Evolution of moonwort ferns (Botrychium, Ophioglossaceae) on local to global scales Thèse présentée à la Faculté des sciences Institut de biologie Laboratoire de génétique évolutive Université de Neuchâtel, Suisse Pour l’obtention du grade de DOCTEUR ÈS SCIENCES Par Benjamin Dauphin Présenté aux membres du jury de thèse: P.D. Dr Grant Jason, directeur de thèse et président du jury Prof. Daniel Croll, rapporteur Prof. Donald Farrar, rapporteur Prof. Felix Kessler, rapporteur Dr Michael Kessler, examinateur Prof. Carl Rothfels, examinateur Soutenue le 17 octobre 2017 1 2 Faculté des Sciences Secrétariat-décanat de Faculté Rue Emile-Argand 11 2000 Neuchâtel – Suisse Tél : + 41 (0)32 718 21 00 E-mail : [email protected] IMPRIMATUR POUR THESE DE DOCTORAT La Faculté des sciences de l'Université de Neuchâtel autorise l'impression de la présente thèse soutenue par Monsieur Benjamin DAUPHIN Titre: “Evolution of moonwort ferns (Botrychium, Ophioglossaceae) on local to global scales” sur le rapport des membres du jury composé comme suit: ñ MER Jason Grant, directeur de thèse, Université de Neuchâtel ñ Prof. Daniel Croll, Université de Neuchâtel ñ Prof. Donald R. Farrar, Iowa State University, USA ñ Prof. Felix Kessler, Université de Neuchâtel ñ Dr Michael Kessler, Universität Zürich ñ Prof. Carl Rothfels, University of California, Berkeley, USA Neuchâtel, le 9 novembre 2017 Le Doyen, Prof. R. Bshary Imprimatur pour thèse de doctorat www.unine.ch/sciences 2 «Fais de ta vie un rêve, et d’un rêve, une réalité» Antoine de Saint-Exupéry (1900–1944) 3 4 Acknowledgments This PhD was an intense and marvelous life experience for me. I am grateful to my spouse Lisa who has provided me emotional support and full confidence during the completion of this dissertation. A very special gratitude goes out to my parents Marie-Christine and Daniel who encouraged and supported me during my university studies, especially at a time when I had other interests than science. I thank my friends for your unconditional support despite the strange world in which I work. For three years, I was lucky to work in such a scientific environment and lead a project that really meant a lot to me. Several people and institutions have contributed greatly in the success of this project and I would like to express my sincerely gratitude in the simplest words. I warmly thank you Jason for giving me confidence to carry out this project and introducing me to the fern world. Also, thanks a lot for your patience. I know, sometimes it was demanding… Thank you Don for everything! Carl, Berkeley is a captivating city, and I am lucky to have studied there on the fascinating topic of polyploidy. Many thanks for your hospitality in California and your precious collaboration, especially your tricks and secrets about the PacBio seq and the use of PURC. I am also grateful to members of the Evolutionary Genetics lab at the University of Neuchâtel, the Genetic Diversity Center from ETH Zürich, and the Rothfels lab at the University of California at Berkeley, with a special mention to Olivier Bachmann, Forrest Freund, Stella Huynh, Ingrid Jordon- Thaden, Silvia Kobel, Aria Minder, Vinciane Mossion, Frédéric Sandoz, Julien Vieu, Guillaume Wos, and Niklaus Zemp. It was fantastic to work with each of them, especially on NGS data! Without funding, we can’t go very far in academic research. Therefore, I thank the Swiss National Science Foundation, the University of Neuchâtel, and the University of California at Berkeley for supporting this thesis. Thanks to everyone for your positive spirit! 5 6 Abstract Plants have long fascinated biologists by their ability to form cryptic species, hybrids between distinct species, large and stable genome sizes, and a wide variety of mating systems among taxa that have driven to the current plant biodiversity. More than 450 million year ago, vascular plants emerged and colonized the land. Rapidly, they dispersed to explore new habitats and diversified in suitable environments to form over 300,000 extant species. A better understanding of past evolutionary forces that led to the current plant morphologies, ecologies, and genetic diversity is critical for predicting their evolution, especially with the global changes ongoing. In this context, the main goals of this thesis were to investigate the phylogenetic relationships among the early divergent Botrychium taxa and how allopolyploidy and the alternation of mating systems have led to the speciation of these species. In a multidisciplinary framework, we combined phylogenetics with population genetics and flow cytometry to provide relevant cellular and molecular data for exploring key biological mechanisms taking place at local or global scale. As the backbone of our project, we reconstructed the plastid phylogeny of the genus Botrychium based on a worldwide sampling to investigate relationships among diploid taxa and the maternal origins of allopolyploids. As a second step, we estimated the genome sizes of Botrychium diploids and polyploids to study fluctuations of DNA amounts after allopolyploidization and between ploidy levels. Then, we applied PacBio sequencing to infer a nuclear phylogeny for revealing the evolutionary history for both, the maternal and the paternal lineages of allopolyploids, and drawing the timescale of their bi-parental inheritance based on divergence time estimates. Focusing at a local and regional scale, we genotyped B. lunaria populations in Alps using co-dominant allozymes and ddRADseq data to identify the predominant mode of reproduction as well as its demographic history during the last glacial maximum. We found unexpected results and major discoveries for the understanding of the biology of early vascular plants. For the species diversity itself, we identified thirteen possibly new taxa and characterized the exceptional haplotype diversity occurring in the Lunaria complex. Also, we presented evidence for multiple origins of several polyploid taxa and highlighted incontestable cases of inter-continental dispersal from North America to Europe and Asia. With flow cytometry, we reported a new hexaploid in Botrychium and described different genome sizes between diploid species of the two major clades Lanceolatum and Lunaria. Besides, our results supported the genome size stability after allopolyploidization, therefore rejecting the scenario of genome downsizing widespread accepted for angiosperms. Probably the most striking outcome of our phylogenetic investigations is the recurrent allopolyploidy in that genus and the strong bias of parental donors in the formation of allopolyploid taxa. Furthermore, our divergence time estimates revealed the recent and rapid 7 speciation via allopolyploidy in the last two million years, which constitutes a first case of radiation in the old lineage of Ophioglossaceae. For our population genetics study, we found unprecedented genetic diversity within B. lunaria populations with a large number of heterozygotes that supports the outcrossing mating system. Thus, we presented the capabilities of dispersion and diversification of these Botrychium species to better understand the ancestral vascular plant mating system. Being a key element of this speciation model, we found a genetic signature indicative of a refuge for B. lunaria in the central Alps during the last glacial maximum, which has hosted individuals having a high allele richness that was secondarily dispersed after deglaciation with the maintenance of outcrossing in alpine grasslands. Undeniably, the genus Botrychium offers a unique opportunity to address the role of allopolyploidy and the importance of alternation of mating systems in plant speciation. This work is intended to be the starting point for further studies in evolutionary biology that ultimately will provide a better understanding of the life style of these enigmatic fern species. Key words: Botrychium; ddRAD sequencing; divergence time dating; ferns; flow cytometry; genome size; low-copy makers; mating system; molecular phylogeny; Ophioglossaceae; PacBio; polyploidy; polyploid network; population genetics; PURC; reticulate evolution. 8 Résumé Les plantes ont depuis longtemps fasciné les biologistes par leur capacité à former des espèces cryptiques, des hybrides entre des espèces distinctes, de grandes tailles de génomes stables, et une large variété de systèmes reproducteurs parmi les taxons qui ont conduit à l’actuelle biodiversité des plantes. Il y a plus de 450 millions d’années, les plantes vasculaires ont émergé et colonisé la terre. Rapidement, elles se sont dispersées pour explorer de nouveaux habitats et se sont diversifiées dans des environnements favorables pour former plus de 300 000 espèces vivantes. Une meilleure compréhension des forces évolutives passées qui ont mené aux morphologies, aux écologies, et aux diversités génétiques actuelles des plantes est essentielle pour prédire leur évolution, particulièrement dans le contexte des changements globaux. Ainsi, les objectifs principaux de cette thèse consistaient à étudier les relations phylogénétiques parmi les taxons de Botrychium et comment l’allopolyploïdie et l’alternance des systèmes reproducteurs ont mené à la spéciation de ces espèces. Dans un cadre multidisciplinaire, nous avons combiné la phylogénétique avec la génétique des populations et la cytométrie en flux pour produire des données cellulaires et moléculaires pertinentes pour l’exploration des mécanismes biologiques clefs prenant place à des échelles locales ou globales. Etant les fondements