University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2003 COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES Kelly D. Craven University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Craven, Kelly D., "COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES" (2003). University of Kentucky Doctoral Dissertations. 431. https://uknowledge.uky.edu/gradschool_diss/431 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Kelly D. Craven The Graduate School University of Kentucky 2003 COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES ________________________________ ABSTRACT OF DISSERTATION ________________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Kelly Doyle Craven Lexington, Kentucky Director: Dr. C.L. Schardl, Professor of Plant Pathology Lexington, Kentucky 2003 Copyright © Kelly Doyle Craven 2003 ABSTRACT OF DISSERTATION COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES Symbioses between cool-season grasses (Subfamily Pooideae) and endophytic fungi in the genera Epichloë and Neotyphodium straddle a continuum of interactions from antagonistic to highly mutualistic. Although these two genera of endophytes are closely related, Neotyphodium endophytes are strictly seed-transmitted and provide many physiological and defensive benefits to their hosts, while Epichloë spp. have an obligately sexual contagious stage wherein host inflorescences are replaced by fungal sexual structures (stromata), effectively sterilizing the plant. Between these two extremes of interactions are Epichloë spp. with a “mixed” strategy, where some grass tillers are sterilized while others develop normally and yield healthy endophyte-infected seeds. These symbioses offer a unique opportunity to dissect evolutionary mechanisms that may drive movement along this continuum. The research presented characterizes distinct hybridization processes in endophytes and grasses that result in the generation of astounding genetic diversity for the symbiosis. Interspecific hybridization via hyphal anatomosis is a common feature of Neotyphodium endophytes, and may promote mutualism by combining suites of defensive alkaloid genes and ameliorating the adverse evolutionary effects of an asexual lifestyle. My results demonstrate that several genetically distinct hybrid endophytes infect grass species in tribe Poeae. Further, I show that a highly mutualistic asexual endophyte infecting tall fescue (=Festuca arundinaceum Schreb.), Neotyphodium coenophialum, also infects two closely related and interfertile relatives of this host. My findings suggest that this seed-borne endophyte may have been introgressed into these grasses through sexual grass hybridization events. These findings highlight interspecific hybridization as a means of generating tremendous genetic variability in both endophytes and their hosts, thus magnifying the adaptive evolutionary potential of these symbioses. Further, I establish a phylogenetic framework for grasses naturally harboring Epichloë and Neotyphodium endophytes. I show that patterns of genetic divergence among grass lineages are emulated by those of their fungal symbionts. These results suggest that endophytes have co-evolved with grasses in subfamily Pooideae, and may have played a critical role in the evolutionary success and radiation of this group of grasses. KEYWORDS: Coevolution, Epichloë, Interspecific hybridization, Neotyphodium, Pooideae Kelly Doyle Craven December 16, 2003 COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES By Kelly Doyle Craven Christopher L. Schardl, Ph.D Director of Dissertation Lisa Vaillancourt, Ph.D Director of Graduate Studies December 16, 2003 Date RULES FOR USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgements. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky. DISSERTATION Kelly Doyle Craven The Graduate School University of Kentucky 2003 COEVOLUTION AND GENETIC DIVERSITY IN GRASS-ENDOPHYTE SYMBIOSES _____________________________ DISSERTATION _____________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture at the University of Kentucky By Kelly Doyle Craven Lexington, Kentucky Director: Dr. C.L. Schardl, Professor of Plant Pathology Lexington, Kentucky 2003 Copyright © Kelly Doyle Craven 2003 ACKNOWLEDGMENTS The following dissertation, while an individual work, benefited from the insights and direction of several people. I am indebted to each of my committee members for their guidance and advice. I have learned a great deal from each, and will carry their lessons with me as I pursue a rewarding career in plant pathology. From Dr. Tim Phillips I have gained a much broader knowledge of the grass family, and a profound fascination for their extraordinary biology. Dr. Lisa Vaillancourt has taught me a lot about fungi in general, and her passion and skill for teaching has opened up this previously unexplored avenue for me. Dr. Mark Farman has always provided very insightful comments on my work, and has taught me how to critically analyze my own scientific research, as well as that of others. I would especially like to thank my major advisor and mentor, Dr. Christopher L. Schardl, for providing me countless exciting opportunities to express my ideas, and for exemplifying the meaning of true scientific excellence. Every aspect of my graduate career has been molded by him, and I am a much better scientist for it. I am incredibly thankful for my wonderful wife, Erin, who runs by my side as we chase our dreams and makes me forget every negative thought I have ever had about myself. She has made my life truly complete. I am thankful for the love and support my parents, Doyle and Josephine, have given me throughout my life and for choosing me as their son. Finally, I would like to thank the faculty, staff, and graduate students for their help and friendship throughout my time in Kentucky. They are truly the best of the best, and I wish them all happiness and success. iii TABLE OF CONTENTS Acknowledgments………………………………………………………………………..iii List of Tables……………………………………………………………………………...v List of Figures……………………………………………………………………….........vi List of Files.......................................................................................................................viii Chapter One: Introduction Introduction………………………………………………………………………..1 Grass evolution...............………………………………………………………….5 Hybridization processes in the fungi........………………………………………..15 Chapter Two: Survey of endophyte-infected grasses in the tribe Poeae Introduction………………………………………………………………………26 Materials and Methods…………………………………………………………...29 Results……………………………………………………………………………32 Discussion……………………………………………….…….............................35 Chapter Three: The tall fescue endophyte, Neotyphodium coenophialum Introduction………………………………………………………………………67 Materials and Methods………………………………………………………...…71 Results……………………………………………………………………………75 Discussion…...........................…….……………………………………………..77 Chapter Four: Co-evolutionary relationships between cool-season grasses and symbiotic fungal endophytes Introduction……………………………………………………………………....99 Materials and Methods……………………………………………………….....106 Results…………………………………………………………………………..111 Discussion…...........................…….……………………………………………118 Chapter Five: Concluding remarks Fundamental importance of the grass-endophyte system..……………………..150 Appendix One..................................................................................................................157 References……………………………………………………………………………....170 Vita……………………………………………………………………………………...192 iv LIST OF TABLES Table 2.1, Endophyte infected grasses and fungal isolates................................................40 Table 2.2, Oligonucleotide primers used in chapter two...................................................41 Table 2.3, Endophyte isolates and GenBank accession numbers for tub2 and tef1 sequences...........................................................................................................................43 Table 2.4, Morphological characteristics of endophytes infecting Poeae grasses examined in this study.......................................................................................................45 Table 3.1, Endophyte isolates and host plants examined in this study..............................83 Table 3.2, Oligonucleotide primers used in chapter three.................................................84 Table 3.3, Microsatellite genotypes