PHYLOGENY RECONSTRUCTION AND CONSERVATION GENETICS OF Conradina AND RELATED SOUTHEASTERN U.S. ENDEMIC MINTS (LAMIACEAE) By CHRISTINE E. EDWARDS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1 © 2007 Christine E. Edwards 2 ACKNOWLEDGMENTS I thank my advisors, Pamela Soltis and Douglas Soltis, and my committee members, David Reed and Walter Judd, for support and guidance throughout my PhD program. I also thank former committee members Rebecca Kimball and Doria Gordon; current and former Soltis lab members, including Chuck Bell, Luiz Oliviera, Matt Gitzendanner, Andrew Doust, Josh Clayton, Michael Moore, Ashley Morris, Monica Arakaki, Sam Brockington and Vaughn Symonds for their interesting discussions, help with experimental design, lab procedures, and analytical methods; Kent Perkins, Norris Williams, and the FLAS herbarium staff and students for assistance with collections and loans; Phil Cantino, Jay Walker, Kurt Neubig, Richard Abbott and Luiz Oliviera for plant material; David Lefkowitz, Amber Pouncey, Zera Damji, Suneel Modani, and Aisha Goodman for assistance with lab work; Alan Prather and Rachel Williams for access to unpublished data; and Claude Bailey, Brian Wender, Eric Tillman, Gretchen Ionta, Sam Brockington, Chuck Bell, Jason Ulev, Lloyd and Mary Edwards, Ann Cox, David Lefkowitz, and Pam and Doug Soltis for assistance with field work. I thank my family, friends, and lab members for moral support and encouragement throughout my dissertation. Funding for this project was provided by the Florida Native Plant Society, Florida Division of Forestry’s Florida Statewide Endangered and Threatened Plant Conservation Program, the Garden Club of America’s Catherine Beattie Fellowship, Sigma XI Grants-in-Aid of Research, a Graduate Student Research Award from the American Society of Plant Taxonomists, and a Botanical Society of America Genetics Section Award. 3 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................3 LIST OF TABLES...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT...................................................................................................................................10 CHAPTER 1 GENERAL INTRODUCTION..............................................................................................12 2 MOLECULAR PHYLOGENY OF CONRADINA AND OTHER SCRUB MINTS (LAMIACEAE) FROM THE SOUTHEASTERN USA: EVIDENCE FOR HYBRIDIZATION IN PLEISTOCENE REFUGIA?............................................................15 Introduction.............................................................................................................................15 Materials and Methods ...........................................................................................................20 Sampling and Outgroups .................................................................................................20 DNA Extraction, PCR Amplification, and Sequencing ..................................................20 DNA Sequence Alignment and Phylogenetic Analysis ..................................................22 Congruence Tests and Hypothesis Testing......................................................................24 Analysis of Spatial Genetic Structure..............................................................................25 Results.....................................................................................................................................26 Monophyly of the SE Scrub Mint Clade .........................................................................26 ITS Sequence Characteristics and Phylogeny Reconstruction........................................29 Congruence Tests ............................................................................................................30 Analyses of the Relationship between Geographic and Genetic Distance......................31 Discussion...............................................................................................................................32 The Monophyly of Conradina and Causes of Discordance between ITS and Plastid Sequence Data..............................................................................................................32 Other Generic-Level Relationships Regarding the SE Scrub Mint Clade ......................35 Relationships within Conradina......................................................................................37 Taxonomic Questions within Conradina: Status of C. brevifolia and the Santa Rosa Populations...................................................................................................................38 3 PHYLOGENY OF CONRADINA AND RELATED SOUTHEASTERN SCRUB MINTS (LAMIACEAE) BASED ON GAPC GENE SEQUENCES.....................................50 Introduction.............................................................................................................................50 Materials and Methods ...........................................................................................................53 Initial Isolation of GapC in the SE Scrub Mint Clade.....................................................53 Determination of GapC Copy Number ...........................................................................54 4 Taxon Sampling for Phylogeny Reconstruction Using GapC-1 and GapC-2 ................57 Analyses of Recombination.............................................................................................58 Separate Analyses of GapC-1 and GapC-2, Congruence Analyses, and Combined Phylogenetic Analyses.................................................................................................59 Results.....................................................................................................................................62 GapC Copy Number in New World Mentheae ...............................................................62 Analyses of Recombination.............................................................................................63 Phylogenetic Analysis of GapC-1 and GapC-2 ..............................................................63 Congruence Analyses ......................................................................................................65 Combined Phylogenetic Analyses...................................................................................66 Removal of Single Partitions...........................................................................................67 Discussion...............................................................................................................................68 Copy Number and Evolution of GapC in New World Mentheae ...................................68 Separate Phylogenetic Analysis of GapC-1 and GapC-2................................................69 Combined Analyses—Congruence and the Effect of Concatenation Approach.............70 Combined Phylogenetic Analyses—Relationships in the SE Scrub Mint Clade............73 Combined Phylogenetic Analyses–Relationships among Conradina Species................75 4 COMBINED PHYLOGENETIC ANALYSIS OF HETEROZYGOUS NUCLEAR LOCI: AN EXAMPLE USING CONRADINA AND RELATED MINTS (LAMIACEAE) ......................................................................................................................84 Introduction.............................................................................................................................84 Materials and Methods ...........................................................................................................86 Characteristics of Data Matrices .....................................................................................86 Methods of Concatenating Multiple Heterozygous Nuclear Genes................................87 Methods of Phylogenetic Analysis..................................................................................92 Results.....................................................................................................................................93 Discussion...............................................................................................................................96 5 ISOLATION, CHARACTERIZATION, AND CROSS-SPECIES AMPLIFICATIONS OF MICROSATELLITE LOCI FROM CONRADINA (LAMIACEAE).............................108 Introduction...........................................................................................................................108 Materials and Methods, Results, and Conclusions ...............................................................108 6 PATTERNS OF GENETIC STRUCTURE BASED ON MICROSATELLITE LOCI REVEAL SPECIES COHESION AND SHARED ANCESTRAL POLYMORPHISM RATHER THAN HYBRIDIZATION IN CONRADINA.....................................................113 Introduction...........................................................................................................................113 Materials and Methods .........................................................................................................117