OUT OF THE BUSHES AND INTO THE TREES: ALTERNATIVE APPROACHES FOR RESOLVING THE PHYLOGENY OF LAMIACEAE By GRANT THOMAS GODDEN 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 2014 © 2014 Grant Thomas Godden To my father, Clesson Dale Godden Jr., who would have been proud to see me complete this journey, and to Mr. Tea and Skippyjon Jones, who sat patiently by my side and offered friendship along the way ACKNOWLEDGMENTS I would like to express my deepest gratitude for the consistent support of my advisor, Dr. Pamela Soltis, whose generous allocation of time, innovative advice, encouragement, and mentorship positively shaped my research and professional development. I also offer my thanks to Dr. J. Gordon Burleigh, Dr. Bryan Drew, Dr. Ingrid Jordon-Thaden, Dr. Stephen Smith, and the members of my committee—Dr. Nicoletta Cellinese, Dr. Walter Judd, Dr. Matias Kirst, and Dr. Douglas Soltis—for their helpful advice, guidance, and research support. I also acknowledge the many individuals who helped make possible my field research activities in the United States and abroad. I wish to extend a special thank you to Dr. Angelica Cibrian Jaramillo, who kindly hosted me in her laboratory at the National Laboratory of Genomics for Biodiversity (Langebio) and helped me acquire collecting permits and resources in Mexico. Additional thanks belong to Francisco Mancilla Barboza, Gerardo Balandran, and Praxaedis (Adan) Sinaca for their field assistance in Northeastern Mexico; my collecting trip was a great success thanks to your resourcefulness and on-site support. Thank you to Patricia Manning, Dr. Michael Powell, Dr. Alan Prather, Dr. Donovan Bailey, Dr. Timothy Lowrey, Robert Savinski, Dr. Leslie Landrum, Dr. Tina Ayers, the US Forest Service, and the US Bureau of Land Management for their assistance with field research activities in the Southwestern United States. Lastly, I wish to thank Dr. Selene Baez, Dr. Omar Torres-Carvajal, Dr. C. Lorena Endara, Dr. Claudia Segovia Salcedo, and Rhonda Arthur for their support of my field research activities in Ecuador. Lastly, thank you to Dr. Matthew Gitzendanner, Dr. Michael Moore, and Dr. David Tank for their guidance and training in the areas of library preparation, next-generation 4 sequencing, and data processing. To Drs. Steven Manchester and Evgeny Mavrodiev: thank you for your assistance sourcing and translating fossil descriptions, respectively. And lastly, thank you to my friends in Gainesville and colleagues in the Soltis Lab, Florida Museum of Natural History, and University of Florida, particularly Jared Cellon, Kim Schleissing, Claudia Segovia Salcedo, Dr. C. Lorena Endara, Dr. Igor Ignatovich, Dr. Gretchen Ionta, Dr. Ingrid Jordon-Thaden, and Dr. Maribeth Latvis. Your friendship and support throughout this journey were vital to my success. Financial support for this research was made possible by a grant from the National Science Foundation (DEB-1210671), with additional funding from the University of Florida Graduate Student Research Abroad Program, The Herb Society of America, The Community Foundation Desert Legacy Fund, the California Native Plant Society, and the Botanical Society of America. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 13 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 15 2 INTERPRETING COMPLEX PATTERNS IN LAMIACEAE PHYLOGENY ............. 19 Introduction ............................................................................................................. 19 Current Status of Lamiaceae Taxonomy and Phylogeny ................................. 19 Interpreting Complex Patterns in Lamiaceae Phylogeny .................................. 22 Materials and Methods............................................................................................ 22 Supermatrix Phylogenetic Inference ................................................................. 22 Phylogenetic Inference ..................................................................................... 23 Assessment of Phylogenetic Sampling Effort ................................................... 24 Divergence Time Estimation ............................................................................. 24 Diversification Dynamics in Lamiaceae ............................................................ 26 Results .................................................................................................................... 27 Descriptive Data ............................................................................................... 27 Phylogenetic Inference ..................................................................................... 29 Divergence Times ............................................................................................ 30 Diversification Dynamics .................................................................................. 30 Discussion .............................................................................................................. 31 Phylogenetic Relationships in Lamiaceae ........................................................ 31 Evolutionary Timing and Tempo in Lamiaceae ................................................. 32 Limitations Imposed by Opportunistic Sampling ............................................... 33 Towards Fully Resolving Relationships in Lamiaceae ...................................... 35 3 MAKING NEXT-GENERATION SEQUENCING WORK FOR YOU: APPROACHES AND PRACTICAL CONSIDERATIONS FOR MARKER DEVELOPMENT AND PHYLOGENETICS ............................................................. 62 Introduction ............................................................................................................. 62 NGS and New Markers for Phylogenetic Studies ................................................... 66 Organellar Genomes and Gene Space as Markers .......................................... 66 Nuclear Markers ............................................................................................... 67 Cost Considerations for NGS Projects .................................................................... 68 Experimental Pathways for NGS Phylogenetics ..................................................... 71 6 Transcriptomes vs. Genomes ........................................................................... 72 Pathways for Phylogenetic Marker Development ............................................. 74 Marker Development Pipeline for Targeted Sequencing ........................................ 76 Data Mining ...................................................................................................... 77 Tests for Orthology ........................................................................................... 78 Primer Development for Targeted NGS ........................................................... 79 Targeted Sequencing Approaches for Phylogenetics ............................................. 82 Amplicon Approaches for Targeted Sequencing .............................................. 82 Hybridization Approaches for Targeted Sequencing ........................................ 87 Processing of NGS Data for Phylogenetic Marker Development ............................ 89 NGS Data: File Types and Conversion Tools ................................................... 89 Quality Control and Trimming of NGS Data ...................................................... 90 Genomic and Transcriptomic Assembly ........................................................... 92 Data Mining for Orthologous Markers ............................................................... 94 Case Study: 1KP Pipeline ................................................................................ 96 Discussion .............................................................................................................. 97 4 RESOLVING RELATIONSHIPS WITH ORGANELLAR PHYLOGENOMIC DATA: A CASE STUDY WITH HEDEOMA AND ALLIED GENERA FROM MENTHINAE (LAMIACEAE) ................................................................................. 114 Introduction ........................................................................................................... 114 Phylogenetic Relationships within Hedeoma and Among Closely Allied Genera of Menthinae .................................................................................. 115 A Model System for Evaluating Organellar Phylogenomic Approaches ......... 117 Materials and Methods.......................................................................................... 118 Plant Materials and Taxonomic Sampling ...................................................... 118 DNA Extraction, Shearing, and Genomic Library Preparation ........................ 119 Plastid Genome Enrichment and Sequencing ................................................ 121 Data Processing, Mining, and Assembly .......................................................