ABSTRACT MODLISZEWSKI, JENNIFER LOUISE. The formation and maintenance of a hybrid zone of Aesculus L. (Sapindaceae) in the southeastern United States. (Under the direction of Qiu- Yun (Jenny) Xiang) Hybrid zones have long been touted by plant evolutionary biologists as an unrivaled phenomenon through which the mechanisms of evolution may be studied as they are occurring. Speciation, introgression, and adaptation may all occur as a result of their formation and thus their study may provide new insight into how these processes occur. The purpose of this study is to determine the manner in which a broad hybrid zone of Aesculus was formed, through the use of chloroplast DNA analysis. This hybrid zone encompasses parts of central and northern Georgia and includes hybrids among three species of Aesculus sect. Pavia: Aesculus flava, A. pavia, and A. sylvatica. These species currently have distinct geographic distributions, with both A. pavia and A. flava absent from the hybrid zone. Previous hypotheses have purported that the Aesculus hybrid zone was formed through either 1) secondary contact of previously isolated species or 2) recurrent long-distance pollen dispersal via the ruby-throated hummingbird, Archilochus colubris. It is also possible that the zone may have originated through a combination of both of these forces. This study consists of two parts, the first involving the verification of maternal inheritance of chloroplasts in Aesculus, and the second involving the assessment of the hypothesis of historical secondary contact, based on patterns of cpDNA variation in hybrid and parental populations. Verification of the inheritance of chloroplasts was accomplished through the sequencing of the matK gene from the parents and progeny of 17 crosses among various Aesculus species. The relative contribution of historical secondary contact to the formation of the hybrid zone was accomplished through PCR-RFLP analysis of three loci in the chloroplast genome: matK, trnD-trnT, and trnH-trnK, from 29 natural populations of Aesculus located within, adjacent to, and broadly separate from the hybrid zone. Haplotypes identified from RFLP analysis of the three loci were sequenced and subjected to phylogenetic and population genetic analyses. Results from the sequencing of the matK gene from controlled crosses verified that chloroplasts are inherited maternally in Aesculus, as in most angiosperms. Twenty-one unique haplotypes were identified via analysis of RFLPs, indicating that the chloroplast genome of Aesculus is highly polymorphic. Phylogenetic and population genetic analyses of sequence and restriction site data revealed that cpDNA haplotypes do not correlate with either spatial or taxonomic boundaries. Haplotypes of A. pavia, a species that is presently physically absent from the hybrid zone, were detected in hybrid populations. Additionally, most populations were fixed for a single haplotype that was unique to that population. These results suggest that while secondary contact has played a historical role in the formation of the hybrid zone, current gene flow via seeds is highly restricted among populations. Furthermore, multiple types of cpDNA, originating from the last common ancestor of the group, are maintained in hybridizing species, suggesting that the common ancestor of species of sect. Pavia was polymorphic. Comparison of these results to those of previous analysis of allozyme markers suggests that intermittent long-distance pollen dispersal has helped to maintain the hybrid zone while localized gene flow due to secondary contact of divergent species was responsible for the initial formation of the hybrid zone. Future studies of the Aesculus hybrid zone might focus on the fitness of hybrid individuals as a determining factor in the continued maintenance of the hybrid zone, as well as genotype-by-environment interactions that may be influencing the physical shape and geographic location of the Aesculus hybrid zone. This study could lead to the development of a new hybrid zone model, which would be particularly well-suited for plants capable of long-distance dispersal. THE FORMATION AND MAINTENANCE OF A HYBRID ZONE OF AESCULUS L. (SAPINDACEAE) IN THE SOUTHEASTERN UNITED STATES by Jennifer Louise Modliszewski A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science BOTANY Raleigh 2005 Approved by: _____________________ _____________________ Dr. Philip Awadalla Dr. Thomas R. Wentworth _____________________ _____________________ Dr. Michael D. Purugganan Dr. Qiu-Yun (Jenny) Xiang Co-Chair of the Advisory Committee Co-Chair of the Advisory Committee BIOGRAPHY Jennifer Louise Modliszewski was born in Manassas, VA on 5 March 1980 to Darlene and Louis Modliszewski. Jennifer graduated from Liberty High School in Bealeton, Virginia in June of 1998 as the valedictorian of her senior class. She received a Park Scholarship to attend North Carolina State University, where she went on to receive a Bachelor of Science in both botany and horticultural science. As an undergraduate, Jennifer worked as an undergraduate research assistant in the molecular evolutionary genetics laboratory of Michael Purugganan. Additionally, during the summer of 2001, she worked as an intern at the United States National Arboretum in Washington, DC, where she studied the genetic diversity of a rare plant, Gaylussacia brachycera. In the fall of 2002, Jennifer enrolled in the Department of Botany at North Carolina State University to pursue a master of science, with interests in population genetics and evolutionary ecology. Jennifer is married, and met her husband, Neil Jacobs, while an undergraduate at North Carolina State University. After graduation, she plans to continue her education in the field of population genetics and evolutionary ecology. ii ACKNOWLEDGMENTS I would like to begin by expressing my deepest gratitude to all of my committee members for their guidance, support and understanding. In particular, I would like to thank Dr. Qiu-Yun (Jenny) Xiang, my major advisor, for her understanding and patience with me, and for her attempts to create a familial atmosphere within the lab. I would like to thank Dr. Michael Purugganan for not seeming to mind that every time that I came to his office to talk to him about something, I ended up crying, although not because of anything that he had to say. While here, he has been a major source of inspiration, motivation, and worldly wisdom for me. And of course, the everyday enthusiasm and cheer of Dr. Tom Wentworth made my course of study here a much brighter one. To Dr. Awadalla, I can only say that I wish you would have been ‘on the boat’ from the beginning. I also thank Dr. Becky Boston for making my decision regarding my course of study here at NCSU an easier one. I would also like to extend special thanks and acknowledgments to collaborators that worked with me on the cpDNA analysis of the hybrid zone: Claude dePamphilis, Daniel Crawford, David Thomas, and Chuanzhu Fan. In particular, Claude contributed many insightful comments and helpful additions to the manuscript. In addition, David Thomas and Dr. Tom Wentworth were, thankfully, extremely meticulous in their editing of the manuscript. Charlotte Chan of the Holden Arboretum and Susan Wiegrefe of the Morton Arboretum provided materials for cpDNA inheritance analysis, and thus deserve many thanks. Additionally, Todd Lasseigne and the J. C. Raulston Arboretum allowed me to perform the initial crossing experiments. The research presented here was supported by a iii Faculty Research grant from Idaho State University and a Faculty Research and Development grant from NCSU, both awarded to QYJX. My graduate experience at NCSU would have been a much more stressful one, were it not for the help of Sue Vitello. Without her help, I might have been giving my thesis seminar on the brickyard. I would also like to thank my fellow lab-mates, Chuanzhu Fan, Wenheng Zhang, and Melinda Peters, for offering advice and help, both scientific and otherwise. Even though many of our shared experiences here have mostly been of the stressful sort, I felt fortunate to have true peers to look to for advice and support. Additionally, I recognize the laboratory trouble-shooting abilities of David Thomas, which have made my time in laboratory well spent. My parents, Louis and Darlene, deserve much acknowledgment and kudos for the careful upbringing of all of their children, and for their (successful) attempts to instill in us the highest of moral values and appreciation for that which we have. I will carry these values with me, in both scientific endeavors and life in general. I thank my father for his demands for perfection and attention to detail, or in other words, his embodiment of the philosophy that if “…you are going to do a shoddy job of something there is no point in doing it at all…”. I would also like to thank my mother for her unending willingness to listen, even when I didn’t want to talk. Finally, I would like to thank my husband, Neil, for always believing in me and for never failing to tell me so whenever I needed to hear it. Unfortunately for him, at times this was quite often. iv CONTENTS List of Tables . vii List of Figures . viii List of Abbreviations . ix Chapter 1 – Introduction — Hybridization and the Aesculus Hybrid Zone . 1 Background . 1 What is a hybrid? What is a hybrid zone? . 1 Significance of the study of hybrid zones . 1 How are hybrid zones formed? How are hybrid zones maintained? — Hybrid zone models . 3 Aesculus L. (Sapindaceae) . 4 Biogeography of Aesculus . 4 North American Aesculus . 4 Floral morphology of Aesculus . 5 General description of the hybrid zone . 6 Hybrid forms . 7 Studies Presented in Subsequent Chapters . 8 Chloroplast DNA inheritance analysis . 8 Analysis of cpDNA restriction site haplotypes . 9 Chapter 2 – Ancestral Chloroplast Polymorphism and Historical Secondary Contact in a Broad Hybrid Zone of Aesculus (Sapindaceae) . 11 Abstract . 11 Introduction . 13 Materials and Methods .