Dean H Leavitt and Dr. David A McClellan, Integrative Biology

DNA sequences provide a wealth of information that allows systematists to better understand the phylogenetic history of the earth’s current biota. Some genes are better suited for phylogenetic studies than others, and one that has been commonly used is the mitochondrial gene cytochrome b. Cytochrome b (cyt-b) is a membrane-bound protein that is the central catalytic subunit of the Q-cycle in the electron transport chain (Zhang et al., 1998). The structure and function of cyt-b has been studied extensively and is well understood (Degli Esposti et al., 1993). Changes within cyt-b have been studied at the nucleotide, amino acid and domain levels. Previous studies have shown the distinct functional domains to have differing evolutionary rates because of the different imposed constraints that influence the evolution of the domains (Irwin et al. 1991; Griffiths, 1997; McClellan and McCracken, 2001).

Elapidae is a family of venomous snakes characterized by fixed front fangs. Well-known members include mambas, cobras, sea snakes and coral snakes. This diverse family is a fascinating group to study for a number of reasons. For example, within this group multiple radiations of sea snakes have evolved marine adaptations independently, including both oviparous and viviparous clades. The diverse properties and effects of their neurotoxic venom have been investigated in physiology. Coral snakes are involved in a complex and intriguing mimicry systems in Neotropical snake communities.

But while molecular and statistical methods are used extensively to create phylogenetic trees, little research is conducted on the selective influences that cause proteins to evolve. My project was to not only examine the evolutionary relationships of this family by deriving a phylogeny, but to use that knowledge to investigate the selective forces acting on the different domains of the cytochrome b gene.

For this project complete sequences from a previous study were used to reconstruct a cladogram for the snake family Elapidae. The data were analyzed by neighbor-joining, maximumlikelihood, and Bayesian posterior-probability methods. Perhaps due to the different methods employed, the results of these analyses vary from the most recent study published on elapid phylogeny (Slowinski and Keogh, 2000). The primary conclusions of the phylogenetic analysis include: (1) coral snakes are recovered as the sister taxon of the marine/Australo-Papuan radiation; (2) the “laticaudine” sea kraits are basal to the remaining marine/Australo-Papuan taxa; and (3) further work is needed to resolve relationships among the genera Dendoraspis, Ophiophagus, Elapsoidea and Bungarus.

I found the results of the phylogenetic analysis to be the most interesting because they unexpectedly and significantly differed from the current understanding of elapid evolution. Future work clearly remains to be done to understand the taxonomic relationships of these snakes. Follow-up analyses could include a more thorough sampling of taxa as well as using different genes. The main obstacle I encountered during my research was waiting for the neighbor-joining analysis to finish. The computer required about three months to complete it, and I could not begin to investigate the molecular evolution until I had finished the phylogenetic analysis. And unfortunately when the neighbor-joining analysis was done, it did little to resolve the relationships among the different taxa.

To investigate molecular evolution of cyt-b, a substitution analysis was performed as in McClellan and McCracken (2001) using the dynamics implied by the topology of the phylogenetic tree. Briefly, each branch of the tree was analyzed by comparing the sequence at each branch terminus with its immediate ancestral sequence. Using the program TreeSAAP, version 1.0 (McClellan and Woolley, 2002), the estimated number and kind of substitutions was inferred from the reconstructed ancestral sequences. TreeSAAP determines the characteristics of each inferred nonsynonymous substitution, including codon position, type of base exchange, and exact location on the protein of each. These changes were also classified as to the functional domain in which each was located, as well as the magnitude of inferred changes relative to thirty-one physicochemical amino acid properties. These results were statistically analyzed relative to predicted distributions based on the expectations of completely random amino acid replacement. Those amino acid properties that significantly deviated from random expectations were further analyzed for the inherent order indicative of selective influences. These influences were further characterized by their magnitude and direction.

Patterns of evolution of cytochrome b in Elapidae are similar to what has been reported in other groups that have been studied in detail (e.g., mammals), in which the intermembrane region is the most constrained of the three domains. The matrix region experienced positive selection, which deviates from the results of other published studies. Whether these results indicate that the matrix domain in these snakes is behaving uniquely in exhibiting positive selection cannot yet be determined.

This project was one of the most significant learning experiences of my undergraduate studies. While I was a little disappointed with somewhat ambiguous results, I definitely hope to have future opportunities to continue my investigation of the evolution of this incredible group of snakes.

References

• Degli Esposti, M., S. De Vries, M. Crimi, A. Ghelli, T. Paternello, and A. Meyer. 1993 Mitochondrial cytochrome b: evolution and structure of the protein. Biochem. Biophys. Acta 1143:243-271.
• Griffiths, C. S. 1997. Correlation of functional domains and rates of nucleotide substitution in cytochrome b. Mol. Phylogenet. Evol. 7:352-365.
• Irwin, D. M., T. D. Kocher, and A. C. Wilson. 1991. Evolution of the cytochrome b gene in mammals. J. Mol. Evol. 32:128-144.
• McClellan, D. A., and K. G. McCracken. 2001. Estimating the influence of selection on the variable amino acid sites of the cytochrome b protein functional domains. Mol. Biol. Evol. 18:917-925.
• McClellan, D.A., and Woolley, S. (2002) TreeSAAP (Selection on Amino Acid Properties from a Phylogenetic perspective), version 1.0. Brigham Young University, Provo, UT.
• Slowinski, J. B. and J. S. Keogh. 2000. Phylogenetic relationships of Elapid snakes based on Cytochrome b mtDNA sequences. Mol. Phylogenet. Evol. 15:157-164.
• Swofford, D. L. 1998. PAUP*. Phylogenetic analysis using parsimony (*and other methods). Version 4. Sinauer, Sunderland, Mass.
• Zhang, Z., L. Huang, V. M. Shulmeister, Y.-I. Chi, K. K. Kim, L.-W. Hung, A. R. Crofts, E. A. Berry, and S.- H. Kim. 1998. Electron transfer by domain movement in cytochrome bc1. Nature 392:677-684.