HYBRIDIZATION, GENOME DUPLICATION, AND CHEMICAL DIVERSIFICATION IN THE EVOLUTION OF CALENDULA L. (COMPOSITAE) A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Olofron Plume January 2015 © 2015 Olofron Plume HYBRIDIZATION, GENOME DUPLICATION, AND CHEMICAL DIVERSIFICATION IN THE EVOLUTION OF CALENDULA L. (COMPOSITAE) Olofron Plume, Ph. D. Cornell University 2015 Hybridization and polyploidy are common in plants. Both processes can have extensive genomic consequences, and resulting morphological, biochemical, and reproductive changes may drive speciation. The effects of hybridization and polyploidy on speciation and biochemical diversity were explored in Calendula, a small, circum-Mediterranean genus in Compositae. Calendula officinalis (pot marigold), the best known species, has been cultivated for centuries for ornamental and medicinal use. Calendula is remarkable for the wide range of chromosome numbers (2n=14, 18, 30, 32, 44, and ~85), likely resulting from hybridization between species with different chromosome numbers followed by genome duplication. Hypotheses of species origins were tested using evidence from three non-coding chloroplast regions (atpIatpH, petLpsbE, and ndhFrpl32), nuclear ribosomal DNA (ITS), and two putatively low-copy nuclear markers (Chs and A39). Analyses of these markers provided support for a division of the genus into annual and perennial polyploid complexes, multiple origins of most polyploid taxa, and a single origin of C. officinalis. A39 was duplicated once in Calendula. Nine or more duplications of Chs were inferred from analyses of Calendula sequences with others from Compositae. Variation of four classes of phenolic (caffeic acid derivatives and flavonoid glycosides) and isoprenoid (monoterpenes and sesquiterpenes) compounds was investigated within the context of the annual and perennial polyploid complexes. Arising from different biosynthetic pathways, and highly diverse in plants, phenolics and isoprenoids offered different perspectives on the effects of speciation, hybridization, and polyploidy on chemical diversification. All four classes varied quantitatively and/or qualitatively across Calendula species. For three of four compound classes, proportions of compounds within each class were relatively steady within taxa or within tissue types but varied across taxa or tissue types, suggesting that particular blends of compounds may have evolutionary and ecological significance in Calendula. Neither the number of compounds detected nor bulk concentration of any compound class increased consistently with ploidy, but both were higher in floral tissue than in leaf tissue. Phenolic profiles were more consistent with hypotheses of evolutionary relationships than were isoprenoid profiles. Compound profiles in the annual polyploids showed additive compound complements or intermediate proportions of dominant compounds relative to putative progenitors. BIOGRAPHICAL SKETCH Olofron Plume received a B.A. in English Literature from the University of Colorado in 1997, an M. A. in Foreign Language Education from Soka University of America in 2002, and a second B. A. in Ecology and Evolutionary Biology from the University of Colorado in 2006. iii To Cindy and Cleo iv ACKNOWLEDGEMENTS Firstly, I want to thank my major advisor, Dr. Jeff Doyle, for his steadfast encouragement, attention to detail, constructive criticism, availability, and flexibility (e.g., four years of weekly meetings over Skype!) no matter what I tried to take on or where I was in the world, and to my entire committee, including Dr. Andre Kessler and Dr. Melissa Luckow, whose commitment to me and to my research was (and is) unflagging. I would like to thank to all of the faculty, staff, and students of the Department of Plant Biology and the L.H. Bailey Hortorium, and particularly Dr. Jerold Davis, Dr. Alejandra Gandolfo, Dr. Kevin Nixon, Dr. Jim Reveal, Dr. Bill Crepet, Sue Sherman-Broyles, Jane Doyle, Anna Stalter, and Peter Fraissinet, each of whom always made time for a conversation, a pep talk, or to share expertise and offer guidance; Peter Fraissinet for his translation of German keys to Calendula taxa; Bob Dirig who gave the best and most inspired tour of an herbarium ever when I was choosing a graduate program; Karin Jantz for keeping me on track toward graduation; Tara Nihil for managing all of my grants; and my fellow graduate students in the L.H. Bailey Hortorium who never ceased to inspire and motivate me, particularly Cynthia Skema, Shannon Straub, Mariana Yazbek, Janelle Burke, Jim Cohen, Jeremy Coate, Caroline Kellogg, Dan Ilut, Tee Havananda, Mary Futey, Simon Gunner, Gwynne Lim, and Adrian Powell. I would also like to thank all the members of the Kessler Lab in the Department of Ecology and Evolutionary Biology, and particularly Rayko Halitschke for his assistance with HPLC and GC/MS analyses. Many thanks also to the staff in the Plant Science and Gutterman greenhouses for caring for my living collection of Calendula plants, and thanks to members of the Doyle Lab and Nancy Roberts for helping me collect nearly 300 samples on ice, over the course of two mornings, for use in chemical analyses. As I prepare to leave Cornell, I fear I may never again find such a vibrant and inspiring community of scientists and scholars, nor ever v again have access to such a depth and breadth of botanical and ecological knowledge and resources all in one place. Many people and institutions outside of Cornell contributed samples, offered field and herbarium assistance, or otherwise provided space and/or resources for the completion of this work. I am grateful for donations of germplasm and tissue for my research from the North Central Regional Plant Introduction Station of the U. S. National Plant Germplasm System, which has an extensive collection of Calendula germplasm, from the Israel Plant Gene Bank, and from colleagues Dr. Paulo Silveira (Universidade de Aveiro, Portugal), Dr. Angelo Troia (Universitá degli Studi di Palermo, Italy), and Dr. Alan Wood (Agricultural Research Council, South Africa). I offer extra thanks to Dr. Paulo Silveira for bringing me on a collecting expedition in southern Spain and for providing taxonomic advice and cytological observations over the course of my studies; to Dr. Francesco Raimondo and Dr. Gianniantonio Domina for providing lodging and facilitating my herbarium work at PAL; to Dr. Francesco Raimondo, Dr. Gianniantonio Domina, and Dr. Angelo Troia, Dr. Cynthia Skema, and several other researchers at the Orto Botanico di Palermo for accompanying me on collecting expeditions in Sicily, to Dr. Angelo Troia for involving me in his studies on hybridization between species of Calendula in Sicily and in his efforts to save the threatened sea marigold (Calendula maritima), to Dr. Mohamed Fennane and Dr. Jalal El Oualidi (Institut Scientifique de Rabat, Morocco) for facilitating my field and herbarium work at RAB and for allowing destructive sampling from some specimens, to Hamid Khamr (Institut Scientifique de Rabat) for accompanying me in the field in the vicinity of Rabat, and finally to Mariana Yazbek for taking me on collecting expeditions throughout Lebanon. I offer a very big thank you to Massey University (Palmerston vi North, New Zealand) and the lab of Dr. Jennifer Tate and Dr. Vaughan Symmonds for hosting me as a visiting student during a portion of this work. I owe an immense debt of gratitude to my family. Thank you to my parents, my sister, and my grandmother for instilling in me a fierce love of exploring and learning, whether it be within the walls of academia or elsewhere. Thank you to my in-laws who supported me and cheered me on to the finish. Thank you to my wife who has taught me much about science and even more about life, who inspired me, encouraged me, and endured my process every step of the way, who believed in me even when I did not, and who never let me give up. Finally, thank you to our daughter, who made all of this worthwhile and who continues to help me keep things in perspective. I gratefully acknowledge funding for this work from a National Science Foundation Doctoral Dissertation Improvement Grant (DEB-0909832), from the Botanical Society of America and the American Society of Plant Taxonomists Graduate Research and Travel Awards, and from the Harold E. Moore Fund. vii TABLE OF CONTENTS Biographical Sketch ....................................................................................................................... iii Dedication ...................................................................................................................................... iv Acknowledgments............................................................................................................................v Table of Contents ......................................................................................................................... viii List of Figures ................................................................................................................................ xi List of Tables ............................................................................................................................... xiii Chapter 1 – Hybridization and polyploidy in the evolution of Calendula L. (Compositae): evidence from the chloroplast and ITS ........................................................................................1