ABSTRACT MORPHOLOGY AND INTROGRESSIVE HYBRIDIZATION IN NORTH AMERICAN DIPHASIASTRUM by Laura L. Klein Although interspecific hybridization in plants often results in sterility, occasionally hybrids show evidence of fertility. Because of their reproductive competence, such F1’s have the ability to interbreed with each other and with their parents, forming introgressive offspring that are morphologically intermediate between the original parental forms. North American Diphasiastrum complanatum, D. digitatum, and D. tristachyum exhibit evidence of such introgressive hybridization. To test for introgression, morphological measurements for parental and putative hybrid taxa were collected and analyzed using principle components analyses and hybrid indices. Spore fertility was also analyzed within the study set. Results support the presence of introgression among all three pair combinations. Although not infrequent, the numbers of hybrid forms are far exceeded by parental forms and so there are no compelling reasons not to recognize these three taxa at the species level. MORPHOLOGY AND INTROGRESSIVE HYBRIDIZATION IN NORTH AMERICAN DIPHASIASTRUM A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Science Department of Botany by Laura L. Klein Miami University, Oxford, Ohio 2012 Advisor _________________________ R. James Hickey Reader ________________________ Richard C. Moore Reader ________________________ Michael A. Vincent CONTENTS Introduction . .1 Materials and Methods . 8 Results . .12 Discussion . .28 Appendix A . .39 Appendix B . .43 Appendix C . .45 Appendix D . .46 Appendix E . .47 Appendix F . .48 ii LIST OF TABLES Table 1: List of characters used in analysis of morphological variation. .11 Table 2: Summary character statistics for parental taxa. 14 Table 3: Summary character statistics for hybrid taxa. .17 Table 4: Summary of spore fertility data. .26 iii LIST OF FIGURES Figure 1: Lycopodiaceae phylogeny. 4 Figure 2: North American distribution ranges for Diphasiastrum complanatum, D. digtatum, and D. tristachyum. 5 Figure 3: Bayesian majority rule concensus phylogram of Diphasiastrum using chloroplast data. 7 Figure 4: Illustration of characters used in present study. 10 Figure 5: Principle components analysis (PCA) of morphological characters measured for Diphasiastrum complanatum, D. digitatum, and D. tristachyum. .16 Figure 6: PCA of morphological characters measured for Diphasiastrum complanatum, D. digitatum, D. tristachyum, D. xhabereri, D. xverecundum, and D. xzeilleri. 20 Figure 7: PCA of morphological characters measured for Diphasiastrum complanatum, D. digitatum, D. tristachyum, and D. xhabereri. 22 Figure 8: PCA of morphological characters measured for Diphasiastrum complanatum, D. digitatum, D. tristachyum, and D. xverecundum. .23 Figure 9: PCA of morphological characters measured for Diphasiastrum complanatum, D. digitatum, D. tristachyum, and D. xzeilleri. .24 Figure 10: Hybrid indices of parental and putative hybrid taxa for all species pairs. .25 Figure 11: Percent fertility for individuals scored using hybrid indices for each of the three species pairs as seen in Fig. 10. .27 Figure 12: Taxonomic distribution of the 1423 herbarium specimens borrowed for this study. 28 iv ACKNOWLEDGEMENTS My time at Miami University has truly been one of the most educational and rewarding experiences of my life. I attribute this to all the wonderful people I’ve met along the way. I am indebted to my undergraduate mentors, Tom Lammers and Bob Wise, who introduced me to the world of botany, shared tips for being a successful grad student, and taught me an important lesson: “be curious.” Such wisdom has been necessary to my success in academia. Thank you to Barb Wilson and Vickie Sandlin for making my transition into and residence in the botany department easy and enjoyable. My fellow botanists and I were lucky to have such dedicated, helpful women on our side! Mike Vincent and Rich Moore have been insightful committee members, as well as invaluable resources. Mike, a master taxonomist and excellent educator, has shared his perspicuity as a steadfast lunch companion. Rich has helped cultivate my knowledge as an evolutionary biologist and shared his perspective as a newly tenured professor with good humor. I am fortunate to have worked with my magnificent lab group. Special thanks goes to Mirabai McCarthy, who helped me transition into graduate life at Miami, but also became my surrogate family. Sushma Shrestha, Li Zheng, and Tia Ahlquist have also been valued friends and colleagues, always available to discuss plants, commiserate over research difficulties, or offer encouragement. Thanks especially to Stephen Barr, who helped me collect spore fertility data. My thesis would not be as impactful without his hard work. An occasional social life helped keep me focused and sane during my Masters. A big thanks goes out to my biking buddies and my cohort broomball team – go Rad Scientists! I’m especially grateful for my frequent dinner companions: Michael Oxendine and Samantha Rumschlag. Whether it was dancing, swimming, or sharing the struggles all young scientists go through, we are successful with help from each other. I attribute my strength and dedication to the support of my loving family. Care packages assembled by Grandma Lois, my sister, Jessica, and my aunts provided remembrances of home. Grandma Rita’s faithful letters were a weekly reminder that I am missed. I have boundless gratitude for my parents’ unwavering love, comfort, and generosity, which eased the burdens that seemed to build during graduate school. I love you all very much! Lastly, thank you, Jim Hickey, for luring me into the world of Lycopodium. More than just imparting on me an appreciation for ancestral plant lineages, you’ve taught me how to think like a scientist and educator. You have helped to develop my confidence as a researcher and imparted on me the importance of frequent reflection (sans cigarettes). Most importantly, I will take your advice as I face future challenges, and JUST DO IT. v INTRODUCTION To provide context for speciation, evolutionary studies, or biological discussions in general, biologists use species concepts. The biological species concept (Mayr 1969 and DeQueiroz 2007) is one of the most commonly used species concepts, as it clearly defines species as populations that are reproductively isolated, an intuitively comprehensible construct. However, the boundaries established by the biological species concept are critically questioned when two species are able to interbreed. Many botanists have shown that hybridization is widespread among plant species (Anderson 1936, Conant and Cooperdriver 1980, Rieseberg 1997, Walker 1958, Wagner 1951). Hybrids can often be identified by intermediate morphology, abortive pollen grains or spores, failure to produce fruit or reach sporangial maturation, or other indications of disrupted meiosis (Anderson 1936, 1949, 1953, Barrington et al. 1989, Wagner 1962, 1968, 1987, Wagner et al. 1986). Additive biochemical signatures in isozymes and flavanoids (Barrington et al. 1989) or intermediate DNA content (Aagaard 2009 and Bennert et al. 2011) have also been used as evidence of hybridization. Although most hybrids are sterile because of reproductive abnormalities, many fern species are theorized to have originated via hybridization (Barrington et al. 1989, Caluff 2002, Knobloch 1976, Manton 1950, Soltis and Soltis 2009). Fertility is often achieved through polyploidy. By doubling their chromosome set, allopolyploid species reestablish complimentary homologues (Barrington et al. 1989). In some cases, interspecific hybrids may retain full or partial fertility (Conant and Cooperdriver 1980, Rieseberg et al. 1996, Walker 1958, Wilce 1965). Geographic isolation of the fertile F1 may form new allohomoploid species (Barrington et al. 1989, Caluff 2002, Conant and Cooper-Driver 1980). Anderson (1936, 1949, 1953) noted that many fertile hybrids not only reproduce but may also backcross with parent species. Most of these hybrids with intermediate characteristics were in populations near parent species in newly created niches, such as man-made or natural secondary successional zones. Anderson and Hubricht (1938) described this phenomenon as introgressive hybridization: “…the gradual infiltration of the germplasm of one species into another as the result of hybridization and repeated backcrossing” (Anderson 1936). Anderson (1936, 1949) further concluded that the result 1 of this crossing was increased variation in the parental genome. Introgressive hybrids typically have homoploid parental species and repeated backcrossing often results in populations that intergrade indistinguishably into the parental taxa (Anderson 1949, Heiser 1949). To aid in the study and identification of introgressive hybrids, Anderson developed a hybrid index, which describes and quantifies morphological intermediacy (Anderson 1936). The first step in using this index is to identify a set (N) of clearly differentiating characters, whether quantitative or qualitative, for the parental species. All character states for one parent are arbitrarily designated as 0, while the character states for the other parent are scored as 1. In this way, an individual specimen of the first species would have a summative character score of 0, whereas a specimen of the other parent would have a summative score of N. Hybrid individuals would be predicted to have a summative score