Investigations of morphological and molecular variation in wild and cultivated violets (Viola; Violaceae) DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Daniel William Howard Robarts, B.S. Graduate Program in Evolution, Ecology and Organismal Biology The Ohio State University 2013 Dissertation Committee: Andrea D. Wolfe, Advisor Pablo Jourdan Laura Kubatko Harvey Ballard Copyright by Daniel William Howard Robarts 2013 Abstract The genus Viola is a large and diverse group of flowering plants. The objectives of this study were to develop and explore new morphological and molecular tools with horticultural and systematic applications. Chapter 1 employed digital image analysis software, Tomato Analyzer, for flower morphology analysis of a 127 accession collection of sect. Melanium violets ("pansy group"). Seventy-seven traits associated with shape, size, and color were scored separately as categorical or continuous variables. The qualitative analysis was favored, capturing more of the variation and receiving higher bootstrap support in cluster analysis dendrograms. Cluster and ordination analyses indicated that the presence of blotch was the primary grouping factor, and secondarily, measures of color and shape (e.g., petal width). Uniformity across accessions of some hybrid morphotypes (e.g., "white with blotch") led to tight clustering across analyses. There were no significant correlations between clustering patterns and accessions' originating country or parent company, as had been previously reported. Chapter 2 builds on the morphological analysis described in Chapter 1 by utilizing sequence-related amplified polymorphism (SRAP) markers to further characterize the collection of Melanium violets. Here, SRAP fragments indicated no significant differences between the horticultural classes of violets, though more were generated from ii species types than hybrid types of the same ploidy. Bayesian analysis suggested distinctive structure clusters within the collection, but was obscured by high levels of admixture. Some color forms (white, white with blotch, yellow with blotch, and orange) tended to cluster strongly together. Correlation analysis of morphological and molecular datasets, as well as analysis of a combined dataset, underscored the conclusion that some genetic lines could be generalized by blotch presence and flower color. The relationship between these data may help in optimizing germplasm collection management and development of SRAPs by breeders for marker-assisted selection. Chapter 3 describes the genetic diversity within Viola pedata, a North American perennial violet with a bicolorous floral syndrome similar to that seen in taxa of the pansy group. Four hundred fifty-eight individuals collected from 42 unique populations and one cultivar were sampled in eastern North America, and compared using microsatellite markers. Results confirm that this species is a high order polyploid (>4x), and underlying structure indicates previous refugial populations in the Driftless Area of the upper Midwest during previous glacial maxima. A genetic discontinuity east and west of the Appalachian Mountain range was depicted by neighbor-joining, principal coordinates, and Bayesian approaches, a pattern seen in many other plant taxa. Chapter 4 reviews the uses of SRAP markers in the current literature. Since their inception in 2001, over 300 published articles have described the use of markers, with incidence of use increasing every year. Analysis of comparative studies shows SRAP markers generating similar levels of amplicons and polymorphism as AFLP, but with drastically fewer, more simple steps. In presenting relevant case studies, SRAP markers iii are shown as valuable, but underexplored tools to address hypotheses in topics beyond those in the applied sciences for which they were developed. These research areas include systematics, biogeography, ecology, and conservation. iv Dedication I dedicate this dissertation to my family, with the deepest gratitude and appreciation for their enduring support of my efforts to complete this endeavor. Their consistent patience and faith in me have not waivered through time or distance, and truly allowed me to follow my dreams. I offer special thanks to my partner in life, Claire, who followed me though countless long nights of dissertation toiling as girlfriend, fiancée, wife, and soon-to-be mother of our twin boys. Thank you, especially, for acceptance of my phytomania, and embracing my passion for the natural world. v Acknowledgments While the first page of this document proffers only my name as author, it would not have come into existence without the generous contributions of many. I humbly thank my advisor, Dr. Andrea Wolfe, for her guidance and support though the entirety of this endeavor. Her patience and mentoring have been a true blessing, and helped me develop as an independent researcher. A debt of gratitude is also owed to the financial support of the Ornamental Plant Germplasm Center (OSU-USDA), and its director, Dr. Pablo Jourdan. Without their interest and provision, none of this research would have come to fruition. It is my hope the research described herein may serve to underscore the immense value of this institution and its endeavors. Also, I give thanks to my other advisors, Dr. Harvey Ballard, who was always in my corner and provided expertise in Viola, and to Dr. Laura Kubatko, for lending her guidance in resolving my statistical quandaries. Thanks to all who assisted in locating, acquiring, and maintaining plant materials for analysis, including Bryan Connolly (MA NHESP), Tony Recznicek (UM), Ted Cochrane (UW), Robert Capers (UConn), Steven Carroll (State Arboretum of Virginia), Dennis Bell (ULM), Peter Stefany (PanAmerican Seed), David Snodgrass (OSU), Peter Zale (OSU), and many other researchers, naturalists, and photographers (thank you Flickr friends!). Special thanks to Jose Diaz, fellow WOLFE PACK comrades Aaron Wenzel, Paul Blischak, Shannon Kilkenny, and graduate colleagues including Bruce Ackley, Chia-Hua Lin, and Mike Sovic. Your daily research assistance was critical for my forward progress. vi Vita June 2000 .......................................................White Mountains Regional High School May 2004 .......................................................B.S. Biology, Bates College 2006-2010 ......................................................Graduate Research/Teaching Associate, Department of Horticulture and Crop Science, The Ohio State University 2010 to present ..............................................Graduate Research/Teaching Associate, Department of Evolution, Ecology, and Organismal Biology, The Ohio State University Publications Robarts, D., & Baum, M. J. (2007). Ventromedial hypothalamic nucleus lesions disrupt olfactory mate recognition and receptivity in female ferrets. Hormones and Behavior, 51(1), 104-113. Batterton, M. N., Robarts, D., Woodley, S. K., & Baum, M. J. (2006). Comparison of odor and mating-induced glomerular activation in the main olfactory bulb of estrous female ferrets. Neuroscience Letters 12, 400(3), 224-229. McCormick, C. M., Robarts, D., Kopeikina, K., Kelsey, J. E. (2005). Long-lasting, sex- and age-specific effects of social stressors on corticosterone responses to restraint and on locomotor responses to psychostimulants in rats. Hormones and Behavior, 48(1), 64-74. vii McCormick, C. M., Robarts, D., Gleason, E., Kelsey, J. E. (2004). Stress during adolescence enhances locomotor sensitization to nicotine in adulthood in female, but not male, rats. Hormones and Behavior, 46(4), 458-466. Fields of Study Major Field: Evolution, Ecology and Organizmal Biology Focus: Ornamental Plant Characterization viii Table of Contents Abstract ............................................................................................................................... ii Dedication ........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita .................................................................................................................................... vii Publications ....................................................................................................................... vii Fields of Study ................................................................................................................. viii Table of Contents ............................................................................................................... ix List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiv Chapter 1. Morphological variation in a collection of pansies (Viola sect. Melanium) detected by digital image analysis ...................................................................................... 1 Abstract ........................................................................................................................... 1 Introduction ..................................................................................................................... 3 Methods