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MULTIGENE PHYLOGENETIC ANALYSIS OF PITVIPERS, WITH COMMENTS ON THEIR BIOGEOGRAPHY CHRISTOPHER L. PARKINSON1, JONATHAN A. CAMPBELL2, AND PAUL T. CHIPPINDALE2 ABSTRACT: Recent attempts to determine the phylogenetic relationships among pitvipers (Crotalinae) have continued to improve understanding of their evolutionary history. A robust phylogeny of pitvipers will help elucidate their biogeographical history. Evolutionary relationships of snakes in the subfamily Crotalinae were investigated using multiple mitochondrial gene regions (12S and 16S rDNA, ND4, and cyt-b for a total of 2,341 bp). Representatives of all but two currently recognized pitviper genera (Ermia and Triceratolepidophis) were included. Two members of the Causinae, two genera of the Viperinae, and Azemiops feae, were included as outgroups for a total of 61 taxa. Individual gene analyses resulted in topologies that were largely unresolved, and for which support for many nodes was weak; combining all regions for a “total mitochondrial molecular evidence” approach yielded a well-resolved phylogenetic framework with strong support for many relationships. Combined analyses strongly support monophyly of the New World taxa. A clade containing Gloydius and Ovophis monticola was found to be the sister group of the New World pitvipers in three of four analyses. These results suggest a single invasion into the New World. This emigration event (presumably via the Bering Land Bridge) probably occurred during the early Tertiary or late Cretaceous. A subsequent divergence resulted in a North temperate group and a Neotropical group with at least five subsequent dispersals into South America. All currently proposed New World genera are monophyletic except Bothrops and Sistrurus, which are paraphyletic with regard to Bothriopsis and Sistrurus ravus, respectively. The Old World genus Trimeresurus (sensu lato) is polyphyletic; recognition of Protobothrops and Tropidolaemus, along with Trimeresurus (sensu stricto), yields three monophyletic genera. Ovophis appears to be polyphyletic; however, a more in-depth study is required before taxonomic revisions can reliably be made. A clade containing Calloselasma, Deinagkistrodon, Hypnale, and Tropidolaemus appears to represent the sister group to all other crotalines. The geographic distribution of this group and monophyly of New World pitvipers is consistent with a Eurasian center of origin for the Crotalinae. INTRODUCTION logically distinct monophyletic lineages as separate The snake family Viperidae currently contains four genera, even if these lineages may be phylogenetically subfamilies: Causinae, Viperinae, Azemiopinae, and nested within larger, often unwieldy species groups. Crotalinae (McDiarmid et al., 1999). The phylogenetic Many phylogenetic studies of the group have been relationships among the subfamilies have not been based on morphological data, but morphological determined conclusively, but the prevailing view is approaches are limited by the fact that snakes are that Causinae is sister to a group that consists of morphologically conservative, and much of the mor- Viperinae, Azemiopinae, and Crotalinae, and that phological variation that is present is the result of Viperinae is sister to Azemiopinae + Crotalinae reduction and simplification of homologous structures (McDiarmid et al., 1999). The Crotalinae, characterized (Keogh, 1998). This simplification is often reflected by the presence of heat-sensory loreal pits, contains by varying degrees of convergence, which makes approximately 150 species (McDiarmid et al., 1999). determination of evolutionary relationships difficult. In the last three decades, this group has been the focus Until recently, few workers have addressed inter- of numerous systematic studies, using both morpho- generic relationships within this diverse group of logical and molecular data (e.g., Parkinson, 1999; snakes, with the exceptions of Brattstrom (1964), Salomão et al. 1999; Vidal et al., 1999; Werman, 1999; Burger (1971), and Werman (1992). However, with Malhotra and Thorpe, 2000). Currently, 19–22 generic advances in molecular systematics, numerous mole- names are in use, and approximately 13 of those have cular studies on generic relationships of pitvipers have been recognized within the last 30 years (e.g., Hoge been published within the last few years (e.g., and Romano-Hoge, 1981, 1983; Campbell and Lamar, Parkinson, 1999; Malhotra and Thorpe, 2000). 1992; Werman, 1992; Zang, 1998; Ziegler et al, 2000; Even with the many recent studies, the phylogenetic Gutberlet and Campbell, 2001). The recent trend in relationships within the subfamily Crotalinae are still pitviper systematics has been to recognize morpho- controversial (e.g., Parkinson, 1999; Malhotra and Thorpe, 2000). One reason for the inconsistency among results of the studies is that only two have 1Department of Biology, University of Central Florida, 4000 included most or all of the proposed crotaline genera Central Florida Blvd., Orlando, Florida 32816, USA E-mail: [email protected] (Kraus et al., 1996; Parkinson, 1999). Limited taxo- 2Department of Biology, The University of Texas at Arlington, nomic sampling can be problematic in phylogenetic Arlington, Texas 76019, USA 94 C. Parkinson, J. Campbell, and P. Chippindale analyses (Hillis, 1998), and when only a few repre- philosophical and practical implications of this “total sentatives of a diverse group are sampled, the result- molecular evidence” method have been highly debated ing phylogenies may reflect the taxonomic sampling in the literature (e.g., Bull et al., 1993; Chippindale rather than accurately portraying genealogical rela- and Wiens, 1994; de Queiroz et al., 1995; Huelsenbeck tionships. Another problem may be that the sequence et al., 1994), but in practice it has led to a new syn- data utilized are highly homoplastic for the level of thesis in numerous groups (e.g., Mindell et al., 1999; divergence being studied. Parkinson et al., 1999; van Tuinen et al., 2000). Certain groupings are common among the recent Accordingly, we used a molecular data set based on molecular studies. Several studies indicate that New multiple mitochondrial genes, incorporating both new and Old World Agkistrodon do not form a clade data and sequences obtained from databases, to inves- (Knight et al., 1992; Kraus et al., 1996; Cullings et al., tigate the generic relationships of pitvipers and evaluate 1997; Parkinson et al., 1997; Parkinson, 1999). Hoge competing biogeographical hypotheses that attempt to and Romano-Hoge (1981) proposed Gloydius for the explain the current distribution of pitvipers. Asiatic Agkistrodon, and we recommend adoption of their classification. A close relationship between MATERIALS AND METHODS Calloselasma and Hypnale was found by Kraus et al. Sampling and Laboratory Protocols (1996) and confirmed by Parkinson et al. (1997) and The ingroup contained 56 species of pitvipers, includ- Parkinson (1999). The genus Trimeresurus (sensu ing all genera except Ermia and Triceratolepidophis; lato) is not monophyletic (Kraus et al., 1996; two recently proposed monotypic genera of the Parkinson, 1999; Malhotra and Thorpe, 2000). Trimeresurus complex. We used two species of the However, if one recognizes Protobothrops, as sug- Causinae, two genera of Viperinae, and Azemiops feae gested by Hoge and Romano-Hoge (1983), monophyly (Azemiopinae) as outgroups (see Table 1 for voucher is established for the remaining species of Trimeresurus information and GenBank accession numbers). (but see discussion below regarding Ovophis). Kraus Fragments of four mitochondrial genes were et al. (1996) suggested that New World pitvipers are sequenced for this analysis: 12S and 16S rDNA, monophyletic; Parkinson (1999) and Vidal et al. NADH dehydrogenase subunit 4 (ND4), and (1999) supported their conclusions, although this cytochrome b (cyt-b). Because these genes evolve at clade was not strongly supported in either study. In all different rates and exhibit different levels of variability, analyses by Parkinson (1999), a sister group relation- we expected that in combination they would resolve ship between the rattlesnakes (Crotalus and Sistrurus) relationships at various depths in the phylogenetic tree. and copperheads/moccasins (Agkistrodon) was estab- Genomic DNA was isolated from whole blood, lished, whereas this result was supported in only one liver, or epidermal tissue samples by standard pro- of the analyses by Kraus et al. (1996). Vidal et al. teinase-K digestion followed by organic purification (1999) also found this relationship, but only a single (Knight et al., 1992). The ND4 region was amplified species of each group was used. Few relationships as in Parkinson et al. (2000), and the ribosomal genes within the bothropoid genera are supported by mul- were amplified as in Parkinson et al. (1997) and tiple studies. Exceptions are the close relationship Parkinson (1999). The cyt-b region was amplified between Bothrops (sensu stricto) and Bothriopsis using the primers Gludg (5'-TGA CTT GAA RAA (but see discussion in Parkinson, 1999), the realloca- CCA YCG TTG-3'; Palumbi, 1996) and ATRCB3 tion of Porthidium melanurum into Ophryacus (5'-TGA GAA GTT TTC YGG GTC RTT-3'), fol- (Gutberlet, 1998; Parkinson, 1999), and the recognition lowing the protocol described in Parkinson et al. of Porthidium hyoprora as a lineage that diverged (2000) for the ND4 gene. In a few cases, PCR ampli- early within Bothrops (Kraus et al., 1996; Parkinson, fications could not be sequenced satisfactorily.