Adaptive variation versus vicariance: what drives speciation in Eucalyptus? Susan Rutherford A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Evolution and Ecology Research Centre School of Biological, Earth and Environmental Sciences Faculty of Science October 2017 i ii COPYRIGHT STATEMENT ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... AUTHENTICITY STATEMENT ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date ……………………………………………........................... iii Eucalyptus regnans (commonly known as the ‘Mountain ash’) from Mount Field National Park, Tasmania. Eucalyptus regnans is the tallest flowering plant in the world reaching heights of 90–100 m (Photography: S. Rutherford). iv Statement of contribution of co-authors and declarations of permission to publish Chapters 2 to 5 of this thesis are stand-alone manuscripts that have been written for publication in peer-reviewed journals. Each chapter is self-contained including tables, figures, references and appendices. The terms, ‘we’ and ‘our’, are frequently used as each chapter has co-authors. Additional assistance is acknowledged at the end of each chapter. All photographs presented in the thesis are by S. Rutherford unless otherwise stated. Permission was obtained from V. Klaphake to use images in Klaphake (2012). The contributions by co-authors of each chapter are listed as follows: Chapter 2 Phylogenomics of the green ash eucalypts (Myrtaceae): a tale of reticulate evolution and misidentification Australian Systematic Botany (2016) 28: 326‒354 doi: 10.1071/SB15038 Authors: Rutherford S, Wilson PG, Rossetto M, Bonser SP This study was conceived by SR, PGW and MR. Leaf material for DNA and voucher specimens were collected by SR and PGW. DNA extractions and phylogenetic analyses were performed by SR with advice from PGW. SR wrote the manuscript and PGW, MR and SPB provided comments and guidance. All journals published by CSIRO Publishing (e.g. Australian Systematic Botany) allow authors to reproduce the Accepted version of their manuscript in a thesis with no embargo. v Chapter 3 Seedling response to environmental variability: the role of phenotypic plasticity in the evolution of Eucalyptus species A version of this chapter has been published in American Journal of Botany (2017) 104: 840-857 doi:10.3732/ajb.1600439 Authors: Rutherford S, Bonser SP, Wilson PG, Rossetto M SR, SPB and PGW developed the research questions. SPB and SR designed the study, SR and PGW collected seed and SR conducted the growth experiment. SR performed statistical and phylogenetic analyses, and wrote the manuscript. SPB, MR and PGW provided editorial advice and guidance on the manuscript drafts. After publishing an article in American Journal of Botany, authors reserve the right to use all or part of the article in compilations or other publications of the authors' own works. Chapter 4 Speciation in the presence of gene flow: population genomics of Eucalyptus species along altitudinal and latitudinal gradients in south-eastern Australia Authors: Rutherford S, Rossetto M, Bragg JG, McPherson H, Benson D, Bonser SP, Wilson PG This study was conceived and designed by SR and MR. DB provided advice on selection of study species. SR, PGW and DB collected leaf material for DNA analysis and voucher specimens. SR performed the DNA extractions and population genetic analyses with advice from MR, JGB and HM. JGB wrote codes for the analysis of large SNP datasets in R studio. SR wrote the manuscript with guidance from MR and PGW. SPB, JGB, HM and DB provided editorial advice on manuscript drafts. vi Chapter 5 From environmental niche modelling to phylogenetics and functional traits: understanding ecological specialisation and species diversification in Eucalyptus Authors: Rutherford S, Wilson PD, Wilson PG, Bonser SP, Rossetto M This study was conceived by all authors. PDW provided environmental data for analyses and an R script for calculating geographic range size in R studio. SR performed environmental niche modelling and data analyses with advice from PDW. SR wrote the manuscript and MR, SPB, PGW and PDW provided editorial comments on manuscript drafts. vii Acknowledgments I have been fortunate to have three very good supervisors during my research: Stephen Bonser (UNSW), Maurizio Rossetto (Royal Botanic Garden Sydney) and Peter G. Wilson (Royal Botanic Garden Sydney). It is due to their patience, generosity, support and guidance that this project has been made possible. Thank you to Stephen for all your advice, for welcoming me into your lab and for ensuring that my PhD at UNSW was a great experience. Maurizio, I thank you for your enthusiasm, ideas, encouragement, and for giving me the opportunity to be a part of your team (Evolutionary Ecology) at the Royal Botanic Garden Sydney. Peter, I am so grateful to you for sharing your knowledge of the Australian flora, taxonomy and phylogenetics. This thesis presents findings from a joint project between UNSW and the Royal Botanic Garden Sydney. Financial support for this research was provided by ARC Linkage Grant LP110100721. A grant from the Dahl Trust allowed me to complete a large section of my molecular work, for which I am extremely grateful. During this research, I was also in receipt of an Australian Post-graduate Award (APA) from the Australian Government. Many people from the Royal Botanic Garden Sydney have been very supportive over the last few years. I would like to thank Doug Benson, who often acted as an additional mentor and who was always up for a chat on the green ashes, ecology and the flora of the Blue Mountains. A big thanks to Miguel Garcia from the Daniel Solander Library, who always helped me to hunt down references in the library, and was another person with whom I could discuss all things to do with evolution, history and speciation. I am grateful to Carolyn Connelly from the Molecular Laboratory as it was thanks to her technical knowledge and practical experience that I was able to successfully extract DNA from very difficult eucalypt samples! Thank you Hannah McPherson, who was always available to give advice on genetic analyses. Many thanks to Jason Bragg, who helped me to better understand population genetics and who provided many R scripts that enabled the analysis of DArTseq markers. Also, thank you to Peter D. Wilson who helped me with environmental niche modelling and data analysis. I would like to gratefully acknowledge many others who have given me a helping hand, including viii Marlien van der Merwe, Margaret Heslewood, Joel Cohen, Matt Laurence, Andrew Orme, Trevor Wilson, Bob Coveny, Michael Elgey, Juelian Siow, Richard Johnstone, Abigail Greenfield, Chris Allen, Samantha Yap, Monica Fahey and Marco Duretto. I am also grateful to Louisa Murray, Barbara Briggs, Karen Wilson, Bob Makinson and Barry Conn, who encouraged me to do botanical research in the first place. I have met many kind people from UNSW, who have been very helpful to me over the last few years. I would like to thank Josh Griffiths, Clara Pang, Ali Namazi, Fatih Fazlioglu and Justin Wan. Thank you Josh, Clara and Justin for assisting me in the glasshouse, and thanks to Fatih for helping me with the plasticity index. Many thanks to Geoff McDonnell (glasshouse manager, UNSW) for providing technical support during my common garden experiment. Thank you to Justin for being so supportive while I was writing up my thesis and for reading over a full draft of the thesis. Researchers from other institutions have provided me with advice during various aspects of my research. I am grateful to Dorothy Steane (University of Tasmania), Jean- François Flot (University Libres de Bruxelles), Jason Carling (DArT Pty Ltd, Canberra) and Andrzej Kilian (DArT Pty Ltd, Canberra). Many volunteers have also generously contributed their time to help me with my research, including Lawrence Mou, Stephanie Creer, Emma Oldman, Aaron Smith, Danca Ciric, Christine Smith, Esthel Varma and Brendan Malloy. Last but not least, I would like to thank my family for all the love and support they have given me: my mother, Christine; my father, David; and my sister, Anne. ix Abstract Speciation is a central process in evolutionary biology and is responsible for the diversity of life on Earth. While there has been much progress in evolutionary research over the last 150 years, understanding the many facets of speciation remains a major challenge. In this thesis, I focus on a group of Eucalyptus species called the green ashes (subgenus Eucalyptus section Eucalyptus). The green ashes comprise tall trees on fertile soils (e.g. the tallest flowering plant in the world, Eucalyptus regnans), as well as medium trees and mallees on low nutrient soils. Although Eucalyptus is Gondwanan in origin, fossil and molecular evidence suggest that many eucalypt groups (including the green ashes) diverged within the last 10 million years.