Evolutionary relationships and reproductive ecology of endemic Sorbus species in south west UK: Implications for conservation. Submitted by Tracey Jane Hamston to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Sciences In September 2016 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature: ………………………………………………………….. 1 Abstract The genus Sorbus is an example of a taxonomically complex group (TCG) with diversity derived from hybridisation, polyploidy and apomixis. The focus of this study was to elucidate the evolutionary relationships among nine Sorbus species including endemics of the Devon and north Somerset region of the south west UK, determine main routes of polyploid formation and investigate reproductive sustainability in order to make recommendations for Sorbus conservation. Molecular analysis showed that genetic structure patterns and genotypic diversity support the hypothesis that the study polyploids are a product of rare interspecific hybridisation, of single origins and are maintained through apomictic reproduction. PCoA, Neighbour Joining analysis and parental simulations reveal a reticulated relationship, with diversification the result of hybridisations between sexual diploid Sorbus torminalis and both tetraploid and triploid species. Hybridisation between S. torminalis and tetraploid Sorbus margaretae (subgenus Aria) have likely given rise to the study members of subgenus Tormaria through production of a triploid which has subsequently backcrossed to Sorbus torminalis to form further tetraploids. The discovery of a cryptic hybrid in subgenus Aria also suggests occasional hybridisation events among tetraploids are a possible route for further tetraploid formation These events illustrate key routes of polyploid formation, both illustrating the role of triploids in tetraploid formation via the triploid bridge and the key role in sexual diploids in diversification in Sorbus. Hand pollination experiments showed that self-incompatibility in the triploid species (Sorbus subcuneata) means reliance on congeneric pollen from sympatric tetraploid species for seed production. 2 Reproductive sustainability in this species is severely compromised through spatial isolation from compatible congeners. Our findings are strong support for the development of conservation strategies that aim to safeguard current diversity through actions that increase reproductive sustainability and recruitment opportunities, and promote opportunities for on-going hybridisation for future diversification of Sorbus in this region. 3 Acknowledgments Completing this PhD has been an enormous personal challenge and would certainly not have been possible without the guidance, help and support from many people. This PhD was funded by the Whitley Wildlife Conservation Trust and supported by the National Botanic Garden of Wales. I am hugely grateful to my supervisors; Dr James Cresswell whose calm wisdom kept me going in the right direction; Dr Jamie Stevens for the many discussions and brain storming meetings and Dr Natasha deVere of the National Botanic Garden of Wales for her overwhelming support, interest and unflagging enthusiasm throughout. I would like to thank Dr Amy Plowman, Whitley Wildlife Conservation Trust, who maintained faith in me over the last seven years. I also want to thank Dr Tim Rich, whose expertise in all things Sorbus and eagerness to scale cliffs to collect samples were an inspiration! The support from other members of the Field Conservation and Research department past and present was particularly appreciated. Thanks to, Dave Ellacott and Kirsten Pullen; fellow PhD students Andy Bowkett, Holly Farmer, Jo Newbolt and Kathy Baker for all their encouragement. Over the course of my PhD I was fortunate to spend many days in the field accompanied by a number of people without whom my data collection would not have been possible. I am enormously indebted to Nick Foale for his enthusiastic assistance in this regard. I also thank Libby Houston and Martin Lepsi for their help collecting samples. Thank you to Exmoor Natural History Society, especially Caroline Giddens, National Trust reserve manager Julian Gurney, 4 Marcus Wilde and the vice-county recorders for all their kind permission to carry out field experiments on their sites and provide me with information about Sorbus distribution. Also to Sas, my faithful companion on all my field trips, whose endless enjoyment of Exmoor was catching! There were many people at Exeter who gave me much appreciated advice and technical guidance in the lab as well as moral support; in particular Josie Paris, Philippa Holder, Sarah Louise-Counter, Emma Kennedy, Charles Ikediashi, Patrick Hamilton and especially Andy King. Thank you Mike Fay and Jaume Pellicer who gave me the opportunity to visit the Jodrell Laboratory at Kew and carry out flow cytometry on my samples. Finally my huge gratitude goes to all my family who provided a never ending source of support and tolerance over the years as well as field work assistance; Jon and Jack thank you I couldn’t have done it without you, and thanks to my mum Anne who always had faith in me. 5 CONTENTS Abstract ...................................................................................................... 2 Acknowledgments ........................................................................................ 4 Chapter 1: General Introduction ............................................................... 20 1.1 Plant conservation ............................................................................. 20 1.2 Taxonomic complexity and diversification .......................................... 21 1.3 Hybridisation and polyploidy .............................................................. 22 1.4 Polyploid formation ............................................................................ 23 1.5 Polyploid speciation ........................................................................... 25 1.5.1 Reproductive isolation ............................................................. 26 1.5.2 Evolution of new breeding systems: Polyploidy and apomixis . 27 1.6 Methodological challenges for genetic studies of apomictic polyploids. .......................................................................................... 28 1.6.1 Molecular markers................................................................... 28 1.6.2 Development of molecular methods for this study.....................30 1.6.3 Molecular analysis of mixed ploidy groups containing sexual and apomictic individuals..................................................................31 1.7 Pollination and population sustainability...............................................33 1.8 Sorbus as a model system of a Taxonomically Complex Group ........ 35 1.8.1 Life history ............................................................................... 36 1.8.2 Reproduction in Sorbus ........................................................... 40 1.8.3 Taxonomic complexity of Sorbus ............................................ 42 6 1.8.4 What constitutes a species in Sorbus?.................................... 44 1.8.5 Diversification in Sorbus .......................................................... 45 1.8.6 Conservation status................................................................. 47 1.9 Sorbus in Devon and north Somerset ................................................ 48 1.9.1 Distribution .............................................................................. 48 1.9.2 History and origins of the Devon and north Somerset Sorbus . 53 1.9.3 Conservation and threats ........................................................ 55 1.9.4 Tree identification and sampling................................................58 1.10 Conclusion ....................................................................................... 66 1.11 Thesis aims and structure ................................................................ 66 References ............................................................................................... 69 Chapter 2: Apomixis and hybridisation drives reticulate evolution and phyletic differentiation in Sorbus L.: Implications for conservation .............................................................................. 80 Abstract ................................................................................................ 81 2.1 Introduction ........................................................................................ 82 2.2 Materials and Methods....................................................................... 87 2.2.1 Plant material .......................................................................... 87 2.2.2 DNA extraction and molecular markers ................................... 87 2.2.3 Estimation of ploidy ................................................................. 89 2.2.4 Data analyses ......................................................................... 90 2.3 Results ............................................................................................... 94 7 2.3.1 Microsatellite markers ............................................................