MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 24 (2002) 194–202 www.academicpress.com Snake phylogeny: evidence from nuclear and mitochondrial genes Joseph B. Slowinskia and Robin Lawsonb,* a Department of Herpetology, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118-4599, USA b Osher Foundation Laboratory for Molecular Systematics, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118-4599, USA Received 24 July 2001; received in revised form 3 May 2002 Abstract We constructed phylogenies of snakes from the c-mos and cytochrome b genes using conventional phylogenetic methods as well as the relatively new method of Bayesian inference. For all methods, there was excellent congruence between the c-mos and cy- tochrome b genes, implying a high level of support for the shared clades. Our results agree with previous studies in two important respects: first, that the scolecophidians and alethinophidians are monophyletic sister clades; and second, that the Colubroidea is a monophyletic group with the Acrochordidae as its sister clade. However, our results differ from previous studies in the finding that Loxocemus and Xenopeltis cluster with pythons. An additional noteworthy result from our data is that the genera Exiliboa and Ungaliophis, often placed with Tropidophis (and Trachyboa, not included in the present study) in the Tropidophiidae, are in reality boids. Ó 2002 Elsevier Science (USA). All rights reserved. 1. Introduction Heise et al., 1995) show little similarity to each other or to the morphological studies. Despite considerable recent interest in the evolution Given the continuing uncertainty about snake phy- of snakes (Caldwell and Lee, 1997; Cohn and Tickle, logeny, we inferred phylogenies from a broad sample of 1999; Greene and Cundall, 2000; Lee et al., 1999; snakes using the mitochondrial cytochrome b and nu- Scanlon and Lee, 2000; Tchernov et al., 2000), the clear c-mos genes. Johnson (2001) has shown that the phylogenies of snakes remain poorly known. An exam- cytochrome b gene can be applied to divergences as old ination of recent morphological (Cundall et al., 1993; as the separation of passeriforms from other birds. Saint Kluge, 1991; Rieppel, 1998; Scanlon and Lee, 2000; et al. (1998) have demonstrated the usefulness of the Tchernov et al., 2000) and molecular (Dowling et al., c-mos gene for the relationships of squamate reptiles. 1996; Heise et al., 1995) snake phylogenies reveals con- The data were analyzed with the conventional phylo- siderable differences among these studies. The morpho- genetic methods of maximum parsimony (MP), mini- logical studies (Cundall et al., 1993; Kluge, 1991; mum evolution (ME), and maximum likelihood (ML), Rieppel, 1998; Scanlon and Lee, 2000; Tchernov et al., as well as the relatively new method of Bayesian infer- 2000) do agree in two respects: first, that the blind ence (Yang and Rannala, 1997). snakes, known as scolecophidians, are the sister clade to Being on different chromosomes, the mitochondrial all other snakes, known collectively as the alethinophi- cytochrome b and nuclear c-mos genes have had inde- dians; and second, that within the alethinophidians, the pendent gene histories and form linkage partitions Boidae, Pythonidae, Tropidophiidae, Bolyeriidae, Ac- (Slowinski and Page, 1999). Data from independent rochordidae, and Colubroidea collectively form a genes can be analyzed either by combining all the data monophyletic group to the exclusion of the Aniliidae, or by keeping the genes separate (de Queiroz et al., Anomochilidae, Uropeltidae, Xenopeltidae, and Lo- 1995), approaches that each have their advantages and xocemidae. The molecular studies (Dowling et al., 1996; disadvantages (Slowinski and Page, 1999). When the gene trees relating the sequences from two linkage par- titions are identical, it is advantageous to combine the * Corresponding author. Fax: 415-750-7013. data, assuming that the data are consistent. On the other E-mail address: [email protected] (R. Lawson). hand, if there is evidence that the gene trees relating the 1055-7903/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S1055-7903(02)00239-7 J.B. Slowinski, R. Lawson / Molecular Phylogenetics and Evolution 24 (2002) 194–202 195 sequences from two linkage partitions are not identical, Table 1 combining the data is contraindicated, primarily be- List of taxa sequenced for this study, GenBank Accession Numbers, cause it gives heavier weight to the gene with a larger and tissue sources number of nucleotides (Slowinski and Page, 1999). By Taxon GenBank Source analyzing linkage partitions separately, one gains the Accession No. major advantage of the significance possessed by shared clades on the trees derived from the partitions: there is a Scolecophidia Leptotyphlopidae high probability that shared clades correspond to real Leptotyphlops humilis CAS 190589 clades on the species phylogeny (Miyamoto and Fitch, Typhlopidae 1995). The rationale behind this is very simple: the Ramphotyphlops braminus CAS 184353 probability that two random trees will share any clades Typhlops brachycephalus CAS 200736 is quite low (Hendy et al., 1988). Hence, gene trees from Alethinophidia Aniliidae two linkage partitions that share a number of clades are Anilius scytale /U69738 LSUMZ H-14435/GB clearly non-random. And because these two partitions Cylindrophidae do not share the constraint of a linked gene history, the Cylindrophus ruffus LSUMZ 12363 constraint must be the shared species phylogeny thereby Uropeltidae revealing clades on the species phylogeny. Uropeltis phillipsi LSUMZ H-5788 Xenopeltidae Because of evidence that the cytochrome b and c-mos Xenopeltis unicolor CAS 204861 sequences from our sample of snakes are not related by Loxocemidae identical gene trees, we have analyzed the date sepa- Loxocemus bicolor LSUMZ H-6319 rately. In doing so, we find excellent congruence, which Pythonidae we use as the basis for creating a robust, albeit incom- Antaresia childreni No voucher Python molurus No voucher pletely resolved, phylogeny for the major groups of Boidae snakes. Acrantophis dumerili /U69735 No voucher/GB Boa constrictor No voucher Candoia carinata No voucher 2. Materials and methods Charina bottae CAS 206040 Epicrates striatus /U69799 No voucher/GB Eryx johni CAS 200907 2.1. Specimens examined Eunectes murinus /U69808 No voucher/GB Lichanura trivirgata CAS 200649 We sequenced the mitochondrial cytochrome b and Sanzinia madagascariensis /U69866 No voucher/GB part of the nuclear c-mos genes using a sample of 42 Bolyeriidae Casarea dussumieri /U69755 No voucher/GB snake species, representing two of the three extant Tropidophiidae scolecophidian families and 13 of the 15 extant alethi- Exiliboa placata UTACV R 44894 nophidian families (Table 1). The Anomochilidae Tropidophis haetianus /U69869 No voucher/GB (Cundall et al., 1993) and Xenophidiidae (Wallach and Ungaliophis continentalis /U69870 No voucher/GB Gunther, 1998) were not included because tissues were Acrochordidae Acrochordus granulatus No voucher not available. For the purpose of tree rooting we chose Colubroidea the outgroup method (Watrous and Wheeler, 1981). Colubridae Among extant lizards it is generally accepted that the Cerberus rhynchops CAS 206574 sister taxon to the snakes lies among the angu- Gonyosoma oxycephala No voucher inomorphs, though which is unknown and controversial Helicops angulatus LSUMZ H-3346 Hydrops triangularia LSUMZ H-3105 (Bellairs, 1972; Bellairs and Underwood, 1951; Gorman Hypsiglena torquata CAS 206337 and Gress, 1970). We have included two anguinomorph Leioheterodon modestus LSUMZ H-1991 lizards (Table 1) as outgroups. Meleya unicolor No voucher Oligodon cinereus CAS 205028 2.2. Molecular techniques Pareas macularius CAS 206620 Phyllorhynchus decurtatus No voucher Pseudoxenodon ROM 30627 For some snakes, donors provided us with previously karlschmidti extracted total genomic DNA. However, in most in- Regina rigida CAS 165994 stances, we extracted DNA from liver tissue or shed skin Rhabdophis tigrinus LSUMZ 37418 by the standard method of proteinase K digestion in Xenochrophis punctulatus CAS 201594 Viperidae lysis buffer, followed by phenol/chloroform extraction Crotalus viridis CAS 200713 (see Burbrink et al., 2000, for details). Template DNA Atheris nitschei CAS 201709 for the polymerase chain reaction (PCR) was also pre- Elapidae pared as in Burbrink et al. (2000). We amplified the Bungarus fasciatus CAS 207988 196 J.B. Slowinski, R. Lawson / Molecular Phylogenetics and Evolution 24 (2002) 194–202 Table 1 (continued) chrome b gene varies considerably among snake species Taxon GenBank Source due to terminal indels, we only used the first 1110 sites Accession for the analyses. Because application of the Templeton No. non-parametric test to the most parsimonious c-mos and Notechis ater SAM R 31604 cytochrome b gene tree (see Section 3) reveals that these Outgroups two genes have non-identical histories, we analyzed the Anguidae genes separately for all analyses. Likelihood tests Anguis fragilis CAS 173016 Shinisauridae (Goldman et al., 2000) were not applied to the likeli- Shinisaurus crocodilurus No voucher hood trees because of the time constraint involved in Institutional abbreviations. CAS, California Academy of Sciences, simulating the null distribution. We constructed phy- Department of Herpetology; LSUMZ, Louisiana State University, logenies of snakes using ME, MP, and ML, as well as Museum of Natural Science;