Douglass Brown and Dr. Jack W. Sites Jr., Biology Department
Introduction
Among extant snakes (~3150 species), Caenophidia or “advanced” snakes are a monophyletic group comprising the great majority of species (~2620) of species (Vidal et al. 2010). The American caenophidian snake fauna comprises five families: the Viperidae and Elapidae – both displaying a front fanged venom system- and the colubroidean families Natricidae, Colubridae and Dipsadidae. The latter is one of the largest families of snakes (>700 species), with all living representatives restricted to the New World. Previous higher level studies generally agree on the delineation of three major clades of Dipsadidae broadly consistent in their distribution, a South American clade including the West Indies, (xenodontines sensu stricto), a Central American clade (dipsadines) and a North American clade (North American xenodontiines). Despite the fact that this last group includes only five genera (Carphophis, Contia, Diadophis, Farancia, and Heterodon) and nine species (with the possibility of several cryptic species) herpetologists have disagreed on the phylogenetic and systematic arrangement of these North American snakes for more than 70 years. Past studies have placed these snakes within the Dipsadidae, the Xenodontidae, the Natricidae, the recognition of new families (Carphophinae and the Heterodontinae), or distributed them across multiple families. (Cadle 1984; Pinou et al. 2004; Lawson et al. 2005; Zaher et al. 2009; Vidal et al. 2010) (Figure 1). Because the phylogenetic position of these snakes remains controversial, they have been termed as “relicts” and are assumed to lack closely related living relatives. However, the confusion surrounding these snakes could be due to a limited sampling, the use of limited molecular data or different molecular markers, or different topologies resulting from different analytical methods. Most studies have focused on the use of a single locus (the mitochondrion) or the combination of a mitochondrial data and a single nuclear locus. This is the first study to: (1) include all “relict” genera and species, and (2) incorporate multiple independent loci, to infer the phylogenetic relationships of the North American xenodontines and their relationships to other New World xenodontine and dipsadine snakes.
Laboratory and Phylogenetic Methods
Specimens were either borrowed from museum collections or collected and euthenized humanely in the field (Fig. 2). Total genomic DNA was isolated using either a standard phenol-chloroform protocol or a Qiagen DNeasy kit. The polymerase chain reaction (PCR) was used to amplify fragments of five genes including the complete mitochondrial cytochrome b (cyt b) gene (1117 bp), and four nuclear genes; Cmos (617 bp), NT3 (557 bp), GAPDH (336 bp), and Enolase (201 bp). PCR conditions were generally consistent across loci with adjustments made to annealing temperatures. Approximately 1–2 μl of DNA template were included in 25 microliter PCR reactions containing the following: 2.5 microliters each of 10 X Amplitaq PCR buffer (Perkin Elmer, Boston, Massachusetts), MgCl (25 mM), and 1.0 microliters of deoxynucleotide triphosphates (dNTP’s,10 mM), 1 microliter of each primer (10 microM), and 0.1 microliter of Amplitaq DNA polymerase (5 U/μl). Thermocycler conditions were 95°C for 2 min followed by 35 cycles at 95°C for 30 s, annealing 30 s, and extension at 72°C for 600 s. Annealing temperatures were 47°C (cytochrome b), 52°C (Enolase), and 50°C (NT3, GAPDH, C-mos).
Approximately 3 microliters of cleaned PCR product was sequenced using ABI BigDye chemistry on an ABI 3730 automated sequencer at the BYU Sequencing Center. Alignments were conducted using MUSCLE and edited by eye. For phylogenetic analyses, the best-fitting model of nucleotide substitution was selected for each gene fragment using the Akaike information criterion (AIC) following the procedure outlined by Posada and Buckley (2004), and implemented in jModelTest v0.1.1 (Posada, 2008) (Table. 1). We conducted partitioned Bayesian analyses using MrBAYES v.3.1.2b (Ronquist and Huelsenbeck, 2003) in which the appropriate model was applied to each gene fragment. Each Markov chain was started from a random tree and run for 1.0×107 generations with every 1000th tree sampled from the chain. All sample points prior to reaching the plateau phase were discarded as burn in and the remaining trees combined to find the a posteriori probability estimate of phylogeny. Branch lengths were estimated as means of the posterior probability density.
Results and Discussion
We obtaiend sequence data for twenty five snake species including all five genera of the North American xenodontines. The combined mixed-model analyses produced a 50% majority-rule consensus tree with a harmonic mean of –17964.31 following a burnin of 20000 generations. In contrast to the work of Pineou et al. (2004), our partitioned bayesian analysis inferred the wellsupported monophyletic clade for the North American xenodontines with each genus recovered as monophyletic, validating the sub-family Heterodontinae (sensu Bonaparte). However, the relationships within the Heterodontinae differ from previous studies. Our topology places Diadophis punctatus as the most basal genus, (which is largely in agreement with Cadle 1984), as opposed to previous phylogenetic studies which in inferred Diadophis as nested within the Heterodontinae. Furthermore, our analysis show strong support for a Heterodon(Carphophis+Farancia) clade. These results do not support the newly designated Carphophinae family (Zaher et al. 2009) and based on our results caution against the continued use of their taxonomy.
Future Work
Add additional taxa from both Central and South America. Incorporate additional nuclear genes. Estimate the dates of divergence for each node using fossil calibrations. Examine the evolution of hemipenial morphology. Publish work (expected in review by the end of 2012).
Literature Cited
- Cadle, J.E. 1984. Molecular systematics of Neotropical xenodontine snakes. III. Overview of xenodontine phylogeny and the history of New World snakes. Copeia, 1984:641-652.
- Lawson, R., Slowinski, J.B., Crother, B.I. & Burbrink, F.T. 2005. Phylogeny of the Colubroidea (Serpentes): new evidence from mitochondrial and nuclear genes. Molecular Phylogenetics and Evolution, 37:581-601.
- Pinou, T., Vicario, S., Marschner, M. & Caccone, A. 2004. Relict snakes of North America and their relationships within Caenophidia, using likelihood-based Bayesian methods on mitochondrial sequences. Molecular Phylogenetics and Evolution, 32:563-574.
- Posada, D. 2006. Collapse: describing haplotypes from sequence alignments <http://darwinuvigoes/software/collapse.html>
- Ronquist, F., Huelsenbeck, J.P., 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.
- Vidal, N., Dewynter, M. and & Gower, D.J. 2010. Dissecting the major American snake radiation: A molecular phylogeny of the Dipsadidae Bonaparte (Serpentes, Caenophidia). Comptes Rendus Biologies, 333:48-55.
- Zaher, H., Grazziotin, G.F., Cadle, J.E., Murphy, R.W., Moura-Leite, J.C., Bonatto, S.L. 2009. Molecular phylogeny of advanced snakes (Serpentes, Caenophidia) with an emphasis on South American Xenodontines: a revised classification and descriptions of new taxa. Papeis Avulsos de Zoologia. 49:115-152.