The genetic consequences of ex situ conservation of exceptional plant species A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences of the College of Arts and Sciences by Megan Philpott B.S. Biology, University of Cincinnati, September 2011 Committee Chair: Dr. Theresa Culley i Abstract The preservation of biodiversity is a pressing concern for many global taxa due to threats like climate change, habitat loss, and anthropogenic pressures. While in situ preservation of species in their habitats is ideal, it is not enough as threats often outpace our ability to conserve, and ex situ conservation methods must be used as a supplement to preserve species and their genetic diversity outside of their natural habitat. For exceptional species, those species ineligible for conventional seed banking due to production of little to no seeds or recalcitrant seeds, ex situ conservation must take the form of tissue culture and cryopreservation, the storage of seeds and tissues in liquid nitrogen (LN). The present study investigates the genetic consequences of these methods on exceptional species, from the initial conservation collection, to their tenure in ex situ collections, to their ultimate use in restorations and reintroductions. For Hedeoma todsenii, a federally endangered exceptional species, collections had been focused around populations located in the San Andres mountain range. A three-year study of populations in this range and the geographically distant Sacramento mountain range indicated low genetic diversity and high inbreeding in the species. However, the Sacramento population had a higher diversity and was genetically distinct from the San Andres populations, indicating that future collection efforts should include a greater focus on the Sacramento locality. Next, the genetic effects of long-term tissue culture and cryopreservation were assessed for 18 different plant species stored in LN for up to 23 years. No association was found between the survival of species after LN storage and RNA integrity, indicating that RNA may not be one of the key indicators of survival after storage. However, evidence of genetic change in DNA after cryopreservation and tissue culture was detected in three species, although this did not appear to have an effect on the survival and growth of species. It is recommended that species in long-term tissue culture or cryopreservation ii be regularly monitored for genetic stability. To understand the genetic effects of reintroduction using micropropagation, Minuartia cumberlandensis, a federally endangered exceptional species, was used as a case study. Seven genotypes of M. cumberlandensis were propagated using tissue culture and planted out into a suitable habitat in Daniel Boone National Forest (KY) in 2005. In 2015, 10 years after the initial outplanting, the population had more than tripled in size to over 200 plants. A genetic analysis indicated that the outplanting was genetically very similar to the natural source population despite the genetic bottleneck of only seven founding genotypes. This is likely due to low genetic diversity and clonal growth in the species. This case study shows that micropropagated plants can be successfully used to facilitate reintroductions. In sum, the presented research indicates that the ex situ conservation methods of tissue culture and cryopreservation are suitable for preserving exceptional species. These methods are key to preserving the biodiversity of often-overlooked exceptional plants for future generations. iii Megan Philpott, 2018 iv Acknowledgements This work would not have been possible without the unfailing support of my advisors, Dr. Theresa Culley and Dr. Valerie Pence. During my undergraduate career, the idea of doing plant conservation research was just a dream, and your mentorship and support have turned it into my career. I am filled with gratitude for the time and experience you have given me, and hope I can build on the foundation of plant science research you have given me in years to come. I also thank my committee for their generous contribution of time and expertise to guide me through my graduate studies: Dr. Eric Tepe, for being constantly available for me to bounce ideas and frustrations off of; Sarena Selbo, for grounding me in the critical perspective of applied conservation and management; Dr. Dennis Grogan, for teaching me everything I know about the mechanics of DNA and RNA; and Dr. Steven Rogstad, for giving me the initial spark of interest in plant science. I am personally grateful to Dr. Susan Dunford, for giving me the confidence to pursue this endeavor; Dr. Denis Conover, for teaching me everything I know about native plants; Charles Britt, for his expertise and assistance in the Hedeoma todsenii study; Dr. Mary Chaiken, for lending her expertise to the RNA integrity study; David Taylor, for his assistance with and dedication to Minuartia cumberlandensis; Kristine Lindsey, for unending research and emotional support; Dr. Herman Mays, for giving me my first experience working in a lab; and all the undergraduate students who dedicated their time to helping me complete this research. v My graduate studies were a time of great personal growth, and I could not have finished without the intellectual and emotional support of the members of the Culley Lab, past and present. For pep talks, research discussions, and countless coffee breaks, I am forever indebted to Rob Tunison, Ben Merritt, Jack Stenger, José Barreiro, Becky Fehn, and Dr. Anne-Catherine Vanhove. Finally, I owe most of my success and persistence to my family and my husband, Madison. Madison, I owe you one. vi Table of Contents Abstract ........................................................................................................................................... ii List of Tables ................................................................................................................................. ix List of Figures ................................................................................................................................. x Chapter One: Introduction: An overview of ex situ plant conservation for exceptional species .... 1 Works Cited............................................................................................................................... 13 Chapter Two: Collecting for ex situ conservation: An investigation of the genetic diversity of Hedeoma todsenii Irving (Lamiaceae) in White Sands Missile Range and Lincoln National Forest, New Mexico ................................................................................................ 20 Introduction ............................................................................................................................... 21 Materials & Methods ................................................................................................................. 27 Results ....................................................................................................................................... 30 Discussion ................................................................................................................................. 34 Works Cited............................................................................................................................... 39 Figure Legends .......................................................................................................................... 45 Tables ........................................................................................................................................ 46 Figures ....................................................................................................................................... 50 Chapter Three: Long-term stability of RNA and DNA in ex situ cryopreserved collections: a case study of shoot tips stored for up to 23 years .................................................................. 57 Introduction ............................................................................................................................... 58 Materials and Methods .............................................................................................................. 63 Results ....................................................................................................................................... 68 Discussion ................................................................................................................................. 70 Works Cited............................................................................................................................... 78 Figure Legends .......................................................................................................................... 86 Tables ........................................................................................................................................ 87 Figures ....................................................................................................................................... 91 Chapter Four: Augmentation and reintroduction using ex situ collections: Genetic effects of small founding population size in an experimental outplanting of the endangered perennial Minuartia cumberlandensis (Wofford & Kral) McNeill (Caryophyllaceae) ......... 94 Introduction