This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. New Approaches for the Conservation Genomics of the Genus Cycas L. in Australia By James A. R. Clugston In Fulfilment of the Requirements for the Doctor of Philosophy in Molecular Plant Sciences College of Science and Engineering School of Biological Sciences The University of Edinburgh 2019 Acknowledgements I wish to extend my heartfelt thanks to my incredible supervisors: Nathalie Nagalingum, Greg Kenicer, Murray Henwood and Richard Milne for their support throughout the project. Nathalie for taking me on as a PhD student, and her help in setting me up when we first moved to Australia and her extended support and advice; Greg for unwavering support provided throughout the project no matter where I was in the world; Richard for providing expert advice and support for the research and Murray who agreed to supervise me so late in the process as I moved permanently to Australia and going above and beyond by providing us with a place to stay, helping get us settled and providing such important advice that has been instrumental to the final product of this project. I wish to acknowledge funding received from the Australian Flora Foundation, the Australasian Systematic Botany Society’s Hansjörg Eichler Scientific Research Fund, and The Nature Conservatory – The Thomas Foundation for an Australian Conservation Taxonomy Award. Furthermore, I wish to acknowledge the Biotechnology and Biological Sciences Research Council (BBSRC) UK and the EASTBIO Doctoral Training Partnership for providing me a studentship at the University of Edinburgh. I thank staff at the Royal Botanic Gardens and Domain Trust Sydney, in particular: Carolyn Connolly for her support in the molecular laboratory, Hannah McPherson for helping in the initial quality testing of data and troubleshooting population genetics scripts, Peter Wilson for his help working with the computer systems and allowing me to run additional RADseq analysis and Maureen Phelan and Simon Goodwin for assistance in obtaining DNA samples from the living collections. I also thank the various botanic gardens including Montgomery Botanic Center, University California Berkley Botanic Garden, Darwin Botanic Garden that provided and allowed me to collect plant tissue samples of many species. At the Royal Botanic Garden Edinburgh, I wish to thank Laura Forrest and Michelle Hart for their continued support throughout the project. And lastly Duncan Reddish at the Royal Botanic Garden Edinburgh for his assistance in setting up a special server to run the Cycas calcicola dataset on. i Daren Eaton is acknowledged for assistance with ipyrad: Robert J. Toonen at the University of Hawaii for advice and information about the ezRAD protocol, and Todd McLay at the University of Melbourne for support in quality checking and filtering of RADseq data. Charles Foster at the University of Sydney for his advice and support and Ian Cowie, Nicholas Cuff and David Liddle at the Northern Territory Herbarium for their assistance during my field work and the collection of an additional sample of Cycas armstrongii to strengthen our results. Joe Russack at the California Academy of Sciences for his assistance with using Amazon Web Services (AWS). Athena Lam and Boni Cruz for their assistance in the laboratory at the California Academy of Science and in many cases just going above and beyond their jobs! Joe Perner at Cycad International in Katherine for his help with finding population of Cycas calcicola in the Katherine Region. Louisa Murray for her continued support during the length of the project and helping me and my partner out during our arrival to Sydney. Also, to my friends and colleagues in the office Yola Metti and Trevor Wilson who were always there for support and up for helping out. Markus Ruhsam at the Royal Botanic Garden Edinburgh and Alan Meerow of the United States Department of Agriculture for examining the thesis and providing significant input to help improve the results and outputs of this project. I must also thank my parents and grandfather for supporting me both financially and mentally during this long period and lastly (but not least) I wish to thank my partner Lyndon for his unwavering support, advice and assistance even during the hardest of times and putting up with me in general. ii Declaration I declare that this thesis has been composed solely by myself, James A. R. Clugston, and that it has not been submitted, in whole or in part, in any previous application for a degree. Except where states otherwise by reference or acknowledgment, the work presented is entirely my own. Throughout this thesis I use the word (“we or our”) to denote that all future publication coming from this work will be written by myself and co-authored by my supervisors and/or others. For the purposes of this thesis, all work has been carried out by me, and the final product produced by myself only. Additionally, Chapter - 2 of this final thesis (RADseq as a valuable tool for plants with large genomes—a case study in cycads) has been accepted by Molecular Ecology Resources for publication and available as early access article. James A. R. Clugston 24 October 2019 iii Abstract Cycad species exist as small fragmented populations, therefore understanding their genetic variation is imperative for their conservation to ensure their long-term survival. Genetic data plays a fundamental role in identifying genotypes and detecting populations with the highest genetic diversity. This project uses next generation sequencing (NGS) and restriction associated DNA sequencing (RADseq) to identify thousands of genome-wide polymorphisms from populations of selected cycad species from the Northern Territory, Australia, namely: Cycas armstrongii, Cycas calcicola, Cycas maconochiei ssp. maconochiei and the interspecific hybrid C. armstrongii x maconochiei. RADseq was used to determine intra- and interspecific genetic variation in populations, verify the putative hybrid, recognize populations of conservation priority and determine if botanic garden collections currently represent the genetic diversity inherent in the wild. Cycas calcicola showed very low levels of genetic diversity and high inbreeding, and although there was significant geographic partitioning between populations in the Katherine and Litchfield National Park regions, which correlated with genetic differentiation. Additionally, the results showed that C. calcicola was not genetically, well represented in ex-situ collections. The genomic diversity of Cycas armstrongii, C. maconochiei ssp. maconochiei and C. armstrongii x maconochiei differs from that of C. calcicola and shows very low levels of genetic diversity yet generally with lower levels of inbreeding. The results show little genetic distance between Cycas armstrongii and C. maconochiei ssp. maconochiei, the most likely explanation is that they represent morphological extremes of a single species. The results from RADseq have far reaching significance for the conservation of cycads. In the case of C. calcicola, a far more structured acquisition of genetic material will be required if the full genetic diversity of this species is to be preserved in ex-situ collections. iv Lay Summary More than sixty percent of Cycad species are threatened, with many existing in small and isolated populations. As a result, understanding the diversity of cycads is imperative for their conservation to ensure their long-term survival. In order to understand the diversity of cycads, genetics plays a fundamental role in helping us to identify how populations differ from one another. Australia represents a diversity hotspot for cycads where there are many different species and also many large, clustered and undisturbed populations. This project uses the latest DNA sequencing technologies to understand the genetic diversity of populations of selected cycad species from the Northern Territory in Australia, namely: Cycas armstrongii, C. calcicola, C. maconochiei ssp. maconochiei and a hybrid population C. armstrongii x maconochiei. Our results find that populations of C. calcicola showed evidence of inbreeding and low genetic diversity, and geographic distance, populations in the Katherine and Litchfield National Park regions were confirmed based genetic evidence. The results also showed that genetic diversity of C. calcicola was not well represented in botanic garden collections, presenting conservation concerns. Cycas armstrongii and C. maconochiei ssp. maconochiei populations also showed low levels of genetic diversity in C. calcicola, but less inbreeding. In addition, based on the DNA evidence were found no genetic difference between C. armstrongii and C. maconochiei ssp. maconochiei, despite looking