SYSTEMATICS AND EVOLUTION IN THE TRIBE SCHIZOPETALAE (BRASSICACEAE): A MOLECULAR, MORPHOLOGICAL, AND ECOLOGICAL ANALYSIS OF THE DIVERSIFICATION OF AN ENDEMIC LINEAGE FROM THE ATACAMA DESERT (CHILE) By ©2013 Oscar Fernando Toro Núñez Submitted to the graduate degree program in Ecology and Evolutionary Biology and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy. ________________________________ Chairperson Mark E Mort ________________________________ Daniel J. Crawford ________________________________ Craig C Freeman ________________________________ Jorge Soberón ________________________________ Rafe M. Brown ________________________________ Matthew J. Buechner Date Defended: August 26, 2013 The Dissertation Committee for Oscar Fernando Toro Núñez certifies that this is the approved version of the following dissertation: SYSTEMATICS AND EVOLUTION IN THE TRIBE SCHIZOPETALAE (BRASSICACEAE): A MOLECULAR, MORPHOLOGICAL, AND ECOLOGICAL ANALYSIS OF THE DIVERSIFICATION OF AN ENDEMIC LINEAGE FROM THE ATACAMA DESERT (CHILE) ________________________________ Chairperson Mark E. Mort Date approved: December 19, 2013 ii Abstract As aridity has been identified as an active promoter of diversification in deserts, attempts to test organismal differentiation in the Atacama Desert have resulted particularly challenging. Most limitations are related to the recent origin of the extreme aridity in the Atacama Desert, which have stimulated a rapid process of diversification and obscured evidence of interspecific divergence. Based on its favorable biological attributes and high endemicity, genera from the tribe Schizopetalae (Mathewsia and Schizopetalon) emerge as a practical study group to conduct studies of diversification under rapid and recent diversification. The present dissertation focuses on exploring this issue, 1) solving the phylogenetic relationships in the tribe Schizopetalae, 2) describing patterns of interspecific divergence in a well-defined lineage of Schizopetalon from the Atacama Desert, and 3) searching and testing multiple highly variable nuclear loci for phylogenetic and phylogeographic purposes. The results confirmed the monophyletic status of the tribe Schizopetalae and genus Schizopetalon; nevertheless, genus Mathewsia requires to be redefined because the exclusion of M. nivea. Patterns of interspecific differentiation suggest a process of allopatric divergence promoted by ecological niche differentiation between the Andes and coastal ranges in the Atacama Desert. While this result is consistent with previous hypotheses of divergence by habitat differentiation, elements of hybridization, incomplete lineage sorting, and phenotypic plasticity obscured the identification of species limits and precluded a better inference of lineage isolation. The analysis of available genomic resources demonstrated the suitability of obtaining multiple low copy nuclear loci from already available genomic data in Schizopetalon. However, the use of these markers is yet limited, as the detection of multiple copies implies that further analyses are needed to discard paralogous copies. Overall, this dissertation sets the foundation for more elaborated studies, as more available genomic iii resources and intricate pattern of divergence can result promising to explore the consequences of local patterns of extreme aridity in the diversification and evolution of species of Schizopetalae. Keywords: Schizopetalae, Schizopetalon, Mathewsia, Atacama Desert, systematics, diversification, aridity. iv Acknowledgements I would like to acknowledge the following people, as contributors to this work and for helping in my formation in Biological sciences. I thank M. Mort, D. Crawford, C. Freeman, J. Soberón, and R. Brown as committee members for guiding my progress throughout my graduate studies. I would also like to thank J. Archibald, D. Martinez-Gordillo, N. Barve, L. Campbell, J. Oaks, and especially my friend A. Lira-Noriega, for their invaluable contributions to my knowledge and development of creative ideas to improve this dissertation. I thank the people who assisted me in obtaining herbarium and field collected specimens, including A. Marticorena (CONC), R. Rodriguez (CONC), M. Muñoz (SGO), E. Wood (GH), C. Morse (KANU), Diego Salariato (SI), Federico Luebert, Lira-Cuadra, T. S. Flores-Fernandez, J. Toro-Nuñez, and P. Ramirez-Toro. I also thank all people who contributed in forming my interest in science and the study of South American plants, including my undergraduate advisors E. Ruiz and M. A. Negritto; I. A. Al- Shehbaz for introducing me to the world of South American Brassicaceae: and G. Plunkett and A. Nicolas for allowing me to share field trips to Chile that inspired me to conduct research in the Atacama Desert. I thank all institutions that have provided funding for this research and my studies at KU, including The University of Kansas Graduate School, The University of Kansas Biodiversity Institute, The University of Kansas Department of Ecology and Evolutionary Biology, Sigma Xi, CONICYT, and Fulbright. Finally, I would like to thank my parents (Johanna and Guillermo) for their constant support and encouragement during my studies and I want to dedicate this dissertation to my wife Carola and son Gaspar for their unceasing support and hours of company and joy during the hardships found in the development of this study. v Table of Contents CHAPTER ONE 1 INTRODUCTION 1 Tribe Schizopetalae 2 Chilean Atacama Desert 5 Aridity and diversification of the Atacama flora 7 CHAPTER TWO 15 PHYLOGENETIC RELATIONSHIPS OF MATHEWSIA AND SCHIZOPETALON (BRASSICACEAE) INFERRED FROM NRDNA AND CPDNA REGIONS: TAXONOMIC AND EVOLUTIONARY INSIGHTS FROM AN ATACAMA DESERT ENDEMIC LINEAGE 15 Abstract 15 Introduction 16 Materials and Methods 19 Taxon sampling 19 Phylogenetic analyses 21 Data set and partition congruence 23 Results 24 Analysis of congruence 24 Sequences and data sets 24 nrDNA analyses 26 cpDNA analyses 27 Discussion 28 Monophyly of Mathewsia and Schizopetalon 28 Mathewsia subclades and relationships 30 Schizopetalon subclades and relationships 33 Acknowledgements 38 CHAPTER THREE 48 UNRAVELING PATTERNS OF SPECIES DIVERGENCE IN THE ATACAMA SUBCLADE OF SCHIZOPETALON (BRASSICACEAE) USING MORPHOMETRIC, ECOLOGICAL, AND MOLECULAR APPROACHES 48 Abstract 48 vi Introduction 49 Materials and Methods 51 Morphological data acquisition and quality assessment 51 Interspecific morphometric comparison 53 Species occurrence and environmental data 54 Ecological niche model estimation 56 Assessing species limits using GMYC 58 Results 60 Morphometric variability 60 Ecological Niche Modeling 61 Assessing species limits using GMYC 62 Discussion 63 Are species of the Atacama clade distinguished by morphology? 63 Do species of the Atacama clade have divergent ecological niches? 66 Are species of the Atacama subclade differentiated by genetic – coalescent methods? 68 Can habitat isolation and climatic differentiation explain patterns of morphometric, ecological niche, and genetic differentiation in the Atacama subclade? 69 CHAPTER FOUR 86 DETERMINING PATTERNS OF INTERSPECIFIC DIVERSIFICATION IN THE ATACAMA SUBCLADE OF SCHIZOPETALON (BRASSICACEAE) USING LOW COPY NUCLEAR GENES 86 Abstract 86 Introduction 87 Materials and Methods 90 Specimen and DNA sampling 90 Molecular analyses 92 Results 93 Screening potential low copy nuclear regions 93 Genetic variation and gene trees in ELF8 and MMT introns 94 Discussion 97 Evolutionary and phylogeographic scenarios in the Atacama subclade 100 S. rupestre (groups A, B) 100 S. biseriatum (group C) 101 S. tenuifolium (group D) 101 vii Bibliography 118 Appendices 145 Appendix 1 145 Appendix 2 147 Appendix 3 152 Appendix 4 156 Appendix 5 157 Appendix 6 159 Appendix 7 163 Appendix 8 165 Appendix 9 167 Appendix 10 169 viii LIST OF TABLES CHAPTER 2 Table 1 Statistics from phylogenetic analyses of all data sets. 39 CHAPTER 3 Table 1 Morphological characters for morphometric analysis of Atacama subclade. 72 Table 2 Results from univariate analyses of Atacama subclade. 73 Table 3 Descriptive statistics of morphological characters for Atacama subclade. 74 Table 4 Summary of variable contribution in the estimation of ENM in Atacama subclade. 75 Table 5 Indexes of ecological niche overlap and background similarity test. 76 CHAPTER 4 Table 1 Primers used for scan of LCNG regions. 105 Table 2 Summary statistics of molecular variation from ELF8 and MMT. 106 Table 3 Summary of polymorphism in MMT locus. 107 Table 4 Haplotype identity obtained from parsimony network analysis. 108 ix LIST OF FIGURES CHAPTER 1 Fig. 1 Mathewsia and Schizopetalon. 11 Fig. 2 Geographic Distribution of Mathewsia. 12 Fig. 3 Geographic Distribution of Schizopetalon. 13 Fig. 4 Location of Atacama Desert. 14 CHAPTER 2 Fig. 1 Geographic Distribution of Mathewsia and location of ecoregions. 40 Fig. 2 Geographic Distribution of Schizopetalon. 41 Fig. 3 nrDNA trees inferred with Parsimony (A) and ML (B). 42-43 Fig. 4 cpDNA trees inferred with Parsimony (A) and ML (B). 44-45 CHAPTER 3 Fig. 1 Geographic distribution of species from the Atacama subclade. 77 Fig. 2 Leaf image treatment for morphometric analysis for the Atacama subclade. 78 Fig. 3 PCA of morphological characters for the Atacama subclade. 79-80 Fig.4 Boxplots of univariate variation in morphology for the Atacama subclade 81 Fig. 5 Estimation of optimal habitats of occurrence using ENM for the Atacama subclade. 82 Fig. 6 Leaf area-perimeter variation