ORBIT - Online Repository of Birkbeck Institutional Theses Enabling Open Access to Birkbecks Research Degree output Phylogenetics, genome size evolution and population ge- netics of slipper orchids in the subfamily cypripedioideae (orchidaceae) http://bbktheses.da.ulcc.ac.uk/88/ Version: Full Version Citation: Chochai, Araya (2014) Phylogenetics, genome size evolution and pop- ulation genetics of slipper orchids in the subfamily cypripedioideae (orchidaceae). PhD thesis, Birkbeck, University of London. c 2014 The Author(s) All material available through ORBIT is protected by intellectual property law, including copyright law. Any use made of the contents should comply with the relevant law. Deposit guide Contact: email Phylogenetics, genome size evolution and population genetics of slipper orchids in the subfamily Cypripedioideae (Orchidaceae) Thesis submitted by Araya Chochai For the degree of Doctor of Philosophy School of Science Birkbeck, University of London and Genetic Section, Jodrell Laboratory Royal Botanic Gardens, Kew November, 2013 Declaration I hereby confirm that this thesis is my own work and the material from other sources used in this work has been appropriately and fully acknowledged. Araya Chochai London, November 2013 2 Abstract Slipper orchids (subfamily Cypripedioideae) comprise five genera; Paphiopedilum, Cypripedium, Phragmipedium, Selenipedium, and Mexipedium. Phylogenetic relationships of the genus Paphiopedilum, were studied using nuclear ribosomal ITS and plastid sequence data. The results confirm that Paphiopedilum is monophyletic and support the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum. Four sections of subgenus Paphiopedilum (Pardalopetalum, Cochlopetalum, Paphiopedilum and Barbata) are recovered with strong support for monophyly, concurring with a recent infrageneric treatment. Section Coryopedilum is also recovered with low bootstrap but high posterior probability values. Relationships in Barbata remain unresolved, with short branch lengths and narrow geographical distributions suggesting it may have undergone rapid radiation. Genome sizes were measured for seven taxa in Paphiopedilum and chromosome and genome size data mapped onto the phylogenetic framework, showing no clear trend in increase in chromosome number in the genus. The diploid chromosome number of 2n = 26 in subgenera Parvisepalum and Brachypetalum suggests it is the ancestral condition, with higher chromosome numbers in Cochlopetalum and Barbata pointing to centric fission possibly having occurred independently in these sections. Although species in Barbata have larger genome sizes than other sections, any trend of genome size evolution remains unclear in the genus. Eight primer pairs for plastid microsatellites were designed from consensus sequences generated from different genera, most of them shown to be applicable across the subfamily. High levels of variation in allele size were observed at interspecific levels but at intraspecific level, low levels were observed in Cypripedium calceolus. The application of plastid microsatellites for population genetic analyses in C. calceolus was limited because few of them are polymorphic and low numbers of alleles were detected. Results were generally congruent with a previous study. Within the limits of this data, the plastid haplotype distribution of C. calceolus in western and northern Europe could indicate possible recolonisation routes from three main refugia, following glaciations. Size variation has also been detected in other species in some markers but sampling was sparse. 3 Acknowledgements First of all, I would like to take this opportunity to sincerely thank my supervisors, Dr Mike Fay and Dr Martin Ingrouille for their guidance, support and valuable advice over the duration of my PhD study. I would also like to express my gratitude to Dr Ilia Leitch for her advice and valuable comments. I am also deeply grateful to the staff of the Genetics and Molecular Systematics Sections at the Jodrell Laboratory, RBG Kew, especially, Dr James Clarkson, Dr Dion Devey, Dr Lola Lledó, Robyn Cowan, Laszlo Csiba, Edith Kapinos and Dr Ralf Kynast for their technical support in the laboratory and helpful advice whenever I came across problems. Thank you also to Christopher Ryan and Bala Kompalli (The Tropical Nursery RBG, Kew) for giving access to living plant collections and for generously letting me have the plant material. Many thanks to all of my friends during my study at the Jodrell, especially Jeff Joseph, Benedetta Bernardini, Jacqueline StQuinton, Charlie Harris, Matthew O'Reilly, Bhanubong Bongcheewin, Dr Michele Sanchez, Dr Harris Saslis-Lagudakis, Dr Jaume Pellicer, Dr Laura Kelly, Dr Cynthia Sothers, Dr Elizabeth Baloch, Dr Imalka Kahandawala and Dr Mehdi Zarrei for their advice, support and valuable help, it is deeply appreciated. Finally, I would like to say a heartfelt thank you to all of the members of my family for their encouragement, support and patience. I am sincerely grateful to my husband, Jon Shore, for being supportive, understanding and always standing by me through the good and particularly the difficult times. Without him I would not have come this far to write this page. 4 Table of Contents Declaration .................................................................................................................. 2 Abstract ...................................................................................................................... 3 Acknowledgements .................................................................................................... 4 Table of Contents ....................................................................................................... 5 Index of Tables............................................................................................................ 8 Index of Figures .......................................................................................................... 9 Chapter 1: General Introduction .............................................................................. 10 1.1 The Family Orchidaceae ....................................................................................... 10 1.2 Subfamily Cypripedioideae ................................................................................... 11 1.3 Conservation of the slipper orchid subfamily Cypripedioideae............................... 12 1.4 Genus Paphiopedilum ........................................................................................... 13 1.4.1 Taxonomic history of the genus Paphiopedilum...................................... 14 1.4.2 Ecology and distribution of Paphiopedilum ............................................. 22 1.5 Cypripedium calceolus .......................................................................................... 24 1.6 Phylogenetics and genome size evolution ............................................................ 24 1.7 Population genetics and plant conservation .......................................................... 26 1.8 Thesis structure .................................................................................................... 28 Chapter 2: Molecular phylogenetics of Paphiopedilum based on nuclear ribosomal ITS and plastid sequences ..................................................................... 29 2.1 Introduction ........................................................................................................... 29 2.1.1 Aims of this study ................................................................................... 31 2.2 Materials and methods .......................................................................................... 31 2.2.1 Plant materials........................................................................................ 31 2.2.2 DNA extraction ....................................................................................... 35 2.2.3 Amplification ........................................................................................... 35 2.2.4 Parsimony analysis................................................................................. 37 2.2.5 Bayesian analysis ................................................................................... 37 2.3 Results .................................................................................................................. 37 2.3.1 Alignment of data sets ............................................................................ 37 2.4 Discussion ............................................................................................................ 43 2.4.1 Congruence of ITS and plastid data ....................................................... 43 2.4.2 Phylogenetic relationships in Paphiopedilum .......................................... 43 2.4.2.1 Subgenus Parvisepalum .......................................................... 43 2.4.2.2 Subgenus Brachypetalum ........................................................ 45 5 2.4.2.3 Subgenus Paphiopedilum ........................................................ 46 Chapter 3: Genome size and chromosome number evolution within the genus Paphiopedilum .......................................................................................................... 50 3.1 Introduction ........................................................................................................... 50 3.1.1 Aims of this study ..................................................................................