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(Astragalus Jaegerianus Munz, Fabaceae) with Limited Range Using Microsatellites

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(Astragalus Jaegerianus Munz, Fabaceae) with Limited Range Using Microsatellites California State University, San Bernardino CSUSB ScholarWorks Electronic Theses, Projects, and Dissertations Office of aduateGr Studies 12-2020 POPULATION STRUCTURE OF A FEDERALLY ENDANGERED PLANT (ASTRAGALUS JAEGERIANUS MUNZ, FABACEAE) WITH LIMITED RANGE USING MICROSATELLITES SueAnn Neal Follow this and additional works at: https://scholarworks.lib.csusb.edu/etd Part of the Biodiversity Commons, Desert Ecology Commons, Genetics Commons, Genomics Commons, Molecular Biology Commons, Molecular Genetics Commons, Other Ecology and Evolutionary Biology Commons, and the Population Biology Commons Recommended Citation Neal, SueAnn, "POPULATION STRUCTURE OF A FEDERALLY ENDANGERED PLANT (ASTRAGALUS JAEGERIANUS MUNZ, FABACEAE) WITH LIMITED RANGE USING MICROSATELLITES" (2020). Electronic Theses, Projects, and Dissertations. 1150. https://scholarworks.lib.csusb.edu/etd/1150 This Thesis is brought to you for free and open access by the Office of aduateGr Studies at CSUSB ScholarWorks. It has been accepted for inclusion in Electronic Theses, Projects, and Dissertations by an authorized administrator of CSUSB ScholarWorks. For more information, please contact [email protected]. POPULATION STRUCTURE OF A FEDERALLY ENDANGERED PLANT (ASTRAGALUS JAEGERIANUS MUNZ, FABACEAE) WITH LIMITED RANGE USING MICROSATELLITES A Thesis Presented to the Faculty of California State University, San Bernardino In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology by SueAnn M. Neal December 2020 POPULATION STRUCTURE OF A FEDERALLY ENDANGERED PLANT (ASTRAGALUS JAEGERIANUS MUNZ, FABACEAE) WITH LIMITED RANGE USING MICROSATELLITES A Thesis Presented to the Faculty of California State University, San Bernardino by SueAnn M. Neal December 2020 Approved by: Dr. Anthony Metcalf, Committee Chair, Biology Dr. James Ferrari, Committee Member Dr. David Polcyn, Committee Member © 2020 SueAnn M. Neal ABSTRACT Studies on population genetics examine the relationship and effects of population structure, migration, gene flow and demographic history, and are therefore important in the conservation of endangered species. Astragalus jaegerianus, a critically federally endangered species found in a geographically restricted range is investigated to determine population structure and genetic variation. Previous research on A. jaegerianus focused on DNA sequence data for cpDNA and nrDNA showed no variation. Further research on A. jaegerianus utilizing AFLP’s on the whole genome indicated substantial gene diversity and population structure consistent with geographically widespread species. AFLP research is a cost-effective process to identify levels of genetic diversity but does not allow determination of heterozygosity or homozygosity. However, microsatellite analysis identifies tandem base pair repeats, which are co- dominant markers, and heterozygotes can be determined from homozygotes. Through Illumina paired-end shotgun sequencing, 26 microsatellites were isolated and characterized for A. jaegerianus. Loci were screened across 24 individuals in 5 subpopulations. The 26 identified microsatellites underwent preliminary screening to determine the 7 optimal primers for this study. Of the 176 initial samples, 140 individuals successfully amplified across a minimum of 4 microsatellite loci. Microsatellite analysis supports Walker and Metcalf’s (2008) findings of population structure and genetic variation comparable to geographically widespread plant species. The RST values ranged iii from 0.010 to 0.328, indicating that Brinkman Wash is the most distinct of the populations. In comparing the STRUCTURE analysis to the outcome of the PCoA, the data indicate there are four distinct populations and that Coolgardie Mesa and Lane Mountain appear to be more similar and can be viewed as a single population. Both AFLP and microsatellite analyses support the findings that Astragalus jaegerianus has maintained a high level of genetic diversity despite the limited range and reduced population size. iv ACKNOWLEDGEMENTS I would like to take this opportunity to thank the village that helped in the completion of this thesis project. First, I would like to thank my thesis advisor, Dr. Anthony Metcalf, for agreeing to fund and support this project. I would also like to thank my committee members, Dr. James Ferrari and Dr. David Polcyn for agreeing to assist in the completion of my project. Thank you to George Walker for his previous research on the unique little plant that inspired me to take his research to the next level, and to Stacey Nerkowski for first bringing me into the lab and introducing me to an area of research that would lead me to my master’s thesis. To my amazing family who put up with me through this whole process, although I feel I was very calm, cool and collected. Stop laughing, we all have our delusions. Thank you to my husband Greg Neal, the day I decided it was time to go back to school and find a new path he just rolled with it and told me he was behind me and always took the opportunity to tell me I was capable of great things. When I decided I wanted to go on for my Master’s degree he again rolled with it and was my biggest supporter. Even through the computer crashes, blown up papers and threats of throwing all electronic devices out the window and changing my major to underwater basket weaving he still had my back. He is my rock, my Tech Support, my biggest cheerleader and the best human being I know. I also want to thank my two heathens, Collin and Ethan. They too just rolled with the whole school process and even joined me in the lab for different v parts of my experiments, most of the time even willingly. Thanks to my parents, Don and Jackie Stine, for cheering me on and keeping my Starbucks card fully loaded. Caffeine makes the world go round when in grad school! Thanks to my mom for “dragging” on amazing vacations to celebrate my accomplishments, can’t wait for our next adventure. I owe so much to my lab mates, especially over the last 3 years of this project. Liane Greaver for keeping me sane and laughing, especially when I hit the wall, and for reminding me to just keep swimming. When things went sideways (DNA Analyzers of DEATH, lost samples in the mail…twice) Liane talked me off the cliff and reminded me I don’t look good in prison orange. Caitlin Hazelquist, aka Lab Lackey, you jumped in and helped me push through the tedious process of DNA extraction. I will forever be grateful I wasn’t the only one in the lab to accidentally knock over a sample or two. I also blame you for new found love of Botany gin, thanks for that! Lauren Morrison, aka Lab Elf, the little ray of sunshine in our lab, thank you for bringing your pure enjoyment of biology to the lab and taking the jokes and pranks we threw at you with a smile. I’ve been lucky to have such a truly amazing group to work with and I am forever grateful for their help and the many, many, many trips for coffee. Thank you to my Brunch Squad, Dawn Ladd and Renee Bilbrew, for getting me out of my own head and smiling and nodding when I went on one of my school tangents and being the best cheerleaders a girl could ever ask for. Thanks to Bob Trees for always being on ‘Team Greg’, he needed someone on his side every now and then when I was vi going crazy but seriously it’s time to switch to ‘Team Suzy’. I also owe a huge thanks to the two angels looking out for me; Traci Allen (Princess Bratty Pants The Quilt Streaker) and Jennie Trees (my bestie). I miss you both every day. Lastly, I would like to thank the following organizations for their financial support: California State University, San Bernardino’s Biology Department; California State University, San Bernardino ASI, Inc.; California State University, San Bernardino Office of Student and the Department Defense, U. S. Army. vii DEDICATION This is dedicated to my husband, Greg Neal. You get me like no one ever could and you keep me grounded while still allowing me to soar. BEST HUSBAND EVER. TABLE OF CONTENTS ABSTRACT ......................................................................................................... iii ACKNOWLEDGEMENTS ..................................................................................... v CHAPTER ONE: INTRODUCTION ...................................................................... 1 Overview .................................................................................................... 1 Geography and Habitat Fragmentation ...................................................... 4 Population Genetics ................................................................................. 11 Genetic Structure: Fixation Index (FST) and Slatkin RST ................ 11 Hardy-Weinberg Equilibrium Model ............................................... 12 Gene Flow and Migration .............................................................. 12 Isolation by Distance ..................................................................... 14 Methods for Estimating Genetic Diversity ................................................ 15 Amplified Fragment Length Polymorphism (AFLP) ....................... 16 Microsatellites ............................................................................... 17 CHAPTER TWO: MATERIALS AND METHODS ............................................... 20 Specimen Collection ................................................................................ 20 Molecular Methods .................................................................................. 20 Microsatellite
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