DOES ASEXUALITY CONFER A SHORT TERM ADVANTAGE? A CASE STUDY IN THE FERN MYRIOPTERIS GRACILIS (PTERIDACEAE) A Thesis by David Wickell Bachelor of Science, Wichita State University, 2013 Submitted to the Department of Biological Sciences and the faculty of the Graduate School of Wichita State University in partial fulfillment of the requirements for the degree of Master of Science July 2015 © Copyright 2015 by David Wickell All Rights Reserved DOES ASEXUALITY CONFER A SHORT TERM ADVANTAGE? A CASE STUDY IN THE FERN MYRIOPTERIS GRACILIS (PTERIDACEAE) The following faculty members have examined the final copy of this thesis for form and content, and recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science, with a major in Biological Sciences. ________________________________ James Beck, Committee Chair ________________________________ Mary Liz Jameson, Committee Member ________________________________ Craig Torbenson, Committee Member iii ACKNOWLEDGEMENTS I would like to thank James Beck for guiding my research and generally doing his best to prepare me for a career in the biological sciences. Also, I want to thank Mary Liz Jameson for introducing me to bioinformatics and giving me my start in biological research. I am grateful to Mike Windham and Amanda Grusz for sharing their extensive knowledge of Myriopteris as well as help in the field. I would also like to thank George Yatskievych (Missouri Botanical Garden) for assistance finding M. gracilis in the field and the herbarium as well as Elizabeth Johnson (Garrett Herbarium, Natural History Museum of Utah) and Tiana Rehman (Biological Research Institute of Texas) for their assistance in locating herbarium specimens. Collections from the field were conducted with permission from the New Mexico Bureau of Land Management, Arizona Bureau of land Management, US National Forest Service, University of Central Oklahoma, Virginia Department of Conservation and Recreation, Kansas Department of Wildlife, Parks, and Tourism, Illinois Department of Natural Resources, Texas Parks and Wildlife Department, Kentucky State Nature Preserves Commission, Minnesota Department of Natural Resources, and the Missouri Department of Conservation. Finally, this research would not have been possible without grants from the Kansas Academy of Science, American Society of Plant Taxonomists and Wichita State University. iv ABSTRACT Asexual taxa are generally seen as evolutionary dead ends, relegated to the tips of phylogenies due to elevated extinction rates. Despite this macroevolutionary disadvantage, there is evidence in some cases that asexual reproduction may provide a short-term benefit. This is particularly evident in asexual species that display a wider distribution than their sexual relatives. Alternatively, it is possible that such broad distributions are an illusion created by multiple asexual lineages, each occupying a relatively small area. Myriopteris gracilis Fée (Pteridaceae) is a North American asexual fern species with a particularly large range. In this study we investigate, first, if M. gracilis is exclusively asexual throughout its range and second, whether M. gracilis comprises a single wide-ranging lineage, or multiple, more geographically restricted lineages. Sexuality was assessed by counting spores/sporangium in 502 herbarium specimens from 28 states and provinces in the USA, Canada and Mexico, revealing no cryptic sexual populations. Lineage structure was then assessed with both plastid DNA sequence and Genotyping By Sequencing (GBS) SNP datasets. The plastid data identified two large, roughly eastern and western, groups. Each group was further subdivided by the GBS data, to reveal a complex distribution of asexual lineages of varying geographic range sizes none of which accounted for the total size of the M. gracilis range. Thus, viewed as a single, continuous distribution, they tend to overstate the success of any one lineage in M. gracilis and by extension, asexuality’s contribution to short-term success in other species. v TABLE OF CONTENTS Chapter Page 1. BACKGROUND (REVIEW OF LITERATURE) 1 2. INTRODUCTION 10 3. METHODS 14 Field collections and spore counting 14 DNA extraction and plastid sequencing 15 Plastid sequence alignment and analysis 15 Genotyping by sequencing (GBS) 16 GBS filtering and analysis 17 4. RESULTS 19 Geographic distribution of asexual lineages 19 Plastid dataset 19 Genomic dataset 20 5. CONCLUSIONS AND FUTURE DIRECTIONS 22 The absence of a M. gracilis sexual progenitor 22 Lineage diversity and the appearance of short term success 24 REFERENCES 29 APPENDIX 35 Tables 36 Figures 38 Record M. gracilis specimens spore counted 45 M. gracilis specimens submitted to Sanger sequencing and/or GBS 65 vi LIST OF TABLES Table Page 1. Primers used for amplification and Sanger sequencing of the plastid trnGR region 36 2. Ranks and stability values of significant clusters resulting from PCOMC analysis 37 vii LIST OF FIGURES Figure Page 1. M. gracilis U.S. distribution, noting the location of field collections 38 2. M. gracilis U.S. distribution, noting the location of georeferenced spore-counted individuals 39 3. Plastid trnGR Bayesian 50% consensus phylogram of M. gracilis and six closely- related Myriopteris species 40 4. Plastid trnGR haplotype network 41 5. Geographic distribution of plastid trnGR haplotypes 42 6. Principal coordinates analysis of SNP data indicating significant clusters identified by PCOMC 43 7. Geographic distribution of significant clusters identified by PCOMC 44 viii CHAPTER 1 BACKGROUND AND REVIEW OF LITERATURE: Evolutionary biology has long struggled to explain the origin and proliferation of sexual reproduction among multicellular organisms (Stearns 1990). The preponderance of sexual reproduction is especially surprising given the significant costs associated with it. The most cited and perhaps well-known example of these costs is the two-fold demographic cost of sex (Maynard Smith 1978). While only females are capable of reproducing in a sexual population, every asexual individual is capable of reproduction, allowing asexual populations to effectively double in size every generation. Additionally, sexual reproduction imposes a fitness cost, as females are only able to pass on half of their genes despite the much higher energy cost they incur by reproducing. Even when especially fit genotypes do arise, they are potentially eliminated during meiosis, as both recombination and segregation can disrupt effective combinations of genes that have become ideally suited to a specific environment (Williams 1975). With all else being equal, such significant costs should allow asexuals to outcompete their sexual counterparts. This apparent conflict between the world we observe and the one we would expect has led to the development of several hypotheses that could explain the relative scarcity of asexual species across the tree of life. The majority of these hypotheses focus on processes that occur over longer evolutionary timescales. Muller’s ratchet hypothesizes that genetic recombination is necessary to remove deleterious mutations from the gene pool (Muller 1964). This is accomplished at multiple stages during meiosis, initially in the pairing of homologous chromosomes, where 1 sequence aberrations can inhibit proper pairing and cause meiosis to fail (Heng 2007). Additionally, genetic segregation and recombination both concentrate and redistribute deleterious mutations across a population, increasing the efficacy of purifying selection (Barton and Charlesworth 1998). In the absence of recombination, however, an ever-increasing genetic load of deleterious mutations can eventually drive asexual species to extinction (Kondrashov 1988; Lynch et al. 1993). This sort of “mutational meltdown” has been observed in numerous eukaryotic taxa, notably in yeast (Zeyr et al. 2001), protozoans (Bell 1988), Drosophila (Crow and Simmons 1983) and nematodes (Morran 2009). Alternative hypotheses generally focus on asexual species' failure to adapt alongside rapid changes in various environmental factors (Hakoyama and Iwasa 2004). A popular example is the “Red Queen” hypothesis, where co- evolving parasites exert selective pressure against commonly encountered genotypes. This results in the evolutionary maintenance of sexual reproduction to provide alternative, possibly resistant genotypes (Bell 1982). These dynamics have been demonstrated between reproductively isolated populations of lizards, minnows, snails, nematodes and flax (Moritz et al. 1991; Lively et al. 1990; Lively 1987; Antonovics et al. 2011; Morran et al. 2011; respectively), in which areas experiencing higher parasite loads tend to be inhabited by sexual populations, whereas asexuality is more prevalent in areas with low parasite loads. Ultimately, all of these models imply that the rarity of asexual taxa is due to an ever increasing probability of extirpation as a result of lowered fitness. Being associated with such high rates of extinction leads to obligate asexual species being viewed as evolutionary dead- ends or “twigs on the tree of life” (Agnarsson et al. 2006; Schwander and Crespi 2009). Although such views largely rule out the success of asexuality over large evolutionary time 2 scales, they typically don't make predictions regarding the potential short-term advantage of this strategy. In reality, many studies suggest that asexuality may be a successful approach over relatively short evolutionary time scales. Numerous asexual taxa occupy significantly