MOLECULAR PHYLOGENY OF THE GENUS METROSIDEROS AND POPULATION GENETICS OF SOME NEW ZEALAND SPECIES WITHIN THE GENUS A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy in Biology in the University of Canterbury by Kumilign Asmare Melesse University of Canterbury 2017 Acknowledgment First of all, I would like to thank the almighty God for keeping me healthy and giving me the stamina to pursue and complete my PhD study in the field of genetics, which I have liked most since my school days. I would also like to thank my supervisors Assoc Professor Hazel Chapman from University of Canterbury and Dr Gary Houliston from Landcare Research at Lincoln for their wonderful and uninterrupted supervision and support in every step of my research progress. Without their consistent advice and supervision, the completion of my study would not have been realized. I would like to thank Dr Pieter Pelser from University of Canterbury for extending his unreserved help in gap coding of nucleotide sequence data that were used for the molecular phylogeny work, and also for the valuable discussions I had with him regarding the Bayesian and Maximum Parsimony tree building methods. My thanks also go to Landcare Research at Lincoln for providing me the opportunity to use the molecular lab and the reagents therein for all my PhD research, and also for granting me access to Allan Herbarium to take leaf samples of Metrosideros species for my molecular phylogeny work. I am grateful to Dr Dagmar Goeke, Caroline Mitchell and Katherine Trought for their technical support in the Molecular Lab at Landcare Research, which facilitated my lab work and also made my stay at Landcare Research enjoyable. I would like to thank the University of Canterbury (UC) for granting me the financial support (UC Doctoral Scholarship) to pursue my PhD. Last but not least, I would like to thank Drs Peter de Lange and Jeremy Rolfe from the Department of Conservation for providing leaf samples of Metrosideros bartlettii trees for DNA extraction for genetic diversity analysis. ii Table of Contents Acknowledgment ...................................................................................................................... ii Table of Contents .................................................................................................................... iii List of Figures ......................................................................................................................... vii List of Tables ............................................................................................................................ ix Chapter 1: General Introduction ............................................................................................ 1 1.1 Resolving phylogenetic relationships in the genus Metrosideros....................................... 3 1.2 Population genetics of Metrosideros bartlettii ................................................................... 5 1.3 Genetic confirmation of hybridisation between Metrosideros excelsa and M. robusta ..... 6 1.4 Summary of key findings .................................................................................................... 7 1.5 Future research directions ................................................................................................. 10 1.6 Implications of myrtle rust on Metrosideros bartlettii conservation ................................ 11 References ................................................................................................................................. 14 Chapter 2: Molecular phylogeny of the the genus Metrosideros ......................................... 27 Abstract ..................................................................................................................................... 27 2.1 Introduction ....................................................................................................................... 27 2.2 Molecular markers, phylogenetic tree building methods, and nucleotide substitution models ............................................................................................................................... 32 2.2.1 Molecular markers used in the study ..................................................................... 32 2.2.2 Phylogenetic tree building methods ....................................................................... 40 2.2.2.1 Distance-based methods........................................................................... 40 2.2.2.2 Character-based methods ......................................................................... 42 2.2.3 Nucleotide substitution models .............................................................................. 46 2.3 Materials and Methods ...................................................................................................... 49 2.3.1 Acquisition and alignment of nucleotide sequence data ........................................ 49 2.3.2 Data analysis .......................................................................................................... 50 iii 2.4 Results ............................................................................................................................... 51 2.4.1 Results of nuclear sequence data ........................................................................... 51 2.4.2 Results of chloroplast data ..................................................................................... 56 2.5 Discussion ......................................................................................................................... 58 2.5.1 Phylogenetic analysis of the genus Metrosideros .................................................. 58 2.5.1.1 Phylogeny of the subgenus Metrosideros ................................................ 58 2.5.1.2 Phylogeny of the subgenus Mearnsia ...................................................... 64 2.5.1.3 Phylogeny of the subgenus Carpolepis.................................................... 67 2.5.1.4 Phylogenetic analysis of Chloroplast data ............................................... 68 2.5.2 Dispersal routes ...................................................................................................... 71 2.5.2.1 Dispersal routes for the subgenus Metrosideros ...................................... 71 2.5.2.2 Dispersal routes for the subgenus Mearnsia ............................................ 75 2.5.2.3 Dispersal conclusions............................................................................... 77 References ................................................................................................................................. 81 Chapter 3: Genetic diversity and structure of Metrosideros bartlettii as revealed by AFLP and SSR markers .................................................................................... 103 Abstract ................................................................................................................................... 103 3.1 Introduction .................................................................................................................... 103 3.1.1 Description of Metrosideros bartlettii ................................................................. 103 3.1.2 Background and significance of the study ........................................................... 106 3.1.3 Molecular markers used in the study ................................................................... 108 3.1.3.1 Amplified Fragment Length Polymorphism (AFLP) ............................ 108 3.1.3.2 Simple Sequence Repeats (SSR) ........................................................... 110 3.2 Materials and Methods .................................................................................................... 111 3.2.1 Plant material ....................................................................................................... 111 3.2.2 DNA Extraction and AFLP reaction .................................................................... 114 iv 3.2.3 SSR Reaction ....................................................................................................... 115 3.3 Data analysis ................................................................................................................... 116 3.3.1 Genetic diversity .................................................................................................. 116 3.3.2 Genetic structure .................................................................................................. 118 3.4 Results ............................................................................................................................. 122 3.4.1 Genetic diversity .................................................................................................. 122 3.4.2 Genetic structure .................................................................................................. 124 3.5 Discussion ....................................................................................................................... 130 3.5.1 Genetic diversity .................................................................................................. 130 3.5.2 Genetic structure .................................................................................................. 138 3.5.3 Implications for M. bartlettii conservation .........................................................