GENETIC RELATIONSHIPS, MORPHOLOGICAL DIVERGENCE AND ECOLOGICAL CORRELATES IN THREE SPECIES OF THE VIOLA CANADENSIS COMPLEX IN WESTERN NORTH AMERICA A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Cheryl S. McCreary November 2005 This dissertation entitled GENETIC RELATIONSHIPS, MORPHOLOGICAL DIVERGENCE AND ECOLOGICAL CORRELATES IN THREE SPECIES OF THE VIOLA CANADENSIS COMPLEX IN WESTERN NORTH AMERICA by CHERYL S. MCCREARY has been approved for the Department of Biological Sciences and the College of Arts and Sciences by Harvey E. Ballard, Jr. Associate Professor of Environmental and Plant Biology Benjamin M. Ogles Interim Dean, College of Arts and Sciences MCREARY, CHERYL S. PHD. November 2005. Biological Sciences. Genetic Relationships, Morphological Divergence and Ecological Correlates in Three Species of the Viola canadensis Complex in Western North America (209 pp.) Director of Dissertation: Harvey E. Ballard, Jr. Viola flettii, Viola cuneata and Viola ocellata are sister species within the Viola canadensis complex (Violaceae). All are endemics of western North America, growing in widely divergent ecological environments. During the summers of 1998, 1999, 2001 and 2002, leaf material for DNA extraction was collected from 26 populations of the three species, including much of their range. Analysis of V. flettii DNA using intersimple sequence repeat (ISSR) markers showed a great deal of diversity with percent polymorphic loci (P) of 65% and a disjunction between northern and southern populations. Statistical analysis of collected ecological data from V. flettii indicated a microhabitat effect of greater elevation and more southernly aspect leading to lowered genetic diversity and population size, respectively. Preserving the genetic diversity in V. flettii by protecting populations in both regions with emphasis on those at more optimal microhabitats will aid in maintaining the current fitness ability of this endemic species. Nested Clade Analysis (NCA) of Polymerase chain reaction - restriction fragment length polymorphisms (PCR-RFLP) data from chloroplast regions of all three species showed no distinct groupings based on taxon assignment, potentially indicating past hybridization and chloroplast capture during the early stages of speciation or subsequently and repeatedly within Pleistocene refugia harboring all three species. Contrary to the evidence suggesting hybridization and interspecific gene flow, ecological, environmental, leaf morphology and leaf angle data all show the three Viola species to be strongly distinct, supporting the idea that the three species are morphologically and ecologically well differentiated. Approved: Harvey E. Ballard, Jr. Associate Professor, Environmental and Plant Biology Acknowledgements I would like to first thank my advisor, Dr. Harvey E. Ballard, Jr., Department of Environmental and Plant Biology. Certainly, this would never have been accomplished without his knowledge, help, and advice. I would like to think my past and present doctoral dissertation committee members (Dr. Kim Brown, Dr. Sarah Wyatt, Dr. Elisar Barbar and Dr. Shiyong Wu), and other faculty of the Department of Environmental and Plant Biology, most especially Dr. Morgan Vis and Dr. Brian McCarthy who were on my original master's thesis committee. Several past and present students in these departments and program were friends and fellow classmates and should be thanked for their companionship and aid. My fellow Ballard lab mates present while I was here (Aurea Cortes-Palomac, Min Feng, Ross McCauley, Jennifer DeMuria, and Maria Claudia Segovia-Salcedo) and although we were always working on variously different things, they provided wonderful help and friendship. I must also thank my two wonderful field assistants, my significant other Christopher Bade, and my brother Scott McCreary, as well as support from my other friends and family. Funding aid was provided by: a grant from the National Geographic Society, a research assistantship via a NSF grant on the Violaceae, and student travel grants from MCB, PBIO and BIOS departments. 6 Table of Contents Page Abstract................................................................................................................................3 Acknowledgements..............................................................................................................5 List of Tables .......................................................................................................................8 List of Figures....................................................................................................................10 Chapter 1. Overview and Introduction...............................................................................12 Chapter 1 Literature Cited .....................................................................................16 Chapter 2. The Conservation Biology of the Olympic Mountain Endemic Viola flettii ...17 2.1 Abstract............................................................................................................17 2.2 Introduction......................................................................................................19 2.3 Methods............................................................................................................27 2.4 Results..............................................................................................................31 2.5 Discussion........................................................................................................34 Chapter 2 Tables and Figures ................................................................................40 Chapter 2 Literature Cited .....................................................................................48 Chapter 3. Interpretation of Historical Events in the Evolution of Three Closely Related Violets................................................................................................................................52 3.1 Abstract............................................................................................................52 3.2 Introduction......................................................................................................53 3.3 Methods............................................................................................................64 3.4 Results..............................................................................................................70 3.5 Discussion........................................................................................................75 Chapter 3 Tables and Figures ................................................................................84 Chapter 3 Literature Cited .....................................................................................94 Chapter 4. Ecological Differentiation between Three Viola Species and its Relationship to Evolution and Speciation...............................................................................................99 4.1 Abstract............................................................................................................99 4.2 Introduction....................................................................................................100 4.3 Methods..........................................................................................................111 4.4 Results............................................................................................................115 4.5 Discussion......................................................................................................118 Chapter 4 Tables and Figures ..............................................................................123 Chapter 4 Literature Cited ...................................................................................135 Chapter 5. Overall Conclusions .......................................................................................138 7 Appendices.......................................................................................................................144 1. Ecological Data for Viola flettii .......................................................................144 2. Genetic (ISSR) Data for Viola flettii................................................................148 3. Phylogeographic (Chloroplast PCR-RFLP) Data ............................................164 4. Ecological Factors Recorded for Three Species ..............................................169 5. Morphological Variables Measured on Three Species ....................................206 8 List of Tables Table Page 2.1 Location and ecological data collected for V. flettii populations.................................40 2.2 Polymorphic loci (P) and ΦST for V. flettii populations...............................................40 2.3 AMOVA results for V. flettii ISSR data with and without regions included...............41 2.4 Pearson and Spearman correclations for V. flettii data ................................................42 3.1 List of populations of V. cuneata, V. flettii and V. ocellata collected for the project..84 3.2 Geographic analyis of the Nested Clade Analysis for all three species.......................85 3.3 Polymorphic sites, and ΦST values for populations of V. cuneata, V. flettii and V. ocellata based on PCR-RFLP data ....................................................................................86