GENETICS AND EVOLUTION OF MULTIPLE DOMESTICATED SQUASHES AND PUMPKINS (Cucurbita, Cucurbitaceae) By HEATHER ROSE KATES 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 2017 © 2017 Heather Rose Kates To Patrick and Tomás ACKNOWLEDGMENTS I am grateful to my advisors Douglas E. Soltis and Pamela S. Soltis for their encouragement, enthusiasm for discovery, and generosity. I thank the members of my committee, Nico Cellinese, Matias Kirst, and Brad Barbazuk, for their valuable feedback and support of my dissertation work. I thank my first mentor Michael J. Moore for his continued support and for introducing me to botany and to hard work. I am thankful to Matt Johnson, Norman Wickett, Elliot Gardner, Fernando Lopez, Guillermo Sanchez, Annette Fahrenkrog, Colin Khoury, and Daniel Barrerra for their collaborative efforts on the dissertation work presented here. I am also thankful to my lab mates and colleagues at the University of Florida, especially Mathew A. Gitzendanner for his patient helpfulness. Finally, I thank Rebecca L. Stubbs, Andrew A. Crowl, Gregory W. Stull, Richard Hodel, and Kelly Speer for everything. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 9 LIST OF FIGURES ........................................................................................................ 10 ABSTRACT ................................................................................................................... 12 CHAPTER 1 GENERAL INTRODUCTION .................................................................................. 14 2 ORIGINS, AGRICULTURAL IMPORTANCE, AND BREEDING CHALLENGES OF PUMPKINS, SQUASHES, AND GOURDS (Cucurbita) AND THE USEFULNESS OF STUDYING Cucurbita CROP WILD RELATIVES .................... 20 Introduction ............................................................................................................. 20 Origin and Historical Use of the Pumpkins, Squashes, and Gourds ................. 20 Cucurbita pepo ........................................................................................... 23 Cucurbita argyrosperma ............................................................................ 24 Cucurbita maxima ...................................................................................... 24 Cucurbita moschata ................................................................................... 24 Cucurbita ficifolia ........................................................................................ 25 Current Agricultural and Economic Importance of Pumpkins, Squashes, and Gourds .......................................................................................................... 26 Cucurbita pepo ........................................................................................... 27 Cucurbita argyrosperma ............................................................................ 29 Cucurbita maxima ...................................................................................... 30 Cucurbita moschata ................................................................................... 31 Cucurbita ficifolia ........................................................................................ 31 Challenges in Cultivation of Squashes, Pumpkins, and Gourds ....................... 32 Viral, bacterial, and fungal diseases ................................................................. 32 Non-disease challenges to cultivation ........................................................ 35 The effect of climate change on challenges to cultivation .......................... 36 Crop Wild Relatives of Pumpkins, Squashes, and Gourds ..................................... 37 Primary Genepool CWR ................................................................................... 38 Cucurbita argyrosperma ssp. sororia (L. H. Bailey) L. Merrick & D. M. Bates ...................................................................................................... 38 Cucurbita pepo L. (wild) ............................................................................. 39 Secondary Genepool CWR .............................................................................. 41 Cucurbita lundelliana L. H. Bailey .............................................................. 41 Cucurbita okeechobeensis (Small) L. H. Bailey ......................................... 42 Cucurbita pedatifolia L.H. Bailey and C. radicans Naudin .......................... 43 Tertiary Genepool CWR ................................................................................... 44 5 Cucurbita foetidissima Kunth ..................................................................... 45 Cucurbita palmata S. Watson, C. digitata A. Gray, C. cordata S. Watson . 45 Wild Utilized Species .............................................................................................. 47 Conservation Status of CWR and WUS .................................................................. 48 In situ ................................................................................................................ 48 Ex situ............................................................................................................... 52 3 EVOLUTIONARY AND DOMESTICATION HISTORY OF Cucurbita (PUMPKIN AND SQUASH) SPECIES INFERRED FROM 44 NUCLEAR LOCI ....................... 65 Introduction ............................................................................................................. 65 Materials and Methods............................................................................................ 69 Plant Material and DNA Extraction ................................................................... 69 Selection of Nuclear Loci .................................................................................. 70 Microfluidic PCR Amplification and Illumina Sequencing ................................. 70 Assembly of Sequencing Reads into Targeted Nuclear Loci ............................ 72 Sequence Alignment and Phylogenetic Analyses ............................................ 73 Ancestral State Reconstruction ........................................................................ 75 Results .................................................................................................................... 76 Sequence Data ................................................................................................. 76 Phylogeny of Cucurbita Based on CA-ML and Species Tree Methods ............ 77 Ancestral Character State Reconstruction........................................................ 79 Differences in Topology and Support among the Gene Trees, CA-ML Tree, and Species Trees ........................................................................................ 80 Discussion .............................................................................................................. 81 New Cucurbita Species Topology .................................................................... 81 Evolution of Mesophytic Habit in Cucurbita Species ........................................ 84 Phylogenetic Resolution within Clades of Crops and their Wild Relatives ........ 85 Conclusions ............................................................................................................ 87 4 ALLELE PHASING HAS MINIMAL IMPACT ON PHYLOGENETIC RECONSTRUCTION FROM TARGETED NUCLEAR GENE SEQUENCES IN A CASE STUDY OF Artocarpus (Moraceae) ............................................................. 97 Introduction ............................................................................................................. 97 Methods ................................................................................................................ 100 Datasets ......................................................................................................... 100 Phylogenetic Analyses ................................................................................... 103 Assessing Gene Tree Resolution, Topological Incongruence, and Allele Coalescence ............................................................................................... 106 Results .................................................................................................................. 108 Assemblies and Alignments ........................................................................... 108 Allele Coalescence, Sequence Heterozygosity, and Gene Tree Resolution .. 109 Levels of Gene Tree/Species Tree Discordance across Methods of Data Assembly ..................................................................................................... 110 Topological Incongruence among Data Sets and Phylogenetic Inference ..... 111 6 Comparison of Support for Relationships Reconstructed Using Different Datasets ...................................................................................................... 113 Phylogenetic Analysis of Genes with High Levels of Deep Coalescence ....... 114 Discussion ............................................................................................................ 115 Highly Heterozygous Genes and Individuals Do Not Appear Disproportionately Responsible