Molecular Systematics of Australian and New Guinea Pythons
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zbl -úìa 'aa'.iü Molecular Systematics of Australian and New Guinea Pythons Lesley H. Rawlings A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Department of Genetics The University of Adelaide March 2001 rll Abstract Mitochondrial control region and cytochrome b gene sequence comparisons were used to investigate the molecular systematics of pythons. Duplicate, non-tandem copies of control region sequence from single mitochondrial genomes were compared for divergent genera and within populations of pythons. There was almost total sequence identity between the two regions within individuals but there were diflerences in copy number of an -88 bp repeat between the two regions within individuals. Structures such as conserved sequence blocks and termination sequences previously documented in vertebrate control regions were present and there was a \RNAIL paralogue and a large 5' haþin in the Australasian pythons that was not present in the Afro-Asiatic pythons, boids, colubrids or viperids. A range of squamate reptiles was tested for the presence of the duplication, which was found to be absent in all the squamate reptiles tested and present in all the snake lineages tested except Scolecophidians. Phylogenetic analyses of python species suggested that the genus Python is paraphyletic and the Afro-Asian Python species formed the sister lineage to all other pythons. Aspidites was nested amongst the Australo-Papuan pythons, albeit without strong statistical support for this arrangement. There was low genetic divergence among all taxa, and there was monophyly of the genera Antaresia, Aspidites and Ziasis only' Two studies focused on phylogenetic relationships at the population level. Analyses of Morelia viridis nucleotide sequences and allozyme loci revealed that in New Guinea, populations north of the central cordillera were genetically different from those of the south. The Australian population was closely related to the southern form. Analysis of control region sequences of Liasis and Apodora concluded that Apodora papuana was the sister lineage to all Liasis and that Liasis fuscuslL. mackloti and L. olivaceus are separate lineages. There was strong support for the recognition of two lineages ("eastem" and "western") of water pythons in Australia. iv To conclude, the genetic split found in green pythons across the central cordillera of New Guinea was investigated further. To test if the most recent episodes of the Plio- Pleistocene climatic oscillations have had an effect on genetic subdivision of the lowland rainforest fauna, molecular phylogenies were estimated for six vertebrate taxa. The distribution of cytochrome b lineages and allozyme fixed diflerences demonstrated a pattern of historical genetic subdivision that correlates with populations distributed either side of the central cordillera. Under a molecular clock assumptior¡ the magnitude of sequence divergence within the six taxa suggests that uplift of the central cordillera acting in concert with the earlier climatic oscillations of the Plio-Pleistocene was the most likely factor involved in genetic subdivision. V Acknowledgments If I have seen further, it is by standing of the shoulders of giants. NEWTON, Sir Isaac (l'642'L727) If anyone had asked me in 1990 what I thought I might be doing in the year 2001, I am sure that finishing a PhD about pythons would not have been anywhere near the possibilities of my imaginings. Living in Asia and caring for three small children was a world away from tertiary educational pursuits. When the door of opportunity opened, I stepped through into undergraduate studies and then into the challenge of the python project. Ultimately, the challenge was to maintain a balance between family life and the demands of the project/thesis and for better or worse, the challenge has been met. This thesis and the work that it reports is not representative of my o\illl eflorts alone. Like Isaac Newton, I need to acknowledge a multitude of people who have far more experience and expertise than I, in the fields of herpetolog¡ systematics and molecular evolution. As always with science, I have just added a few more pieces to a giant puzzle. As well as those who are experts in their fields, I have also become aware of the tremendous support from an army of encouragers. The unflagging support and genuine interest of my supervisor, Steve Donnellan has been invaluable to keep the components of this project moving forward. His professionalisn¡ in-depth knowledge of the field of molecular evolution and vast range of contacts in fauna research and government areas gave me a great start into this project. There are so many other "bits and pieces" that didn't make it into the thesis that I hope will frnd their usefulness someday, that are legacies of Steve's contagious enthusiasm for the python project. I was fortunate among PhD students in that Steve was always willing to spend time explaining the nuances of terminology and statistical methodology that are part of molecular evolution. Sometimes, my brain \À/as very slow in grasping hold of these ideas. I am also grateful for the encouragement and opportunities provided to attend conferences and present my work. These stretched and challenged my reserved nature in ways that were vi painful, but profitable. I especially appreciated the opportunity to pursue a tangential dabble into the forensic science of fauna enforcement using the sequencing methods and sample database I had established for green pythons. Seeing a direct application resulting from this work was very rewarding and involvement in court proceedings was enlightening. In the long terrr¡ I have appreciated the encouragement to try new methods when they appeared in the literature and the encouragement to persist with the difficult museum specimens that have been important to the studY. I especially want to thank my beautiful family, Malcokn, Nathan, Joel and Lauren and my mother-in-law, Rita. They released me from a stereo-type and family background that said that women of my age, family status, education and work history did not start a university education and certainly not in science. They accompanied me with interest and eager participation through my undergraduate days of making sundials, counting lichens and catching slaters. They have been my biggest fans and encouragers and without their support, I would not have wanted to continue. They believed that I could go on, and they have constantly encouraged me to make it to the end, when sometimes I doubted myself. They have demonstrated pride in my achievement and in these last few months, have been willing to bear the cost of my monopoly of the computer without complaining and my single- mindedness, by picking up the domestic slack. I am looking forward to enjoying our family fun again! I must also thank our many friends, who think I'm crazy for setting out on such a task, but who have always offered interest and encouragement and cheered me on to the fTnish. Even Jennie, who can hardly mention the word "snake" without becoming a neurotic wreck has been brave enough to show polite interest in Monty, our pet carpet python and oflered words of encouragement when I needed them. Special thanks go to Roxanne, Dave and Steve H. who have patiently listened through four years of triumphs and self-doubts with love, acceptance, encouragement and prayer. I'm looking forward to the party! Throughout my time in the Evolutionary Biology Unit, there has been a very happy vlt blend of hard work and fun with fellow EBUers. Agatha, Brenda, Brian, Caroline, Cat, Chris, David, Debbie, Jan, Kathy, Kylie, Kym, Leanne, Malcokn, Mark' Mike, Penny, Ralph, Remko, Sarah, Scott, Steve C. and Terry have all added to a great time. Agatha, Jan, Steve C., Terry and Jenny Thurmer have all given wonderful moral support during times when I wasn't sure that I could keep going, particularly following the death my mother. From the purely practical requirement to obtain tissue samples, the list of those who have assisted in the field and amongst the museum pickles is very long. The SA museum's python sampling across Australia is now very extensive and even if I had been given the funding to make wide-ranging field trips, I could not have covered such a vast geographic area myself. I would like to thank A. Allison, K. Aplin, D. Armstrong, D.G. Barker, G. Bedford, S. Bielke, F. Bonnacorso, M. Bonnett, J. Bredl, B. Bush, A. Carey, M.Chambers, N. Charles, S. Cooke, P. Couper, M. Cunningham, F. Delpiva, S. Donnellan, A. Emmett, D. Farlarn, T. Flannery, R. Foster, G. Fyfe, G. Gillespie, R. Hancock' T. Herbert, J-M. Hero, G. Hollis, R. How, S. Hudson, G. Husband, M. Hutchinson, S. Irwin, R & J. Jarman, C' Kishinami, M. Kearney, P. Kendrick, M. King, P. Krauss, D. Leyder¡ D. McCrae, J. McKenzie, W. Mannion, R. Martin, G. & S. Maynes, J. McKenzie, M. McCoy, T. Morley, L. Naylor, D. Neijelske, M. O'Brien, D. O'Connor, H. Owens, D. Pearson, P. Quill, T. Reardon, J. Reid, S. Richards, D. Robinson, P. Robinsofl, M. Ryan, R. Sadlier, C. Schneider, T. Schwaner M. L Sidik, L. Smith, S. Stone, D. Storch, S. Sutherland, G. Torr, R. Turnbull, L. Valentine and F. Yuwono for providing materials. These are people who have an appreciation for pythons from many varied perspectives, some are researchers, some are fauna rescuers, some are keepers and some just have an affection for their friend who lives in the garden shed. I would like to thank the Trustees of the D.R. Strank's Travel Fellowship for awarding me the opportunity to travel to Indonesia. I thank S.N. Prijono, director of the Bogor museum and I. Sidik, curator of reptiles, for their assistance and a very special thank you to Hari S.