Studies in the genus Fritillaria L. (Liliaceae) Peter D. Day, BSc. October 2017 Submitted in partial fulfilment of the requirements of the Degree of Doctor of Philosophy Supervisors: Prof. Andrew R. Leitch Dr Ilia J. Leitch Statement of Originality I, Peter Donal Day, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: October 31st 2017 Details of collaboration and publications. Chapter 2 is published in Day et al. (2014): Day PD, Berger M, Hill L, Fay MF, Leitch AR, Leitch IJ, Kelly LJ (2014). Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae). Molecular Phylogenetics and Evolution, 80: 11-19. Martyn Rix and Takato Natsui provided plant material; Jeremy Broome, Richard Kernick and Kit Strange cultivated and supplied plant material from the RBG Kew Living Collection; Jaumé Pellicer helped with figures; Yasmin Milner and Matthew O Reilly produced some of the plastid sequences used in this study; Donald Fraser helped with the LCNG screen; László Csiba and Edith Kapinos provided education about DNA extractions; Sven Buerki gave guidance on using software program PAUPRat; Christine 2 Leon provided guidance on medicinal usage of Fritillaria; Haris Saslis-Lagoudakis indulged in useful discussions about bioprospecting. All authors contributed to editing and commenting on the original manuscript. 3 Abstract Genus Fritillaria consists of approximately 140 species of bulbous herbaceous perennials with a bicontinental distribution (Eurasia, North America) across the temperate zone of the Northern Hemisphere. The genus includes species of horticultural commercial value and those used in Traditional Chinese Medicine (TCM) preparations. Since the mid-16th century species within genus Fritillaria have attracted much attention as ornamental plants in the Middle East and Europe. The genus has received much attention by geneticists in recent years because it includes species with an exceptionally large range of genome sizes, including a subgenus containing some of the largest diploid genome sizes so far recorded in plants. This thesis first presents the most comprehensive analysis of the phylogenetic relationships between the species to date, published in 2014 in Molecular Phylogenetics and Evolution. The work reveals two distinctive clades, one containing a small number of species from N. America and N. E. Asia and the other, with the majority of species, found in Europe, N. Africa, Middle East, China and Japan. This phylogenetic distribution of Fritillaria species indicates that two independent shifts towards giant genomes have taken place during evolution of the genus. One particularly charismatic species occurring across Eurasia is the Snake’s-head Fritillary (F. meleagris) with both horticultural and conservation importance. The thesis presents new genetic markers to analyse the relationships between populations of F. meleagris across Eurasia. Three distinct populations were found, in Northern Europe/Scandinavia, Eastern Europe and the Russian Federation. All the English populations are almost certainly derived from populations originally occurring in Northern Europe and likely introduced into England through multiple introductions. However, its origin in the British Isles remains a mystery, although recent research within this project has thrown new light on the history of its first record and description in France and its early presence in Britain. 4 Contents 1. General Introduction 16 1.1 Genus Fritillaria (Liliaceae) 16 1.2 Why study genus Fritillaria? 16 1.3 Traditional Chinese Medicine (TCM) 17 1.4 History of Fritillaria species in Europe 18 1.5 Why study Fritillaria meleagris? 18 1.6 Species characteristics 19 1.7 Habitats 20 1.8 Life cycle of F. meleagris 21 1.9 Outlook on this thesis 22 2. Evolutionary relationships in the medicinally important genus Fritillaria L. 23 2.1 Summary 23 2.2 Introduction 24 2.3 Materials and methods 26 2.3.1 Taxon sampling 27 2.3.2 DNA isolation 27 2.3.3 Amplification and sequencing of plastid regions 27 2.3.4 Amplification and sequencing of low-copy nuclear gene (LCNG) regions 27 2.4 Sequence alignment and dataset optimisation 28 2.5 Phylogenetic analyses 29 2.6 Results 30 2.6.1 Combined plastid analyses 30 2.6.2 LCNG screen 33 5 2.7 Discussion 34 2.7.1 Relationships between Fritillaria subgenera 34 2.7.2 Polyphyly of subgenus Fritillaria and phylogenetic distribution of species used in Traditional Chinese Medicine (TCM) 37 2.7.3 Evidence for species non-monophyly 39 2.8 Conclusions 39 3. The introduction of Fritillaria to Europe 41 3.1 Introduction 41 3.2 Materials and methods 42 3.2.1 Herbals 42 3.2.2 Institutions, libraries, online databases and archives 42 3.2.3 Academics, researchers and authors 44 3.2.4 Searching for Fritillaria from the late 15th century 45 3.3 Results 45 3.3.1 Searching for Fritillaria 45 3.3.2 First descriptions and depictions of Fritillaria 46 3.3.3 The tulip 48 3.3.4 Fritillaria meleagris in the mid to late 16th century 49 3.4 Discussion 54 4. Microsatellite development and analysis for F. meleagris 56 4.1 Introduction 56 4.1.1 Large genome size constraints 57 4.2 Materials and methods 58 4.2.1 DNA template sample acquisition and assay 58 4.2.2 454 sequence production and quality assessment 58 4.2.3 Microsatellite discovery and primer pair design 59 4.2.4 Population leaf sample collection 60 4.2.5 DNA isolation from a test panel of population samples 61 6 4.2.6 M13-tailed primer method and procedure 61 4.2.7 Primer pair screening 62 4.2.8 PCR protocol 62 4.2.9 Optimisation of template DNA quantity and primer Tm for PCR 63 4.2.10 Genotype recovery from panel individuals 64 4.2.11 Problems of inconsistency in allele sizing and genotype recovery 64 4.2.12 Genotype recovery process 65 4.2.13 Consistent and logical allele boundary sizing 65 4.2.14 Software programs used for genotype development 66 4.3 Results 66 4.3.1 DNA template measurements of purity and concentration 66 4.3.2 454 sequence production and quality assessment 66 4.3.3 Microsatellite discovery and primer pair design 67 4.3.4 Primer pair screening and selection 67 4.3.5 Optimising template DNA quantity and primer Tm for PCR 68 4.3.6 Test panel alleles and genotyping 68 4.3.7 Problems encountered during the genotyping process 70 4.4 Discussion 71 5. Genetic diversity in Eurasian populations of F. meleagris 74 5.1 Introduction 74 5.2 Materials and methods 74 5.2.1 Methodology of approach 74 5.2.2 Distribution and status of F. meleagris 75 5.2.3 Determining which populations to sample 75 5.2.4 Collecting leaf sample material 79 5.2.5 Sampling and sample collection strategy 79 5.2.6 Completion of DNA extractions and genotype recovery 80 7 5.2.7 Validation of collected leaf and voucher samples 80 5.3 Descriptive and inferential statistics 81 5.3.1 Hardy-Weinberg genotypic equilibrium (HWE) 82 5.3.2 Linkage disequilibrium (LD) 82 5.3.3 Null alleles, large allele dropout and private alleles 83 5.3.4 Locus heterozygosity and polymorphism 85 5.3.5 Population bottlenecks 85 5.3.6 Principal coordinates analysis (PCoA) 86 5.3.7 Mantel Test 87 5.3.8 STRUCTURE analysis 88 5.3.9 Software programs used for population structure analysis 93 5.4 Results 93 5.4.1 Population sizes 93 5.4.2 Validation of collected samples to be of F. meleagris origin 93 5.4.3 Leaf sample DNA isolation and genotype recovery 93 5.4.4 Hardy-Weinberg equilibrium 94 5.4.5 Linkage disequilibrium 95 5.4.6 Alleles, private alleles and null alleles 95 5.4.7 Locus heterozygosity and polymorphism 96 5.4.8 Population bottlenecks 96 5.4.9 Principal coordinates analysis 97 5.4.10 Mantel Test 99 5.4.11 STRUCTURE analysis 100 5.5 Discussion 104 5.5.1 Movement of bulbs 106 5.5.2 History of F. meleagris in the British Isles 109 5.5.3 Conservation implication 111 5.6 Data archiving 113 8 6. Conclusions and future research 114 6.1 Thesis aims 114 6.2 Future work 115 Bibliography 118 Appendix A Supplementary Tables 152 Appendix B Supplementary Figures 165 9 List of Figures 1.1 Data from the Plant DNA C-values database 17 1.2 F. meleagris, red form 19 1.3 F. meleagris, colour morph 19 2.1 Ninety-five percent majority rule consensus tree 32 2.2 Distribution of Fritillaria in Asia 33 3.1 ‘Hiacinto de homero’ Giallo 82. BNM 48 3.2 ‘Lilium susianum’ Giallo 83. BNM 48 3.3 ‘Tulipa turcarum’ Conrad Gessner c. 1559 48 3.4 ‘Tulipa turcarum’ Conrad Gessner (1561) 49 3.5 Fragment of letter from Capperon to Clusius 49 3.6 ‘Fritillaria’ named by Capperon 49 3.7 F.