A Systematic Revision of the Viola pedatifida Group and Evidence for the Recognition of Viola virginiana, a New Narrow Endemic of the Virginia Shale Barrens A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Bethany A. Zumwalde August 2015 © 2015 Bethany A. Zumwalde. All Rights Reserved 2 This thesis titled A Systematic Revision of the Viola pedatifida Group and Evidence for the Recognition of Viola virginiana, a New Narrow Endemic of the Virginia Shale Barrens by BETHANY A. ZUMWALDE has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by Harvey E. Ballard, Jr. Associate Professor of Environmental and Plant Biology Robert Frank Dean, College of Arts and Sciences 3 ABSTRACT ZUMWALDE, BETHANY A., M.S., August 2015, Environmental and Plant Biology A Systematic Revision of the Viola pedatifida Group and Evidence for the Recognition of Viola virginiana, a New Narrow Endemic of the Virginia Shale Barrens Director of Thesis: Harvey E. Ballard, Jr. The genus Viola (Violaceae) encompasses 1,000-1,100 species and includes one of the largest North American groups, the "acaulescent blue" violets (subsection Boreali- Americanae). In the subsection, the Viola pedatifida species group is distinctive in producing only divided leaf blades. Presently the group consists of Viola pedatifida G. Don., Viola brittoniana Pollard, and Viola subsinuata (Greene) Greene. Recent fieldwork in the mountains of Virginia suggested that disjunct populations of V. pedatifida were different than Midwestern populations. Macromorphological, micromorphological, genetic, and ecological data were analyzed to compare taxa. Phenetic analyses of leaf, flower, cleistogamous capsule and seed variables and scanning electron microscopic examinations of seed coats and petal trichomes distinguished four species. Genetic studies using four microsatellite loci and microhabitat data indicated genetic and ecological differentiation among the four species. All evidence support recognition of the Virginia violet as a narrowly endemic species, Viola virginiana Zumwalde & H. E. Ballard. 4 DEDICATION This thesis is dedicated to my sister, Amanda Huff 5 ACKNOWLEDGMENTS I wish to express my appreciation to my advisor, Dr. Harvey Ballard, for his infinite support, advice, and scientific expertise. I would also like to thank my committee members, Drs. Morgan Vis and David Rosenthal, for their assistance and valuable input. I would like to especially recognize Benjamin Gahagen for his helpful guidance and direction on many fronts of my graduate work. Thank you to Harlan Svoboda for his endless support, nomenclatural expertise, and all of his assistance in the micromorphological section of my research. I would like to recognize Dr. Martin Kordesch and the Department of Physics at Ohio University for granting me access to use their scanning electron microscope and materials. I thank Ohio University and the Graduate Student Senate at Ohio University for providing funding for my research. I would like to recognize and thank those who have contributed their various time and efforts to my research: Donnie Day, Toddrick Roth, Chris Benson, Bailey Hunter, David Robson, Colin Kruse, Anne Sternberger, Alexander Meyers, and Erik Anderson. Also, thank you to John Townsend for his collaboration, monitoring of the new species, and for granting me access and use of pictures. Thank you to Tom Wieboldt, Otto Gockman, Steve Young, Craig Freeman, and Irvine Wilson for sending specimens for ecological and molecular analyses. Additional thanks to the Missouri Botanical Garden (MO), Smithsonian Institution (US), New York Botanical Garden (NY), Harvard University (GH), and the Virginia Polytechnic Institute and State University (VPI) for providing herbarium specimens. 6 TABLE OF CONTENTS Page Abstract ............................................................................................................................... 3 Dedication ........................................................................................................................... 4 Acknowledgments............................................................................................................... 5 List of Tables ...................................................................................................................... 8 List of Figures ..................................................................................................................... 9 Chapter 1: Introduction ..................................................................................................... 10 Taxonomic History of the Viola pedatifida Group ....................................................... 13 A New Endemic Species ............................................................................................... 18 Objectives ..................................................................................................................... 20 Chapter 2: Macromorphological phenetic studies ............................................................ 22 Introduction ................................................................................................................... 22 Methods ........................................................................................................................ 22 Results ........................................................................................................................... 26 Leaf Character Analyses ........................................................................................... 26 Floral Character Analyses ......................................................................................... 27 Cleistogamous Capsule and Seed Character Analysis .............................................. 28 Univariate Analyses .................................................................................................. 28 Discussion ..................................................................................................................... 29 Chapter 3: Micromorphological Study ............................................................................. 44 Introduction ................................................................................................................... 44 Methods ........................................................................................................................ 45 Results and Discussion ................................................................................................. 49 Chapter 4: Genetic Study .................................................................................................. 59 Introduction ................................................................................................................... 59 Methods ........................................................................................................................ 60 Results and Discussion ................................................................................................. 63 Chapter 5: Ecological Study ............................................................................................. 68 Introduction ................................................................................................................... 68 7 Methods ........................................................................................................................ 69 Results and Discussion ................................................................................................. 70 Biogeography and Habitat Descriptions ....................................................................... 71 V. brittoniana ............................................................................................................ 71 V. pedatifida .............................................................................................................. 73 V. subsinuata ............................................................................................................. 74 V. virginiana ............................................................................................................. 76 Chapter 6: Taxonomic Revision ....................................................................................... 79 Key to Species of the Viola pedatifida Group .............................................................. 79 Descriptions of the Taxa ............................................................................................... 81 References ......................................................................................................................... 92 Appendix 1: Additional Macromorphological Traits Examined ...................................... 97 Appendix 2: Specimens Examined ................................................................................... 98 Appendix 3: Raw Presence/ Absence (0/1) Data Matrix of Alleles ............................... 110 8 LIST OF TABLES Table 1. A comparison of the major treatments of the V. pedatifida group ..................... 17 Table 2. Variables used in statistical and phenetic analyses ............................................. 25 Table 3. Variable-variate correlation of leaf matrix data .................................................. 33 Table 4. Variable-variate correlation of leaf matrix data following step-wise removal