Hybridisation and phylogenomics of Betula L. (Betulaceae) Nian Wang School of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS Supervisor: Dr Richard J. A. Buggs A thesis submitted to the University of London for the degree of Doctor of Philosophy November 2015 1 Declaration I certify that this thesis and the research conducted here are the product of my own work, and that all ideas and quotation from other works, published or otherwise, are fully acknowledged in accordance with standard refereeing practices in the biological sciences. I acknowledge particular data acquisition and analytical assistance as follows: James Borrell helped do Ecological niche modelling based on which I produced Figure 2.4. Richard Nichols helped develop scripts to do cline analysis using the mixed-effect model, based on which I produced Figure 2.3 in R. Jasmin Zohren helped to write an R script to change mapped loci name for Chaper 5. Nian Wang 2 Acknowledgements First and foremost I would like to express my sincere gratitude to my PhD supervisor Richard J. A. Buggs. Richard, as an outstanding researcher and supervisor, has made my research as smooth as possible. Thanks to him for his guidance, inspiration, patience and weekly discussion, which have benefitted me not only for my PhD at QMUL but also for my career. I owe a big thanks to Prof. Andrew R. Leitch, whose ideas in science are amazing. I extend my sincere thanks to Hugh McAllister, whose knowledge on the taxonomy of birch is second to none, his comments and guidance have been insightful and invaluable. Many thanks to Paul Bartlett, who manages a great Botanic Garden with an excellent collection of birches, for his generosity in providing me with almost all samples I need. I wish to thank Prof. Richard Nichols, as one of my panel members and the organiser of the „Labchat‟, whose comments on my project are insightful. I wish to acknowledge my sincere gratitude to James Borrell for helpful discussions on sciences and help in data analysis; to Jasmin Zohren and Maité Guignard for their help on R scripts; to Laura Kelly for her insightful comments on Chapter 4; to Monika Struebig, who has provided excellent support during the period of my study and has made life in the lab much easier. I also would like to thank the following people in alphabetic order: Hannes Bechler, Peter Brownless, Endymion Cooper, William Crowther, Paul Grogan, Hao-Chih Kuo, Ilia Leitch, Lu Ma, Xiu-Guang Mao, Jaume Pellicer, Martyn Rix, Elizabeth Sollars, Jonathan Stocks, Bruno Vieira, Gemma Worswick, Zhi-Kun Wu and Jie Zeng for various helps. Last but not least I would like to thank my family members: Pei-Gang Wang, Shu-Ping Zhai and Xin-Ying Zhao for their continued support and encouragement. 3 Abstract Hybridisation and polyploidy are important in the evolution of species. The genus Betula L. is an ideal model to study these processes as species of this genus hybridise frequently and polyploid species are common. In this thesis, I investigated the hybridisation of three Betula species in Britain; the phylogenetic relationships and genome size of all known Betula species, and conducted phylogenomic analysis of diploid Betula species. A cline of introgression of microsatellite marker alleles from B. nana was found extending into B. pubescens populations far to the south of the current B. nana range in Britain. We suggest that this genetic pattern is a footprint of a historical decline and/or northwards shift in the range of B. nana populations due to climate warming in the Holocene. The Atkinson Discriminant Function (ADF) is a leaf-morphology metric to distinguish between B. pubescens and B. pendula. We test it on 944 trees sampled across Britain against species‟ discriminations made using 12 microsatellite loci and found that the accuracy of the ADF can be raised to 97.5% by using an ADF of -2 rather than zero as the boundary line between the species. The taxonomy of the genus Betula is debated and a new classification has been proposed in a recent monograph. We evaluated it using ITS and restriction site associated DNA sequencing (RADSeq). The result based on ITS largely agrees with species classification in the recent monograph but with uncertainties. Phylogenomic analysis of 587 RAD loci for Betula diploid species using coalescence-based methods, a concatenation method and binary presence/absence of RAD loci produced well- resolved trees with similar topology. Based on these analyses, we propose a new classification of Betula into four subgenera and seven sections. Further research is needed to infer the parental origins of polyploid species within Betula. 4 Contents Abstract ..................................................................................................................................... 4 Chapter 1 General Introduction ........................................................................................... 10 Introduction to relevant theory ............................................................................................. 10 Introduction to relevant methodologies ................................................................................ 18 Introduction to the study system: Betula (Betulaceae) ......................................................... 22 Chapter 2 Molecular footprints of the Holocene retreat of dwarf birch in Britain ......... 26 Summary............................................................................................................................... 27 Introduction .......................................................................................................................... 28 Materials and Methods ......................................................................................................... 31 Results .................................................................................................................................. 40 Discussion............................................................................................................................. 46 Chapter 3 Is the Atkinson discriminant function a reliable method for distinguishing between Betula pendula and B. pubescens?................................................. 50 Summary............................................................................................................................... 51 Introduction .......................................................................................................................... 52 Materials and Methods ......................................................................................................... 54 Results and Discussion ......................................................................................................... 55 Conclusion ............................................................................................................................ 60 Chapter 4 Molecular phylogeny and genome size evolution of the genus Betula (Betulaceae) ............................................................................................................................. 61 Summary............................................................................................................................... 62 Introduction .......................................................................................................................... 64 Materials and Methods ......................................................................................................... 70 Results .................................................................................................................................. 83 Discussion............................................................................................................................. 92 Concluding remarks.............................................................................................................. 99 Chapter 5 RAD markers and phylogenomics of Betula diploid species .......................... 100 Summary............................................................................................................................. 101 Introduction ........................................................................................................................ 102 Materials and Methods ....................................................................................................... 105 5 Results ................................................................................................................................ 112 Discussion........................................................................................................................... 118 Chapter 6 Conclusions ......................................................................................................... 123 General overview................................................................................................................ 123 The contribution of this thesis ............................................................................................ 123 New questions and future research ..................................................................................... 127 References ............................................................................................................................. 129 Appendix ..............................................................................................................................