RATITE MOLECULAR EVOLUTION, PHYLOGENY AND BIOGEOGRAPHY INFERRED FROM COMPLETE MITOCHONDRIAL GENOMES by Oliver Haddrath A thesis submitted in confonnity with the requirements for the Degree of Masters of Science Graduate Department of Zoology University of Toronto O Copyright by Oliver Haddrath 2000 National Library Biblioth&que nationale 191 .,,da du Canada uisitions and Acquisitions et Services services bibliographiques 395 Welington Street 395. rue WdKngton Ottawa ON KIA ON4 Otîâwâ ON K1A ûN4 Canada Canada The author has granted a non- L'auteur a accordé une iicence non exclusive licence allowing the exclusive permettant A la National Library of Canada to Bihliotheque nationale du Canada de reproduce, loan, distribute or sell reproduire, @ter, distribuer ou copies of diis thesis in microfonn, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fïîm, de reproduction sur papier ou sur format 61ectronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette tbése. thesis nor substantial exûacts fiom it Ni la thèse ni des extraits substantiels may be priated or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Ratite Molecular Evolution, Phylogeny and Biogeography Inferred fiom Complete Mitochoncîrial Genomes. Masters of Science. 2000. Oliver Haddrath Department of Zoology, University of Toronto. The relationships within the ratite birds and their biogeographic history has been debated for over a century. While the monophyly of the ratites has been established, consensus on the branching pattern within the ratite tree has not yet been reached. To examine this problem, I sequenced the complete mitochondnal genomes Rom a representative of each extant ratite lineage, one extinct lineage (moas) and two species of tinaniour. Based on concatenated sequences, the moas were found to be the most basal ratite and the kiwis were found to form a clade with the emu and cassowary. It was las clear whether among the extant ratites the ostrich or the rheas were more basal. Application of a molecular clock found most of the speciation events were otder or very close to the dates of the breakup of Gondwana and so a vicariant origin for these birds could not be rejected. Acknowledgements Any large project undertaken has the input directly or indirectly of many people. This thesis certainly falls into that category. 1 have many people to thank for cornments, ideas and insights. First and foremost 1 would like to thank Madeleine, my wife, for al1 her loving patience, indulgence of my many late nights in the lab and assistance over the last few years. This thesis and degree are shared with her. 1 would also like to thank my family, particularly my parents who instilled a curiosity about the world and an appreciation of science at an early age. Over the course of my years at the Royal Ontario Museum, 1 have worked with many talented people who have al1 had an input on this work. They inclucie Cathy Ayley, Chris Pankewicz, Sue Chopra, Brad Millen, Mark Peck, Jon Barlow, Ross James, Glenn Murphy, and James Dick. Also included in this list are the graduate students with whom 1 have worked with: Mike Demison, Dawn Marshall, Oksana Borowik, Jaime Alvarado, Kevin Seymour, Vicki Friesen, Tim Birt, Paul Wenink, Don Stewart, Anna Toline, Dilara Ally, Tracey Monehan, Carol Ritland, Colette Baril, Marlene Walker, Alessandro Grapputo, Jan Hughes, Tara Paton, Annette Greenslade, Maryann Burbidge, Andrew Given, Nicola Wade, Carol Cooke and Gustavo Ybazeta. I would also like to thank Alejandro Lynch and Pavneet Arora whose help were indispensable with statistical tests and computer programming. During this project many researchers with whom 1 corresponded were generous with their assistance and computer programs. 1 would like to thank Masami Hasegawa, Lars Jermiin, Wen- Hsiung Li, Sudhir Kumar, Nicolas Galtier, Ziheng Yang, Spencer Muse, Peter Lockhart, David Mindell, Peter De Rijk, and Eka Hagelberg. iii For David Irwin, my adjunct supervisor, 1 am grateful for the insights you provided and the depth of your knowledge. 1 reserve my final acknowledgement for Allan Baker, my supervisor. My thanks for giving me such an interesting project and al1 your support and guidance. The project proved to be challenging in ways that we did not anticipate and you were generous in your advice, time and in giving me the latitude to overcome the problems. You set a high standard for yourself and your students and you constantly made me defend my ideas and theories and hopefully I am a better student for it. Table of Contents .. Abstract ............................................................... ......................................................li ... Acknowledgments.................................................................................................... 111 Table of Contents ...................................................................................................... v List of Tables ............................................................................................................ ix .. List of Figures ....................................................................................................... ..XII List of Appendices................................................................................................ .xvi Chapter I : Introduction and Overview of Ratite Systematics Introduction........... ... ......................................................................................... 1 The Objectives of this Thesis ...........................................................................1 I . Organization of the Thesis.......................................................................... .12 Re ferences................................................................................................... .13 Chapter 2: Characterization of the Complete Mitochondrial Genomes of Nine Paleognathous B irds Introduction. ............................................................................................................ 19 Materials & Methods .............................................................................................. -21 Specimen Information.................................................................................... .21 DNA Extraction and Amplification.................................................................. 22 Sequence Analysis .......................................................................................... 25 Results .................... ................... .............................................................................. 24 Structural Features of Paleognathous Mitochondrial Genomes........ 25 Codon Usage and Base Composition ....................................... 29 Amino Acid Composition .........................................................30 RNA Editing ............................................................................ 36 rIWAs.................................................................................. 38 Control Region ........................................................................ 42 Levels of Variation Between Genes .................................................. 44 Discussion ............................................................................................... 46 *@ . Genome Organization............................................................................... 46 Codon Usage and Sequence Composition ............................. .................. 47 RNA Editinp ......................................................................................... 48 References .................................................................................................... 49 Chapter 3: Rates and Patterns of Mitochondrial Genome Evolution in Paleognathous Birds Introduction.......................................................................................................... 58 Materiais & Methods........................................................................................... 60 DNA Isolation. Amplification and Sequencing......................................... 60 Data Analysis ............................................................................................. 61 Estimation and Pattern of Nucleotide Substitution.................................... 62 Substitutional Rate Variation Among Sites and the Rate Constancy Test ..........................................................................................63 Mode1 of Substitution................................................................................ 64 Results ...................................................................................................................64 Patterns of Nucleotide Substitution for the Protein-Coding, rRNA and tRNA Genes ............................................................................-64 Substitutional Biases Among the Four Nucleotides ..................................65 Stationarity.......................... .. .....................................................................72 Rate Variation Among Sites and Among Genes ........................................72 Mode1 of Nucleotide Substitution.............................................................. 76 Discussion .............................................................................................................78