Genetic Diversity and Regenerative Potential of Tilia cordata Miller in the Lincolnshire Limewoods Amanda Julie Mylett A thesis submitted in partial fulfilment of the requirements of the University of Lincoln for the degree of Doctor of Philosophy August 2015 Abstract The Lincolnshire Limewoods are a group of Ancient Semi Natural Woodlands within Central Lincolnshire that include the nationally important Bardney Limewoods National Nature Reserve. The woods, although fragmented and isolated by tracts of agricultural land, are reservoirs of biodiversity and contain large populations of Tilia cordata Mill. The current management aims are to increase the biodiversity within the woods, as well as to extend and improve the connectivity between the woodlands, with new planting. An understanding of the genetic diversity and structure of the Limewoods, both as individual woods and by comparison with woods from other regions of Britain, will help to inform management decisions. A pilot study was undertaken using RAPD markers which demonstrated the potential for these markers to amplify and identify individual T. cordata trees. Dominant markers are less informative than co-dominant markers, especially when trees may be closely related, and to facilitate this study a T. cordata enriched microsatellite library was constructed. The ten microsatellite loci designed for the genetic study amplified both T. cordata and closely related Tilia platyphyllos Scop. and were also able to identify hybridisation between the two species. T. platyphyllos and hybrid trees were detected in eleven of the Lincolnshire Limewoods and were associated with identification of private alleles within the T. cordata populations. The high levels of genetic diversity and low genetic variance which were found show that the Lincolnshire Limewoods’ populations are all similar. Comparison with populations from outside Lincolnshire show similar genetic diversity, with AMOVA i conducted over all populations showing that only 4% of the variation could be allocated between the populations whereas 82% was allocated between the individuals within the populations. Weak isolation by distance was identified and would suggest that the Lincolnshire Limewoods should be treated as a single population group for management purposes. To increase the likelihood that T. cordata is used for replanting schemes, seeds should be taken from woods without hybrid populations and, preferably, should be screened to identify and exclude F1 hybrids. Tissue culture was considered as a potential source of planting stock for the Lincolnshire Limewoods to provide trees of known provenance with potentially rejuvenated characteristics. Preliminary tissue culture investigation showed that the initial treatment and age of the tissue before sterilisation is important in controlling contamination after sterilisation. Tissue collected in the spring, prior to leaf emergence, with the buds allowed to develop under clean laboratory conditions resulted in a reduction in the number of contaminated explants. In explant culture, roots were induced with application of the auxin naphthaleneacetic acid (NAA) and microshoots were induced with a combination of NAA and cytokinin, 6-benzyl-aminopurine (BAP). However, no rooted plantlets were produced. Further investigation into the use of somatic embryos as an explant source should now be considered as a possible way of reducing the chronic contamination that was experienced using axial buds. ii Acknowledgements I would like to thank my supervisors, Dr C. Casey, Dr R. Dixon and Dr S. Brown. I am particularly grateful to Dr Sarah Brown who has been with me throughout this journey, providing help, support, constant encouragement and friendship. Also Dr Ciara Casey, who has been a friend and a mentor. I would like to thank my work colleagues Dr Taghread Hudaib, Dr. Bill Hayes and Rachel Farrow for their support over the years. I am also grateful for the help I received from Anne Goodall, who advised me on all aspects of the Bardney Limewoods, and to members of the Limewoods Working Group, especially Hugh Milner and Rodger Clooney, who collected samples for this study. My thanks go to my children Ian, Joanna, Katie and my sister, who have encouraged me throughout, but especially to my husband Dave, who, from collecting samples to fixing computers, has always been there to help. Thanks must also go to the Forestry Commission, especially Wally Grice, as well as the Lincolnshire Limewoods Project and Liz Fleuty for facilitating access to and providing information about the woods. I would also like to recognise that this research would not have been possible without the support of the Department of Biological Science at the University of Lincoln. iii CONTENTS Abstract i Acknowledgements iii Contents iv Figures ix Tables xv Glossary of Abbreviations xviii CHAPTER ONE General Introduction 1 1.1 Introduction 2 1.2 The Bardney Limewoods 3 1.3 Ancient and Semi-Natural Woodland 8 1.4 The Origin of Ancient Limewoods in Britain 9 1.5 The Characteristics of Tilia cordata Miller 13 1.6 Regeneration of Tilia cordata within the Limewoods 23 1.7 The study of genetic variation and structure in Tilia spp. 28 1.8 Aims of the Study 31 1.9 References 33 CHAPTER TWO Development of DNA Extraction Method and Initial Investigation into Genetic Diversity Using RAPD Markers. 41 2.0 Introduction 42 2.1 DNA Extraction 43 2.1.1 DNA Extraction Introduction 43 2.1.2 DNA Extraction Materials and Methods 45 2.1.2.1 Optimised Extraction Method Based on the CTAB B Protocol 48 iv 2.1.3 DNA Extraction Results 51 2.1.4 DNA Extraction Discussion 54 2.2 RAPD Analysis 56 2.2.1 RAPD Analysis Introduction 56 2.2.2 RAPD Analysis Materials and Methods 57 2.2.3 RAPD Analysis Results 59 2.2.4 RAPD Analysis Discussion 63 2.3 References 65 CHAPTER THREE The Development of Microsatellite Markers for the Investigation of the Population Structure of Tilia cordata Miller (Malvaceae) within the Lincolnshire Limewoods 69 3.1 Introduction 70 3.2 Materials and Methods 74 3.2.1 Microsatellite Library Development 75 3.2.2 Microsatellite Characterisation 84 3.3 Results 86 3.3.1 Genetic Diversity and Cross Amplification between Species 88 3.4 Discussion 94 3.5 References 98 v CHAPTER FOUR The Genetic Variation and Structure of Tilia cordata within the Lincolnshire Limewoods. 104 4.1 Introduction 105 4.2 Materials and Methods 109 4.2.1 Sample collection, DNA extraction and PCR 109 4.2.2 Identification of Clonal Ramets 114 4.2.3 Identification of T. platyphyllos and Occurrence of Hybridisation within the Population Groups 115 4.2.4 Identification of Population Genetic Diversity 117 4.2.5 Population Structure 118 4.2.5.1 Fine scale spatial genetic structure within local populations 122 4.3 Results 123 4.3.1 Identification of Clonal Ramets 123 4.3.2 Identification of T. platyphyllos and Hybridisation within the Populations 127 4.3.3 Genetic Diversity 133 4.3.4 Population structure 139 4.3.4.1 Cryptic Population Structure within the Limewoods 147 4.3.4.2 Fine Scale Spatial Genetic Structure 159 4.4 Discussion 161 4.4.1 Clonal Groups 161 4.4.2 Identification of T. platyphyllos among the T. cordata Populations and Hybridisation between T. cordata and T. platyphyllos 163 4.4.3 Genetic Diversity 166 4.4.4 Population Genetic Structure 169 4.4.5 Identification of Cryptic Population Structure 171 4.4.6 Fine Scale Spatial Genetic Structure 173 4.4.7 Conclusion 174 4.5 References 176 vi CHAPTER FIVE Tissue Culture 188 5.1 Tissue Culture Introduction 189 5.1.1 Explant Selection 190 5.1.2 Aseptic Tissue Sterilisation 193 5.1.3 Tissue Culture Medium 194 5.1.4 Plant Growth Regulators 195 5.1.5 Aim 196 5.2 Tissue Culture Materials and Methods 197 5.2.1 Explant Selection, Preparation and Sterilisation 198 5.2.2 The comparison of two chlorine-based sterilisation solutions 201 5.2.3 Tissue Culture Medium and Plant Growth Regulators 203 5.3 Tissue Culture Results 205 5.3.1 Explant Selection, Preparation and Sterilisation 205 5.3.2 The Comparison of Two Chlorine-Based Sterilisation Solutions 207 5.3.3 Tissue Culture Medium and Plant Growth Regulators 214 5.4 Tissue Culture Discussion 219 5.5 References 228 CHAPTER SIX General Discussion 235 6.1 Discussion 236 6.2 Further work 241 6.3 Conclusion 242 6.4 References 243 vii APPENDICES 247 Appendix 1 Maps of the General Location of all Woods Sampled and Maps of Individual Woods Showing Individual Trees with their Genotype Assignment. 248 Appendix 2 Pairwise Fst derived from AMOVA for: - A. 20 wood populations with T. cordata trees B. 21 wood populations with T. cordata, T. platyphyllos and hybrid trees. 270 Appendix 3 Tissue Culture Media Composition and Comparison 273 viii FIGURES Figure 1.1 Map of the Lincolnshire Limewoods Project Area. This area includes the nine SSSI woods that comprise the Bardney Limewoods National Nature Reserves. 6 Figure 1.2 T. cordata together with Hyacinthoides non-scripta (L.) Rothm. and Anemone nemorosa L.; some of Lincolnshire’s ancient woodland indicator plants (Scotgrove Wood spring 2011). 9 Figure 1.3 Isochrone map of the increasing extent of Tilia pollen in the British Isles over time. The isochrones estimate the date (years BP) of the palynological evidence of the presence of Tilia pollen (Birks, 1989). 12 Figure 1.4 The European distribution of Tilia cordata Mill (EUFORGEN, 2008a) and Tilia platyphyllos Scop.(EUFORGEN, 2008b). 15 Figure 1.5 Mature standard T. cordata in Poterhanworth Wood, Lincolnshire. 16 Figure 1.6 T. cordata coppiced stool less than 5 years old. 17 Figure 1.7 Four dominant trunks of a mature T. cordata coppiced tree. 17 Figure 1.8 Young red barked stems of T. cordata before leaves emerge.