The evolution of the mitochondrial DNA in tuatara (Sphenodon punctatus) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Evolutionary Genetics, Massey University, Auckland, New Zealand

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The enigmatic tuatara from New Zealand, occupies a central place in the evolution of vertebrates and tuatara have changed little morphologically since the Cretaceous period approximately 200 millions of years ago. A central aim of this thesis was to examine rates of molecular evolution in tuatara using entire mitochondrial genomes of both ancient and modern samples. A total of 51 complete mitochondrial genomes from 42 modern (from eight island groups) and 9 ancient samples (from eight locations on mainland) were sequenced using Sanger method. These complete genomes were used to investigate the population genetic structure of tuatara. Diverse phylogenetic analyses suggest that Sphenodon is a monotypic genus. This is in contrast to the suggestion made by Daugherty et al. (1990 b) that there are two species of tuatara. This two species model was subsequently almost universally accepted. The current result illustrates that when species are incorrectly identified scarce conservation resources are inappropriately used to ensure their conservation. Using these complete mitochondrial genomes and by employing three very different analytical methods, I have also estimated molecular evolutionary rates for tuatara. Using modern and ancient complete mitochondrial genomes, I showed that the rates of molecular evolution in tuatara are higher than other vertebrates. This result and the stable morphology of tuatara over tens of millions of years suggest a disconnect between molecular and anatomical evolution, as originally suggested by Allan Wilson in the 1970s. From a biological perspective perhaps this is not surprising, since morphological and molecular evolution are governed by very different biological processes. I then explored the possibility that tuatara might be characterised by high mutation rates. Using Roche 454 next generation DNA sequencing, I recovered seven complete mitochondrial genomes in tuatara. A total of 28 potential heteroplasmies were detected among these genomes. These sites were also shown to be polymorphic among the 42 modern aligned genomes suggesting that they are characterised by high mutation rates. This result suggests that a high level of heteroplasmic sites in tuatara mitochondrial genome contributes to the high molecular rates estimated when comparing modern and ancient genomes.
Appendix D content removed due to copyright restrictions: Mohandsen, E., Mowla, S. J., Noobari, A. H., Hofreiter, M. (2008) "Ancient DNA from human and animal remains from north-west Iran", Journal of Sciences, Islamic Republic of Iran 19(1), 3-8. Hay, J. M., Subramanian, S., Millar, C. D., Mohandesan, E., Lambert, D. M. (2008) "Rapid molecular evolution in a living fossil", Trends in Genetics, 24(3), 106-109. Millar, C. D., Huynen, L., Subramanian, S., Mohandesan, E., Lambert, D. M. (2008) "New developments in ancient genomics", Trends in Ecology and Evolution, 23(7), 386-393. Subramanian, S., Hay, J. M., Mohandesan, E., Millar, C. D., Lambert, D. M. (2008) "Molecular and morphological evolution in tuatara are decoupled", Trends in Genetics, 25(1), 16-18.
Tuatara genetics