This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Systematics of Clematis in Nepal, the evolution of tribe Anemoneae DC. (Ranunculaceae) and Phylogeography and the Dynamics of Speciation in the Himalaya Alan C Elliott Doctor of Philosophy The University of Edinburgh Royal Botanic Garden Edinburgh 2016 The Nepalese endemic Clematis phlebantha L.H.J. Williams. Stainton #4281, 6th June 1963, Suli Gad, West Nepal. Photograph A.D. Stainton. Sibbald Trust care of Royal Botanic Garden Edinburgh. Declaration I hereby declare that the work contained in this thesis is my own unless otherwise acknowledged and cited. This thesis has not in in whole or part been previously presented for any degree. Abstract The genus Clematis L. (Ranunculaceae) was used as a new model group to assess the role of the Himalayan orogeny on generation of biodiversity through investigations of its phylogeny, phylogeography and taxonomy. Although existing checklists include 28 species of Clematis from Nepal, a comprehensive taxonomic revision of available material in herbaria and additional sampling from fieldwork during this study has led to the recognition of 21 species of Clematis in Nepal, including one species (C. kilungensis) not previously recorded from Nepal. Exisiting phylogenetic and taxonomic concepts were tested with the addition of new samples from Nepal. The results highlight the shortcomings of the previous studies which were poorly resolved and indicate the need for a thorough revision of the sectional classification. Despite the increased sampling the results are still equivocal due to poor statistical support along the backbone of the phylogeny. Groups of species in well supported terminal clades are broadly comparable with results from previous studies although there are fewer clearly recognisable and well supported clades. The published dates for the evolution of Clematis were tested and the methodology of the previous study critically reappraised. The results indicate that the genus Clematis is approximately twice as old as previously reported and evolved in the middle Miocene. The phylogeny also demonstrates that, even allowing for poor support for the relationships between groups of species within Clematis, the extant Nepalese species must have multiple independent origins from at least 6 different colonisations. With their occurrence in the Pliocene and Pleistocene, these events are relatively recent in relation to the Himalayan orogeny, and may be linked more to the dispersal ability of Clematis than to the direct effects of the orogeny. Additional Nepalese samples of Koenigia and Meconopsis were added to exisiting datasets and these were reanalysed. The result from Clematis, Koenigia and Meconopsis were appraised in light of the the geocientific literature and previously published phylogeographic studies to create an overview of the drivers behind speciation in the Himalaya. i Acknowledgements I’d like to sincerely thank my supervisors Dr Colin Pendry, Dr Gregory Kenicer and Dr Richard Milne for sharing their time, knowledge and considerable skills in helping me get to this point. At the Royal Botanic Garden Edinburgh I would thank Dr Laura Forest, Dr Michelle Hart and Ruth Hollands, in the molecular laboratory, help, assistance and who all answered the same questions again and again. Thanks to David Knott for permission to sample Clematis from the Living Collection. Thanks to John Mitchell for collecting new samples from central Asia and Dr Sabina Knees for new samples from Oman. Special thanks to Elspeth MacKintosh and Gunnar Ovstebo for propagating and caring for the living material on my behalf. From the library my thanks go to Graham Hardy, Leonie Patterson and Deborah Vaile for their help and assistance on every occasion I ventured in. I am especially grateful to Marnel Scherrenberg who generated the extra Koenigia sequence data as part of her excellent thesis on Koenigia for her 2014 MSc in the Biosiversity and Taxonomy of Plants at the University of Edinburgh and Royal Botanic Garden Edinburgh. There are many other people at the Botanics who have helped me during the past four years and I thank them all. I would like to thank and acknowledge the Japanese Society of Himalayan Botany, especially Professor Hiroshi Ikeda for allowing me to join the 2012 fieldwork to Darchula District in the Far West of Nepal. Thanks also to Dr. Partick Kuss for sharing his knowledge about Pedicularis, alpine plants in general and his good humor so freely when on fieldwork to Balgung, Rukum and Dolpa Districts in 2014. I would like to thank the curators and staff at the herbaria of BM, E, K, KATH, for allowing me to visit, study herbarium specimens and arranging loans. Thanks also go to the curators at LIV and PE who digitised specimens for study. Thank you to all the PhD students based at the Botanics who have come and gone in the past four years. I am indebted to the Scottish Rock Garden Club, Davis Expedition Fund and Alpine Garden Society for fincancial support to participate in fieldwork. Finally and especially I have to thank my wife, Carolyn, who has put up with my absences to go on fieldwork and for tolerating my considerable grumbling over the past four years. ii Contents Chapter 1: Introduction 1 Chapter 2: Revision of Clematis L. (Ranunculaceae) in Nepal 4 Introduction 4 Taxonomic History of Clematis in Nepal 10 General morphology of Clematis 20 Materials and methods 21 Morphology of Clematis in Nepal 22 Key to species 23 1. Clematis acuminata DC. 25 2. Clematis alternata Kitam. & Tamura 29 3. Clematis barbellata Edgew. 32 4. Clematis buchananiana DC. 34 5. Clematis confusa Grey-Wilson 39 6. Clematis connata DC. 41 7. Clematis gouriana Roxb. Ex DC. 46 8. Clematis grata Wall. 48 9. Clematis graveolens Lindl. 52 10. Clematis grewiiflora DC. 56 11. Clematis kilungensis W.T.Wang & M.Y.Fang 58 12. Clematis montana Buch.-Ham. ex DC. 60 13. Clematis napaulensis DC. 66 14. Clematis phlebantha L.J.H.Williams 69 15. Clematis puberula Hook.f & Thomson 72 16. Clematis rehderiana Craib. 74 17. Clematis smilacifolia Wall. 78 18. Clematis tibetana Kuntze 80 18a. Clematis tibetana subsp. brevipes (Tamura) Yonek. 80 18b. Clematis tibetana subsp. vernayi (C.E.C.Fisch.) Grey-Wilson 84 19. Clematis tongluensis (Brühl) Tamura 86 20. Clematis zemuensis W.W.Sm. 88 21. Clematis zeylanica (L.) Poir. 90 iii Uncertain Taxa 93 Chapter 3: The evolution of tribe Anemoneae DC. (Ranunculaceae) 94 Introduction 94 Fossils and dating 97 Dated phylogeny of Clematis 100 Aims and Objectives 102 Materials and Methods 102 Taxon sampling 102 Extraction, PCR and sequencing 119 Analysis 122 Sequence Data 122 Divergence time estimations 124 Visualisation of Trees 125 Results 125 Phylogenetic Relationships 125 Divergence time estimate results 133 Discussion 139 Support across the trees 139 Phylogenetic relationships – across the whole group 139 Tribe Anemoneae 139 Subtribe Anemoninae (Anemone sensu lato) 139 Subtribe Clematidinae (Clematis sensu lato and Anemoclema) 140 Divergence time estimates 140 The age of tribe Anemoneae 140 The age of subtribe Anemoninae 140 The age of subtribe Clematidinae 142 Conclusion 143 Chapter 4: The Phylogeography of Clematis L. (Ranunculaceae) in Nepal 144 Introduction 144 Aims 145 iv Materials and Methods 146 Phylogenetics 146 Divergence time estimates 153 Visualisation of Trees 154 Phylogeogrpahic Analysis – RASP 155 Species Distribution Modelling (SDM) 156 Results 158 Phylogenetic Results 158 Placement of new Nepalese accessions 158 Divergence time estimate results 166 Phylogeograhic RASP results 173 Species Distribution Model (SDM) results 177 Discussion 181 Congruence 181 The genus Clematis 182 Section Chieropsis subsection Montanae 183 Section Meclatis 184 Clematis phlebantha 185 Clematis grata 186 Himalayan Connatae grade 187 Appraisal of Clematis fossils 188 Conclusion 188 Chapter 5: Phylogeography and the Dynamics of Speciation in the 190 Himalaya Himalayan Orogeny and Speciation 190 Introduction 1890 Geoscientific Histories of the Himalaya 190 Tectonics 190 Paleoclimate 192 Conflicts in the literature surrounding the Himalayan-Tibetan Orogeny 194 Phylogeography and the Dynamics of Speciation in the Himalaya 199 Reassessment of existing datasets – Meconopsis 199 Materials and Methods 199 v Additional taxon sampling 200 Analysis 202 Divergence time estimates 206 Visualisation of Trees 207 Results 207 Reassessment of existing datasets – Koenigia 211 Materials and Methods 211 Divergence time estimates 212 Visualisation of Trees 212 Results 215 Discussion 217 Uplift and Tectonics 217 Koenigia 217 Cyananthus 218 Monsoon as a driver 219 Begonia 219 Meconopsis 220 Aridification as a driver 224 Clematis 224 Berberis 224 Long distance dispersal 226 Clematis 226 Ranunculus 226 More recent speciation events 227 Koengia 227 Meconopsis 228 Lathyrus 228 Conclusion 228 Chapter 6 : Final conclusions and further work 230 Taxonomy of Clematis in Nepal 230 The evolution of tribe Anemoneae 231 Phylogeography of Clematis in Nepal 232 Phylogeography and Dynamics of Speciation in the Himalaya 233 vi Publications from this PhD 234 Chapter 7 : References 235 List of Figures Chapter 2 Figure 2.1 Dot map of georeferenced herbarium specimens of Clematis from Nepal at 23 BM, E, KATH, K, P, TI. Figure 2.2 Distribution of Clematis acuminata DC. in Nepal 26 Figure 2.3 Clematis acuminata.