A Molecular Phylogeny of the Lythraceae and Inference of the Evolution of Heterostyly
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A MOLECULAR PHYLOGENY OF THE LYTHRACEAE AND INFERENCE OF THE EVOLUTION OF HETEROSTYLY A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Julie A. Morris August 2007 Dissertation written by Julie A. Morris B.S., Mesa State College, 1995 M.S., Kent State University, 2002 Ph.D., Kent State University, 2007 Approved by __________________________________, Chair, Doctoral Dissertation Committee Andrea L. Case __________________________________, Members, Doctoral Dissertation Committee Andrea E. Schwarzbach __________________________________ W. Randolf Hoeh __________________________________ L. Gwenn Volkert __________________________________ Alison J. Smith Accepted by __________________________________, Chair, Department of Biological Sciences James L. Blank __________________________________, Dean, College of Arts and Sciences John R. D. Stalvey ii TABLE OF CONTENTS LIST OF FIGURES ……………………………………………………………………v LIST OF TABLES ……………………………………………………………………vi ACKNOWLEDGMENTS …………………………………………………………...vii CHAPTER I. A Molecular Phylogeny of the Lythraceae (Myrtales) Based on Combined Analysis of Five Chloroplast Regions and ITS Abstract……………………………………………………………1 Introduction………………………………………………………..3 Methods……………………………………………………………9 Taxon Sampling and DNA Sequencing…………………...9 Data Analysis…………………………………………….15 Results…..………………………………………………………..17 Individual Data Sets……………………………………...17 Combined Data Sets……………………………………...21 Discussion………………………………………………………..29 Data Sets and Analysis………………………………...…29 Relationships Among Taxa………………………………30 Literature Cited…………………………………………………..36 II. A Molecular Phylogeny of Lythrum (Lythraceae): Preliminary Analyses Based on the atpB-rbcL Intergenic Spacer and ITS Abstract…………………………………………………………..39 Introduction………………………………………………………41 Methods…………………………………………………………..48 Taxon Sampling and DNA Sequencing………………….48 Data Analysis…………………………………………….52 Results……………………………………………………………53 Discussion………………………………………………………..60 Peplis…………………………………………………......60 iii Relationships Within Lythrum…………………………...62 North American Clade…………………………...63 L. junceum –L. hyssopifolia Clade……………….65 L. virgatum – L. salicaria Clade…………………66 Conclusions………………………………………………………67 Literature Cited…………………………………………………..69 III. Evolution of Heterostyly in the Lythraceae (Myrtales): Approaches to Ancestral Character State Reconstruction Using a New Molecular Phylogeny Abstract.………………………………………………………….72 Introduction………………………………………………………74 Methods…………………………………………………………..80 Taxon Sampling and Molecular Data……………………80 Ancestral Character State Reconstruction……………….83 Results……………………………………………………………86 Discussion………………………………………………………..96 Limitations of Current Models…………………………...96 Repeated Loss of Heterostyly in Lythraceae………….…99 Literature Cited…………………………………………………104 iv LIST OF FIGURES 1-1. Distribution of the Lythraceae…………………………………………………….5 1-2. Results of Analyses of Individual Data Sets……………………………………..19 1-3. Results of Combined Analysis of Data Sets with No Missing Data……………..23 1-4. Results of Combined Analysis of All Available Data…………………………...25 1-5. Selected Morphological Characters Mapped onto the Phylogeny……………….34 2-1. Botanical Illustration of Lythrum salicaria………………………………………44 2-2. Results of Combined Analysis…………………………………………………...55 2-3. Results of Combined Analysis with Branch Lengths …………………………...59 3-1. Schematic representation of Heterostyly………………………………………...76 3-2. Maximum Likelihood Character Reconstruction………………………………..88 3-3. Maximum Parsimony Optimization; Equally Weighted…………………………91 3-4. Maximum Parsimony Optimization; Weighting Gains to Losses 2:1…………...93 3-5. Maximum Parsimony Optimization; Weighting Gains to Losses 3.14:1………..95 v LIST OF TABLES 1-1. Sample List with Voucher Information and GenBank Accession Numbers…….10 1-2. PCR and Sequencing Primers……………………………………………………14 1-3. Comparative Statistics for Maximum Parsimony Analyses……………………..20 1-4. Summary Statistics for Pair-wise Topology Tests……………………………….26 2-1. Current Species List for Lythrum………………………………………………..46 2-2. Sample List with Voucher Information and GenBank Accession Numbers…….49 2-3. PCR and Sequencing Primers……………………………………………………51 2-4. Comparative Statistics for Maximum Parsimony Analyses……………………..56 3-1. Sample List and Character Coding Information…………………………………81 vi ACKNOWLEDGEMENTS During my work on this dissertation, a substantial number of people have helped me in a great number of ways. In fact, properly acknowledging all of them would likely take almost as much time as the dissertation itself. However (as anyone who knows me will not be surprised to hear), I am preparing these comments with very (very) little time remaining prior to the submission deadline, and so am forced to be brief☺. I am extremely grateful to Shirley Graham, Andrea Schwarzbach, and Andrea Case, without whom this work would not have been possible. Most students would consider themselves fortunate to have had one great advisor during the course of their graduate career, and I was privileged enough to have three!! I should also acknowledge Barbara Andreas and Oscar Rocha who regularly and willingly provided advice and support. All of these people contributed a great deal to my professional development at Kent State University, and I am honored to count them as mentors and friends. Many thanks are also due: to Eric C. for his gifted (and patient) technical assistance in all of my analyses; to Holger and the Vals for making our lab a fun place to work; to Andrea, Diana, Esther, Rajlakshmi, Maria, Jenn C., and Eric F. (and his magic coffee) for holding me together during “the final days”; to Diana, Esther, and Raj for providing defense refreshments that have already become legendary; to Chris, Sharon, Pat, Robin, Gail, and Linda -without whom the biology department would not function; to Chris R. for keeping the green things growing; to Joe, Pam, Min, Ken, Michelle, John, vii Darrel, and Dale for keeping the “good old days” alive; to Jenn C., Justin M., Joel, Eric F., Justin R., Julie P., Steph, Dan, Steve, Doug, and my entire PCC crew for reminding me how to stay young; to Oscar and all of the Costa Rica Crew for an experience I will never forget; to Diana and Brian for being there from start to finish; to Holger, Ana, Jen, Esther, Philip, Laura, Raj, Amiya, Oscar, Maria, Andrea, Pat, and Mason for great friendships, great conversations, and a lot of great food!!; to DW and Jeannie (among “what I will miss most about Kent”) for their friendship and support (including, but not limited to: dog-walking, orchid-watering, door-opening, car-starting, movie-going, and postcard-sending); to Al for all he has done and continues to do, and to Tanya, Porter, Polly, Mark and Evan for all of the laughs and all of the hugs. I have been extraordinarily blessed with the friendships I have made during my time at Kent State, and I will never forget the lab meetings, field days, journal clubs, floral hikes, MEEC meetings, Thanksgiving meals, Darwin parties, kitty rescues, beer fests, Spanish lunches, knitting nerds, Blossom nights, mental breakdowns, afternoon tea times, Ska concerts, pot-lucks, Bronco games, backyard barbecues, bird-watching trips, Wednesday dinners, walks in the woods, AND countless “crawls” down Franklin Ave. Thank you all!!!!!!!! Finally, special thanks are due to Towner’s, Beetle, Rana, and the Boys -the source of my sanity; and to my family –the source of all of my good qualities: to my Dad from whom I gained focus and strength; to my Mom from whom I gained patience and curiosity; and to my brothers from whom I gained my sense of humor. There is nothing that I have accomplished, or will achieve, that would be possible without them. viii CHAPTER 1 A Molecular Phylogeny of Lythraceae (Myrtales) Based on Combined Analysis of Five Chloroplast Regions and ITS Abstract The Lythraceae (Myrtales) comprises approximately 32 genera and 600 species with a worldwide distribution. Members of the family show extreme variation in habit, ranging from tall trees and woody shrubs to small aquatic herbs. This vegetative variation, combined with a fairly generalized floral morphology, has complicated inferences of relationships among these genera based on traditional morphological characters. Previous molecular analyses have been unable to distinguish among various hypotheses of basal relationships within the family, making it difficult to correctly polarize characters for the study of morphological evolution. In this study, molecular data from two newly sequenced chloroplast regions (atpB-rbcL intergenic spacer and the trnK-matK region) are added to previous sequence data (rbcL, trnL-trnF, psaA-ycf3, and ITS) and analyzed using maximum parsimony, maximum likelihood, and Bayesian approaches. Topologies resulting from these analyses are congruent and generally well resolved. The relationships of terminal clades are more stable than in previous analyses, however, statistical support for basal relationships remains relatively low, possibly suggesting an early rapid radiation among these taxa. It is clear that multiple 1 2 convergences in morphological characters and life history traits have occurred during the evolution of this family. 3 Introduction As currently defined, the Lythraceae includes approximately 32 genera and 600 species (Graham et al. 2005). The family has been traditionally classified in the order Myrtales and closely allied with the Onagraceae based on morphological, anatomical, and embryological evidence