Volume 14, Number 1 January 2004 ISSN 0268-0130 THE HERPETOLOGICAL JOURNAL Published by the Indexed in BRITISH HERPETOLOGJCAL SOCIETY Current Contents HERPETOLOGICAL JOURNAL, Vol. 14, pp. 21-33 (2004) REASSESSMENT OF THE VALIDITY AND DIAGNOSIS OF THE PITVIPER TRIMERESUR US VE NUSTUS VOGEL, 1991 ANITA MALHOTRA AND ROGER S. THORPE School of Biological Sciences, University of Wa les Bangor, Bangor, UK Trimeresurus venustus Vogel, I 99 I was described from southern Thailand in I 991 ' and distinguished from the similar T. kanburiensis primarily by the following characters: 21 scale rows at midbody rather than I 9 and less irregular and indented supraoculars. However, very few specimens of T. kanburiensis were known at the time of this description, and the name T. venustus has not been universally accepted. Recently, live specimens from the type locality of T. kanburiensis in western Th ailand have become available, allowing a reassessment of the status -of the southern Thai population. Phylogenetic analysis of two mitochondrial gene regions indicated that specimens from south Thailand are genetically quite distinct from the specimen from the type locality, and the former are more closely related to T. macrops than to T. kanburiensis. We present a multivariate morphometric analysis of the six specimens of T. kanburiensis from the type locality that are now known and twenty specimens from southern Thai land. Despite the small sample size, it is clear that some of the diagnostic characteristics used to define T. venustus are invalid. We conclude that the current evidence indicates that T. venustus is a valid species, and present new diagnostic characters to separate it from T. kanburiensis. Key words: Crotalinae, systematics, Thailand, Tr imeresurus kanburiensis, Yiperidae INTRODUCTI ON However, the name T. venustus has not been widely accepted. Among recent checklists ofvenomous snakes The genus Trimeresurus (Serpentes: Viperidae: it has been listed by Golay et al. (1993) and David & Crotalinae) contains many taxonomically vexing issues lneich (1999) but not by McDiarmi d et al. (I 999) or the that are still awaiting resolution (Malhotra & Thorpe, EMBL taxonomy database (http://www.embl­ 1997, 2000). Recently, molecular data have promised to heidelberg.de/�uetz/familiesNiperidae.html). resolve some of these issues. One example is the status Specimens of T. kanburiensis that were available to of the taxa T. kanburiensis Smith 1943 and T. venustus Vogel for comparison were in poor condition. The Vogel 1991. The holotype of T. kanburiensis (a female) holotype of kanburiensis is in two pieces and clearly was collected in 1938 from Kancbanaburi (then known T. bas a section of body and the tail tip missing (also noted as Kanburi) province in western Thailand (Fig. 1). It by Warrell et al., 1992). The head is very distorted and was at first identified as T. puniceus and only described squashed, torn skin on one side, and any patternhas as a new species in 1943 (Smith, 1943). It remained the with entirely faded. One of the two additional specimens only specimen available for the species until the late available is also almost in three pieces and the colour 1980s. In the interim, confusion developed over the pattern has faded considerably in both (Wanell et al., identity of this species following specimens of another 1992). Warre ll et al. ( 1992) doubted that venustus species T. purpureomaculatus, that occurs in the same T. was a different species to T. kanburiensis, after finding a region, being mistakenly labelled T. kanburiensis in fourth specin1en of kanburiensis from Kanchanaburi books and by dealers in the captive trade (see Warrell et T. province in 1991 (the only male), in which the colour al., 1992, for details) . In the late 1980s, two additional pattern was well preserved and appeared to be identical specimens, also females, were found in Kanchanaburi to that of the southern population. Province (Warrell et al., 1992). Specimens apparently Further doubt was cast on the validity of T. venustus referable to the species were also found in southern when, during fieldwork in Thailand in the 1990s we Thailand, in Nakhon Si Thammarat and Krabi prov­ found several specimens in the vicinity of the Kllao inces. Ironically, some of these specimens found their Luang massif, near Nakhon Si Thammarat (Fig. 1), that way into the captive trade labelled "T. also had 19 scale rows at mid-body. One of these, a purpureomaculatus" (Warrell et al., 1992). Vogel roadkill, bas since been presented to the Natural History ( 1991) described this southern population as T. Musuem, London (BMNH 2002.52). A specimen from venustus, citing the following diagnostic characters to Surat Thani with the same character state was also seen separate it from T. kanburiensis: 21 scale rows at mid­ by the senior author at the Queen Savoabba Memorial body rather than 19; narrower, less indented and Institute. Therefore, the status of T. venustus required divided, supraoculars; slighter body and a distinctive further verification (Malhotra quoted in Gumprecht, brownish-red banded colour pattern. 2002a ). Recently, a number of live specimens of T. Correspondence: A. Malhotra, School of Biological . kanburiensis have become available fromthe type local­ Sciences, University of Wales, Bangor, Gwynedd, LL 57 ity. One ofthese (a female) was sent to the authors by the 2UW UK.E-mail: [email protected] 22 A. MALH OTRA AND R. S. THORPE et al., ,.,.,.,. T;. ttAr:.l-·· ... ... ard protocols (Sambrook 1989). Cytochrome b 100°E• •. t.•'·· N'\ 1. b ;•,. , ( cyt ) sequences were obtained as described in '"'"""'''''e""'"'20'N f:",•.'. 00-f•••:1•ooooooo"''"""'''"'"""'""'""i""'""' .. ...-·� Malhotra & Thorpe (2000), NADH dehydrogenase ,,.._...... � \ · ..'*' ...' ...) (ND4) as described in Parkinson et al. (2000) and l 2S ) : \ ,6<\o \ l { ··� .. ,i small subunit ribosomal RNA (12S) as described in �. ... .. -.f\, l: (\ t•. ./("''· . \ �v • _.. : \. Knight Mindell ( 1993 ). Unincorporated nucleot ides ' :; , fr ""••, ... fi, & \ !/ � \ � � i �, and primers were removed using a variety of conm1er­ ·., : i ''I, cially available kits (e.g. Prep-a-gene [Biorad], Wizard ., l i 'l \ : '· �..,.L mi nicolumns [Promega], or QIAquick columns � : \· ·? [QIAGen]). The double-stranded product was r... ... �� -t sequenced using dye-labelled terminators (ABI ( '. Erawan Natl. Park t1 PRISMTM BigDyeTM Terminator Cycle Sequencing : '· •• -.... ... -·-.. ...... .. ', • \ \''.'•" ...�� .:i ... , Ready Reaction Kit), and subsequently mn on an ABI � Kanchanaburi / -<! ":i Saiyok military camp Prism 377 automated DNA sequencer. • • l � \ I., PHYLOGENETIC ANALYSIS \: ) � . r·1·· J Malhotra & Thorpe (2000) presented a phylogeny of (� Tr imeresurus sensu la to ! � 21 species of ( ) based on cyt b sequences, and evaluated the taxonomic value of certain . '.: .. : moqJhological characteristics against th is tree. On this 10'N ... .. �::�:�.·�.-� /�·;L;;�;�; . ( basis, four species groups were defined within l ;------ Tr imeresurus sensu stricio, which are diagnosed by a ... Nakhon Si Tha.mmarat ::,../ . :l combination oft.he condition of the fi rst upper labial and Th"" nasal scale (fused or separate) and the hemipenial strnc- ' T. '""'"'Ion>!' 1ure . By these criteria, the T kanburiensis!T venustus l • T. venustus complex is a part of the a/bolabris species group, al­ '.""- ·.. A. T. venusius with 19 scale_j l 1"1 / rows at mid-body though i1 is qu it e genetically divergent and apparently 1(fl. o {1•''"°'1' -�-·----· diverged eariy in the history of the species group. \V e FlG. I. Map of Thailand, showing the location of specimens. therefore analyse a number of species from the albolabris Circles represent specimens referable to T venustus (with 21 species group, as well as representatives of scale rows at mid-body); triangles indicate the specimens the other three species groups, together witJ1 one speci­ from the south with 19 scale rows; squares indicate men of T. kanburiensis, and four specimens of T specimens referred to T. kanburiensis. The type locality was venusius, including all three specimens from the Khao not precise, referring only to limestone hills in the vicinity of Kanchanaburi near the Tennaserim border. Note that the Luang area with J 9 scale rows at mid-body. A fu ll list of position of the Saiyok military camp where two specimens of sequences included and their origins is listed in Table 1. T kanburiensis were located, is diffe rent from that in Fig. I The coding sequences were fi rst translated into of Warrell 1992), wh ich showed the posit ion of Saiyok et al. ( amino acid sequences using MEGA version 2 .1 (Kumar village rather than the military camp (which is, confusingly, al, not near Saiyok village). et 2001) to check for the unexpected occurrence of stop codons which might indicate amplification of owner and is the first specimen available for DNA p�uedogenes. The possibility of non-neutral evolutjon analysis. I1 has since been presented to tl1e Natural His­ was tested using a variety of tests implemented in the tory Museum, London (BMNH 2002.51 ). In th is paper, program DnaSP 3.5 1 (Rozas & Rozas, l 999), including we present a phylogenetic analysis ofmj1ochondrial se­ McDonald and Kreitman 's (1991) 1est, Fu and Li 's D* quences to evaluate the relationship of populations and F*, and their modifications foruse with an outgroup venustus referable to T to that from the type locality of sequence (Fu & Li 1993), and Tajima's D (Tajima, kanburiensis. T \V e also evaluate their morphological 1989). simi larity by eonducting a multivariate morphometric We used both unweighted parsimony and Bayesian analysis of scalation, head shape and colour pattern, Markov Chain Monte Carlo (MCMC) approaches to re­ along with an analysis of internal characters (tooth num­ construct phylogenies, using PAUP* 4 .Ob8 (Swofford, bers and position of the major internal organs) to 2001) and MrBay es (Huelsenbeck & Ronquist, 2001) evaluate the diagnostic chara cters proposed by Vogel respectively. We first checked the dataset for homoge­ (1991 ). neity of base composition among taxa, to detect MATERIALS AND METHODS problems with the assumption of a similar underlying substitutional model.