov. is distinguished from all other species of the subgenus Trimeresurus by the following combination of morphological characters:(1) a dark olive-green or bottle green dorsum; (2) dorsal pattern consisting of about 60 reddish-brown or purple blotches, transversally elongate but not reaching the lower part of the flanks, two or three series of dark brown spots forming a discontinuous pattern on the 1 st to 3 rd dorsal scale rows and white vertebral spots present in males, located approximately on every two or four dorsal scales; (3) venter Pauwels et al. 2013Pauwels et al. 2013: 280.-Sumontha et al. 2021: 320.Trimeresurus cf.venustus -Sumontha et al. 2021: 321, fig.9B.Trimeresurus cf.venustus 1 -Idiiatullina et al. 2023: 699, 704.creamish-green with some dark brown spots; (4) tips of the ventral plates both cream and dark brown, forming a discontinuous, alternating pale and dark, ventrolateral stripe; (5) males with a reddish-brown postocular stripe; (6) internasals generally in contact behind the posteror tip of the rostral; (7) single large supraocular scale; (8) iris pale copper, (9) tail brown with dark purplish-brown crossbars; (10) dorsal

Table 2 .
Uncorrected p-distances (percentage)between the sequences of cyt b mtDNA gene of species of the subgenus Trimeresurus Lacépède, 1804 included in the phylogenetic analyses; genetic differentiation of the new species Trimeresurus kraensis sp.nov. is shown in bold.scales in 21-21-15 rows; (11) ventral scales 167 in a single male, 169-171 in females; (12) subcaudal scales 62 in a single male, 52-54 in females, all paired.

Table 4 .
Morphological data on the type series of Trimeresurus kraensis sp.nov.For abbreviations see Material and methods section.