Hybridization Between Castilleja Levisecta and C. Hispida: Implications for Pacific Northwest Prairie Management
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AN ABSTRACT OF THE THESIS OF Isaac Jerome Sandlin III for the degree of Master of Science in Botany and Plant Pathology presented on March 15, 2018. Title: Hybridization between Castilleja levisecta and C. hispida: Implications for Pacific Northwest Prairie Management Abstract approved: ______________________________________________________ Thomas N. Kaye Conservation conflicts may develop on restoration sites with multiple species recovery objectives. For example, on Pacific Northwest prairies, the co-planting of the diploid cytotype of the common native wildflower Castilleja hispida with the endangered wildflower C. levisecta has resulted in putative Castilleja hybrids on restoration sites, prompting fears that genetic swamping could threaten C. levisecta. Because C. hispida is a larval host for the endangered Taylor’s checkerspot butterfly (Euphydryas editha taylori), this situation puts the recovery of both of these species at risk. However, hybrid fertility in this system is unknown. To assess hybrid fertility and introgression in Castilleja hybrids, we conducted a series of controlled reciprocal crosses between C. levisecta and C. hispida, and backcrosses between F1 hybrids and their progenitors. We measured the resulting fruit set, seed set, and seed germination to look at post pollination barriers to reproduction. Because populations of C. hispida can be diploid (2n = 2x = 24), tetraploid (2n = 4x = 48), or hexaploid (2n = 6x = 72), and C. levisecta is only diploid (2n = 2x = 24), this project explores mixed ploidy crosses between the two species. Reproductive isolation from C. levisecta was between 32 – 61% in crosses between C. levisecta and diploid C. hispida, 89 – 100% in C. levisecta and tetraploid C. hispida crosses, and 98 – 99% in C. levisecta and hexaploid C. hispida crosses. Reproductive isolation from C. levisecta was between -22 – 0% in backcrosses between C. levisecta and diploid F1 hybrids, suggesting higher diploid hybrid fitness. Reproductive isolation from C. levisecta was between 99 – 100% in backcrosses between C. levisecta and triploid F1 hybrids, suggesting interploidy crosses act as a barrier to gene flow. In addition, in order to prevent introgression, the eradication of putative hybrids is critically important to the conservation of the C. levisecta genome, but hybrids can appear extremely morphologically close to either parental species, or highly distinct. Because ploidy differences between two interspecific mating partners can be a potent isolation mechanism in plants, land managers might choose to co-plant a polyploid C. hispida cytotype with C. levisecta at recovery sites in order to mitigate hybridization between these two species, but little morphological data exists on C. hispida cytotypes. To better identify hybrids and C. hispida polyploids, we looked at whether measurable differences are detectable in 15 bract, calyx, and floral characteristics of C. levisecta, three cytotypes of C. hispida, and their diploid F1 hybrids, and if so, whether those differences are distinct enough for field technicians to distinguish them. Using multivariate analyses and univariate ANOVA, we found groups of traits that distinguished between hybrids and their progenitors, and C. hispida cytotypes, that could prove useful for field biologists. ©Copyright by Isaac Jerome Sandlin III March 15, 2018 All Rights Reserved Hybridization between Castilleja levisecta and C. hispida: Implications for Pacific Northwest Prairie Management by Isaac Jerome Sandlin III A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented March 15, 2018 Commencement June 2018 Master of Science thesis of Isaac Jerome Sandlin III presented on March 15, 2018 APPROVED: Major Professor, representing Botany and Plant Pathology Head of the Department of Botany and Plant Pathology Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Isaac Jerome Sandlin III, Author ACKNOWLEDGEMENTS I would first like to thank our many collaborators in this project: Peter Dunwiddie, Sierra Smith and the staff of the Center for Natural Lands Management in Olympia, WA for general guidance, help, and sourcing and collecting seeds for the C. hispida parent generation; Jeremy Fant, Kay Havens, Pattie Vitt, Andrea Kramer, and the Plant Science and Conservation Staff at the Chicago Botanic Gardens for guidance and help; Amy Bartow and the staff of the USDA NRCS Plant Materials Center, Corvallis, OR for teaching me how to grow Castilleja; Sean Logan, Gloria O’Brian, James Ervin, and the staff at the Oregon State University greenhouses for keeping my plants warm, happy, and pest free; Ryan Contreras and the good folks at the Contreras Lab for access to and guidance with flow cytometry; and Dale Brown and the staff of the OSU Seed Lab for cold stratification and germination resources, and all other seed-related knowledge and know-how. So many people have provided personal and professional help, assistance, and inspiration to me over the course of this project. Foremost, I’d like to thank my father, Jerry Sandlin, for providing many, many years of support and patience while I pursued a “sorta funny” profession in botany (he’s an engineer...); Caitlin Lawrence and Andrew Esterson for watering my plants, and being great friends; Sarai Carter for being my closest friend and confidant during the hardest times, and helping me count so many seedlings; Danielle Agular, Christina Partipilo, and Camille Eckel for helping with plant care and counting seeds; and all of my fellow graduate students at the Department of Botany and Plant Pathology at OSU, I couldn’t have done this without their support. The opportunity for me to study botany at OSU simply would not have been possible without serious help from colleagues and mentors: Dr. Johnny Randall and the staff at the North Carolina Botanical Garden, who gave me my first job out of university and taught me all about plant conservation; Dr. Ed Guerrant, director of the Rea Selling Berry Seed Bank & Plant Conservation Program, who convinced me that I was indeed cut out for graduate school (and introduced me to my future major professor); and, of course, Dr. Tom Kaye, executive director of the Institute for Applied Ecology, my major professor, and my mentor of many years, who has provided me with endless opportunities. Finally, this thesis is dedicated to TLH, my heart… TABLE OF CONTENTS Page Chapter 1: Introduction ................................................................................................................... 1 Pacific Northwest Prairies .......................................................................................................................... 2 Taylor’s Checkerspot Butterfly .................................................................................................................. 3 The Castilleja System ................................................................................................................................ 4 Research Goals and Objectives .................................................................................................................. 5 Literature Cited .......................................................................................................................................... 6 Chapter 2: Hybridization between Castilleja levisecta and C. hispida: Implications for Rare Plant and Butterfly Management ............................................................................................................. 9 Abstract .................................................................................................................................................... 10 Introduction .............................................................................................................................................. 11 Materials and Methods ............................................................................................................................. 16 PARENT PLANT MATERIAL ............................................................................................. 16 PLOIDY and GENOME SIZING .......................................................................................... 17 SEED GERMINATION ......................................................................................................... 18 GREENHOUSE CULTURE .................................................................................................. 19 CREATION OF F1 HYBRIDS .............................................................................................. 19 ASSESSMENT OF F1 HYBIRD FERTILITY ..................................................................... 21 FITNESS MEASURES .......................................................................................................... 22 Results ...................................................................................................................................................... 27 PLOIDY DIFFERENCES ...................................................................................................... 27 FRUIT SET, SEED SET, and SEED GERMINATION ........................................................ 28 REPRODUCTIVE ISOLATION BETWEEN C. LEVISECTA and F2 HYBRIDS ............... 34 Discussion ...............................................................................................................................................