DETECTING HYBRIDIZATION IN HAWAIIAN CYRTANDRA USING GENOME-WIDE DATA By JOSEPH A. KLEINKOPF A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN PLANT BIOLOGY WASHINGTON STATE UNIVERSITY School of Biological Sciences MAY 2018 © Copyright by JOSEPH A. KLEINKOPF, 2018 All Rights Reserved © Copyright by JOSEPH A. KLEINKOPF, 2018 All Rights Reserved To the Faculty of Washington State University: The members of the Committee appointed to examine thesis of JOSEPH A. KLEINKOPF find it satisfactory and recommend that it be accepted. Eric H. Roalson, Ph.D., Chair Joanna L. Kelley, Ph.D. Larry D. Hufford, Ph.D. Warren L. Wagner, Ph.D. ii ACKNOWLEDGMENTS I would like to first thank my advisor, Eric Roalson. He introduced me to Cyrtandra, a fascinating group of flowering plants with questions aligned to my interests, while encouraging me to identify and pursue my goals beyond graduate school. Secondly, I would like to express my sincerest gratitude to my committee members, Joanna Kelley, Larry Hufford, and Warren Wagner for their support, feedback, and time despite their busy schedules. Additionally, I would like to thank others who helped me along the way. Thank you to Corey Quackenbush for showing me best practices for library preparation and members and associates of the Roalson lab - Lucy Allison, Wade Roberts, Kimberly Hansen, Josh Brindley, Luciano Soares, and Nan Jiang - who helped make every day of graduate school interesting and enjoyable. I am especially grateful to Wade for his guidance and input throughout the research process. Finally, I would like to thank my friends and family, whose enduring support encouraged me to complete this study. iii DETECTING HYBRIDIZATION IN HAWAIIAN CYRTANDRA USING GENOME-WIDE DATA Abstract by Joseph A. Kleinkopf, M.S. Washington State University May 2018 Chair: Eric H. Roalson Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 62 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian lineage of Cyrtandra, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high-throughput sequencing, and modern phylogenomics tools to test 33 Hawaiian Cyrtandra samples (including 21 species, five putative hybrids, and two outgroups) for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species-tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. Based on prior studies of Cyrtandra and other Hawaiian lineages, we expected to see both a classic stepping-stone model, where species relationships group by island from oldest to youngest, and species relationships grouping by morphology. Additionally, we expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and iv species-tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Of the five putative hybrids tested, only one was inferred to incorporate genetic material from both putative parents. The genomic methods utilized here have allowed us to reconstruct better resolved hypotheses and to test for hybridization in the presence of incomplete lineage sorting. Wider sampling within this lineage will allow us to better understand species relationships and gene flow within Hawaiian Cyrtandra in future studies. v TABLE OF CONTENTS Page ACKNOWLEDGMENTS ......................................................................................................... iii ABSTRACT .............................................................................................................................. iv LIST OF APPENDICES ..........................................................................................................viii LIST OF TABLES ..................................................................................................................... ix LIST OF FIGURES .................................................................................................................... x CHAPTER ISLAND SYSTEMS AS NATURAL LABORATORIES FOR INFERENCES OF ECOLOGICAL AND EVOLUTIONARY PROCESSES................................................. 1 Introduction ............................................................................................................... 1 Formation of Island Biota .......................................................................................... 2 The Progression Rule and Adaptive Radiation ........................................................... 3 Biodiversity and Conservation of Plants..................................................................... 5 The Hawaiian Islands................................................................................................. 6 Literature Cited .......................................................................................................... 8 DETECTING HYBRIDIZATION IN HAWAIIAN CYRTANDRA USING GENOME- WIDE DATA ................................................................................................................ 17 Introduction ............................................................................................................. 17 Materials and Methods ............................................................................................. 23 Results ..................................................................................................................... 27 Discussion ............................................................................................................... 33 Conclusions ............................................................................................................. 44 Literature Cited ........................................................................................................ 46 vi SUMMARY OF RESULTS AND FUTURE DIRECTIONS ......................................... 91 Literature Cited ........................................................................................................ 94 vii APPENDICES Page Appendix 1: Sampled specimens ............................................................................................... 58 viii TABLES Page Table 1: Summary of targeted enrichment sequencing and mapped reads .................................. 60 ix FIGURES Page Figure 1: Concatenated maximum likelihood tree ...................................................................... 61 Figure 2: ASTRAL tree for all taxa ........................................................................................... 62 Figure 3: ASTRAL tree for all taxa with quartet scores ............................................................. 63 Figure 4: ASTRID tree for all taxa ............................................................................................ 64 Figure 5: ASTRAL tree for the core dataset .............................................................................. 65 Figure 6: ASTRAL tree for the core dataset with C. calpidicarpa x hawaiensis ......................... 66 Figure 7: ASTRAL tree for the core dataset with C. grayi-1 ...................................................... 67 Figure 8: ASTRAL tree for the core dataset with C. hawaiensis-3 ............................................. 68 Figure 9: ASTRAL tree for the core dataset with C. hawaiensis x grayana ................................ 69 Figure 10: ASTRAL tree for the core dataset with C. longifolia x kauaiensis ............................ 70 Figure 11: ASTRAL tree for the core dataset with C. munroi x grayi ........................................ 71 Figure 12: ASTRAL tree for the core dataset with C. sandwicensis x grandiflora ...................... 72 Figure 13: ASTRID tree for the core dataset.............................................................................. 73 Figure 14: ASTRID tree for the core dataset with C. calpidicarpa x hawaiensis ........................ 74 Figure 15: ASTRID tree for the core dataset with C. grayi-1 ..................................................... 75 Figure 16: ASTRID tree for the core dataset with C. hawaiensis-3 ............................................ 76 Figure 17: ASTRID tree for the core dataset with C. hawaiensis x grayana ............................... 77 Figure 18: ASTRID tree for the core dataset with C. longifolia x kauaiensis.............................. 78 Figure 19: ASTRID tree for the core dataset with C. munroi x grayi.......................................... 79 Figure 20: ASTRID tree for the core dataset with C. sandwicensis x grandiflora ....................... 80 Figure 21: Model selection using negative pseudolikelihood scores........................................... 81 x Figure 22: Introgression model for all taxa ................................................................................ 82 Figure 23: Introgression model for the core dataset ................................................................... 83 Figure 24: Introgression model for the