MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Aaron H. Kennedy Candidate for the Degree: Doctor of Philosophy ______________________________ Director (Dr. Linda E. Watson) ______________________________ Reader (Dr. Nicholas P. Money) ______________________________ Reader (Dr. R. James Hickey) ______________________________ Reader (Dr. D. Lee Taylor) ______________________________ Graduate School Representative (Dr. David J. Berg) ABSTRACT PHYLOGENY AND EVOLUTION OF MYCORRHIZAL ASSOCIATIONS IN THE MYCO-HETEROTROPHIC HEXALECTRIS RAF. (ORCHIDACEAE : EPIDENDROIDEAE) by: Aaron H. Kennedy Some plant species have abandoned an autotrophic life style and obtain their carbon and mineral nutrition exclusively from mycorrhizal fungi. Although myco-heterotrophic species have evolved in many plant families, they are most common in the Orchidaceae. Several myco- heterotrophic orchid species have been shown to associate with a very narrow range of ectomycorrhizal forming fungi, revealing a high degree of mycorrhizal specificity. However, these studies have often investigated single or few, often unrelated, species without support for their monophyly or knowledge of their phylogenetic relationships. Using primarily molecular methods and phylogenetic analyses, this dissertation investigates i) the monophyly and circumscription of Hexalectris species, ii) interspecific phylogenetic relationships within Hexalectris, iii) the identities of the mycorrhizal fungi that associate with each Hexalectris species, iv) the breadth of mycorrhizal associations within Hexalectris and within each of its species, and v) uses a Hexalectris phylogeny as a framework for investigating mycorrhizal specificity and patterns of associations. The monophyly of H. warnockii, H. grandiflora, H. brevicaulis, and H. nitida, plus the H. spicata species complex, are well supported. The remaining species are not monophyletic, prompting the recircumscription of H. spicata s.l. as H. spicata and H. arizonica, H. revoluta s.l. as H. revoluta and H. colemanii, and H. fallax as a synonym of H. parviflora. Extreme specificity with ectomycorrhizal agaricomycete fungi was identified in each Hexalectris species. Hexalectris warnockii associates exclusively with members of the Thelephoraceae; H. brevicaulis and H. grandiflora associate exclusively with members of Russulaceae and Sebacinaceae subgroup A; the remaining species, all members of the H. spicata complex, associate strictly with members of Sebacinaceae subgroup A. Optimizing these associations onto a Hexalectris phylogeny reveals that, with one exception, each Hexalectris species associates with a different clade or group of fungal clades, and that shifts in association from one group of fungi to another occurred during each speciation event in the evolutionary history of this genus. PHYLOGENY AND EVOLUTION OF MYCORRHIZAL ASSOCIATIONS IN THE MYCO-HETEROTROPHIC HEXALECTRIS RAF. (ORCHIDACEAE : EPIDENDROIDEAE) A Dissertation Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Botany By Aaron H. Kennedy Miami University Oxford, Ohio 2009 Dissertation Director: Dr. Linda E. Watson © Aaron H. Kennedy 2009 Contents Chapter 1. Introduction……………………………………………………1 Chapter 2. Species delimitations and phylogenetic relationships in the myco-heterotrophic Hexalectris Raf. (Orchidaceae : Epidendroideae)…………….........…………………………...10 Chapter 3. Mycorrhizal specificity and patterns of host associations in the myco-heterotrophic Hexalectris (Orchidaceae : Epidendroideae)……………………........................................48 Chapter 4. Discussion: Summary and Future Directions….......................82 iii List of Tables Chapter 2 Table 1: Primers used for PCR amplification and sequencing of DNA regions included in phylogenetic analyses………….15 Table 2: Summary of individual and combined matrix partition characteristics...........................................................................20 Table 3: Summary of ITS cloning results...............................................26 Table 4: Summary of nucleotide positions suggesting additive inheritance of ITS sequences in H. revoluta var. colemanii from H. fallax and H. spicata var. arizonica…………………26 Chapter 3 Table 1: Collection location and voucher information for each Hexalectris accession included in this study………….......54-55 Table 2: A list of fungal taxa that were identified from within the roots of each Hexalectris species based on MegaBlast searches of GenBank................................................................60 Table 3: Ascomycete fungi that were identified from within the roots of Hexalectris species based on MegaBlast searches of GenBank…………………………………………62 Table 4: Summary of relative mycorrhizal specificity and phylogenetic breadth of association within the Sebacinaceae for each Hexalectris species…………………...63 iv List of Figures Chapter 2 Figure 1: Geographic distributions of Hexalectris species……….....12-13 Figure 2: Scatter plots of model corrected- versus uncorrected genetic distance of all pairwise comparisons involving Hexalectris accessions in the combined plastid matrix for accD (A) and psbA (B)……………………………21 Figure 3: Single most parsimonious phylogram and 50% majority rule Bayesian Inference tree resulting from analyses of ITS............................................................................................22 Figure 4: Strict consensus of eight most parsimonious trees and 50% majority rule Bayesian Inference tree resulting from analyses of the combined plastid matrix including psbA…......23 Figure 5: Single most parsimonious phylogram and 50% majority rule Bayesian Inference tree resulting from analyses of the combined plastid matrix excluding psbA……………………..24 Chapter 3 Figure 1: 50% majority rule Bayesian Inference tree resulting from the analysis of Sebacinaceae.....................................................61 Figure 2: 50% majority rule Bayesian Inference tree resulting from the analysis of Ceratobasidiaceae.............................................64 Figure 3: 50% majority rule Bayesian Inference tree resulting from the analysis of Russulaceae......................................................66 Figure 4: 50% majority rule Bayesian Inference tree resulting from the analysis of Thelephoraceae.................................................68 Figure 5: Phylogeny of Hexalectris with mycorrhizal associations optimized as character states……...………………………….69 v Acknowledgments I am particularly thankful to my wife Kellie Kennedy who has provided me with a great deal of support and encouragement through the PhD process. Much of Kellie’s support has involved shouldering a large share of our personal, household, and parental responsibilities. Remarkably, she has confronted these happily and with amazing grace. For this, I am sincerely grateful and forever in her debt. I also thank our daughter Ava who, although is only 22 months old and may not currently understand the contributions that she has made to her family’s success and happiness, has, if not only through her being, immeasurably improved the lives of her parents and provided meaning to their work. I also thank my mother and father who have never wavered in their emotional, philosophical, and even financial, support of my search for happiness. Also, thank you to my grandfather, J. Edward Hill, for his sincere interest and pride in my accomplishments. I thank my dissertation committee, Drs. Linda Watson, Jim Hickey, Lee Taylor, Nicholas Money, and David Berg, who have supplied me with insight, support, and necessary criticism during my studies, research, and writing. I am particularly thankful to my major advisor. Dr. Watson has very thoughtfully mentored me throughout the PhD process, carefully and accurately balancing her intervention and my independence. Also, special thanks to Dr. Taylor for his willingness to serve as a committee member despite his distance as a professor at the University of Alaska, Fairbanks, and for sharing his valuable knowledge regarding orchid mycorrhizal associations. I thank the American Orchid Society, Miami University Department of Botany, Willard Sherman Turrell Herbarium (MU), and the Ohio Garden Club for financial support of this dissertation research. Thank you to the following for access to land, and collection permission: Big Bend and Guadalupe Mountains National Parks, Briarwood - Caroline Dormon Nature Center (Natchitoches Parish, LA), Cedar Ridge Preserve (Audubon Society; Dallas, TX), Coronado National Forest, Indiana and Ohio Departments of Natural Resources, North Carolina Department of Environment and Natural Resources, The Nature Conservancy (TNC) of Ohio and The Edge of Appalachia Preserve, TNC of Texas and Davis Mountains Preserve, and University of West Alabama. I am grateful to many for their assistance with collecting Hexalectris species, but give special thanks to Pablo Carrillo-Reyes and Aaron Rodríguez, who not only very generously spent multiple weeks with me in the field in Mexico, but also took me into their homes and introduced me to their beautiful country. I am also thankful to Paul Martin Brown, Marcy Brown-Marsden, Ronald Coleman, Frank Farruggia, Rick Gardner, Roberto González-Tamayo, John Karges, Allison Leavitt, Larry Magrath, Al Schotz, Joe Sirotnak, Victoria Sosa, Bonnie Stolp, and Michael Vincent for assistance with collections and permits. Thank you to Neil Winn