MATING SYSTEM AND THE EVOLUTION OF STAMEN MORPHOLOGY IN THE MUSTARD FAMILY (BRASSICACEAE) By Anne Michelle Royer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Biology – Doctor of Philosophy Ecology, Evolutionary Biology and Behavior – Dual Major 2014 ABSTRACT MATING SYSTEM AND THE EVOLUTION OF STAMEN MORPHOLOGY IN THE MUSTARD FAMILY (BRASSICACEAE) By Anne Michelle Royer Biotic diversity is characterized by patterns of both divergence and similarity. Both natural selection and constraint may operate to conserve a trait across related species, depending on the ecology of the species. My dissertation investigates the roles of these evolutionary forces in maintaining a family-diagnostic stamen morphology. Flowers in the Brassicaceae (mustard plant family) are characterized by four long and two short stamens within a flower (Zomlefer 1994). Although found in >95% of the genera in the family (Endress 1992), the reason(s) for the widespread conservation of this morphology are not known. Existing adaptive hypotheses for the evolution and the maintenance of tetradynamy address how the trait could increase fitness for outcrossing species. While there is some evidence that tetradynamy is adaptive in self-incompatiBle members of the Brassicaceae (e.g. Kudo 2003; Conner et al. 2009), how it functions is not clear. Additionally, there have Been multiple independent losses of self- incompatiBility in the family (Lloyd 1965; MaBle et al. 2005), followed in some cases by the evolution of high self-pollination rates (Preston 1986). In these highly selfing species, the maintenance of short stamens, which general appear too short to pollinate the stigma within a flower (Müller 1961), is particularly mysterious. My dissertation unites approaches from evolution, ecology, and genetics to understand the maintenance of short stamens in the Brassicaceae across the full range of mating systems. I investigate the function and morphology of short stamens in three species: the obligately outcrossing wild radish (Raphanus raphanistrum), the highly selfing model plant Arabidopsis thaliana, and A. thaliana’s sister species, A. lyrata, which includes both outcrossing and selfing populations. In the outcrossing species Raphanus raphanistrum, I used experimental manipulations in arrays exposed to pollination in the field to show that having more stamens increases male fitness, and female fitness is also affected By stamen treatment. There were some indications that short stamens were more attractive to pollinators at high overall pollinator visitation rates. In the highly selfing model plant Arabidopsis thaliana, I showed that short stamens do not significantly increase fitness. I found many populations, particularly near the southern end of the geographic range, have partially lost the short stamens. Genetic mapping revealed three QTL controlling the number of short stamens, with strong epistasis greatly reducing their individual effects. Ongoing evolutionary loss of non-adaptive short stamens in Arabidopsis thaliana may Be slowed By gene interactions, low genetic variance in the north, and an inBreeding mating system. Finally, I investigated the evolution of floral morphology in selfing and outcrossing populations of the mixed-mating Arabidopsis lyrata. I found that while relative investment in female fitness has increased as predicted in selfing populations, predicted changes in size and evolution of short stamens have not yet occurred. This may Be consistent with recent evolution of selfing, continued facilitation of pollination By insects, and/or constraint on the evolution of short stamens. For Toby, who taught me I’m capable of things I wouldn’t have believed possible, and Matt, who supported and loved me unconditionally. iv ACKNOWLEDGMENTS I’m grateful first to Jeff Conner for his tireless dedication as an advisor. He has been an exceptional editor, an encouraging mentor, and an inspiration with his continuing enthusiasm for the unfolding questions being pursued in his lab. He always made sure I had what I needed to do the work I wanted to do, and I rarely had to wait to get requested feedback. My other three committee members have also played essential roles in my development. Doug Schemske hosted me in his lab as a new graduate student on campus, eventually supporting me in ongoing collaborations with his lab that have been some of my most productive projects. I’ve benefitted immeasurably from the time he’s spent pushing me to think harder and follow my dreams. Jen Lau offered me support and mentoring at KBS, especially since our sons were born and in the last year when Jeff was on sabbatical. Ian Dworkin also offered an important unique perspective on my research, with particular contributions to the genetic mapping section and several of the future directions that will hopefully develop in the next few years. Financial support for the work in this dissertation came from NSF grants to Jeff and Doug and MSU funds to me, particularly KBS Lauff and Porter funds. The BEACON and GK-12 programs supported me for several years and helped me achieve my goals of integrating teaching and research. Thanks to Tom Getty for involving me in both programs and being the most encouraging faculty member at KBS. Louise Mead at BEACON was also a key mentor in my education pursuits, and my students 2008-2014 have inspired and motivated me. v Jeff’s excellence as an advisor includes his ability to bring together a great group of people. I was lucky to spend much of my time at KBS with my lab-sister Raffica LaRosa (a comrade in good times and bad, research and personal life) and lab manager extraordinaire Cindy Mills, who served as a sounding board and compatriot in years of counting stamens and running gels. Other characters that have also played important parts in my time in the Conner lab include Sam Slowinski (2009 REU and coauthor on my radish work), “younger” labmates Sam Perez and Amanda Charbonneau, 2013 REU Marvin Osborne, who counted many pollen grains, and a long list of other summer undergraduates Jeff supported to help me with my work over the years. My colleagues at Kellogg Biological Station made it a fantastic place to work through their enthusiasm for scientific discussions, side projects, and all other kinds of fun. In no particular order, I’m particularly indebted to Colin Kremer, Mike Grillo, Todd Robinson, Lauren Kinsman, Emily Grman, Rachel Prunier, Idelle Cooper, Liz Schultheis, Kane Keller, Casey terHorst, and Tomomi Suwa. Amanda Posto at Indiana University was also an influential friend and collaborator. On a personal level, I want to thank my yoga teacher Karina Mirsky and the community at Sangha Yoga in Kalamazoo for getting me through grad school happier and healthier than I was when I started. Finally, I’m grateful to my family for believing in me and supporting me in so many ways. vi TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES x CHAPTER 1 1 INTRODUCTION 1 Background 1 Organization of Dissertation 2 CHAPTER 2 5 STAMEN FUNCTION IN WILD RADISH DEPENDS ON POLLINATOR VISITATION RATE 5 Introduction 5 Methods 9 Field experiment 9 Male and female fitness estimates 12 Analysis 14 Results 15 Slow release hypothesis 15 Trait specialization – long stamen attraction hypothesis 21 Discussion 23 Acknowledgements 29 CHAPTER 3 30 ONGOING LOSS OF A CONSERVED TRAIT: LACK OF FUNCTION, LATITUDINAL PATTERNS, AND GENETIC CONSTRAINTS 30 Introduction 30 Methods 32 Results 33 Discussion 39 Acknowledgements 40 CHAPTER 4 42 EARLY EVOLUTION OF SELFING IN ARABIDOPSIS LYRATA INCLUDES CHANGES IN SEX ALLOCATION BUT NOT FLOWER SIZE 42 Introduction 42 Methods 47 Field common garden 48 Greenhouse common garden 49 Floral measurements 51 Results 53 Discussion 63 vii Acknowledgements 65 CHAPTER 5 66 SUMMARY AND FUTURE DIRECTIONS 66 Summary 66 Future Directions 66 APPENDIX 69 Growth conditions for Arabidopsis thaliana 70 Experiment: function of A. thaliana short stamens in selfing 71 Geographic variation in A. thaliana short stamen production 78 QTL mapping 78 A. thaliana candidate gene search 92 LITERATURE CITED 93 viii LIST OF TABLES Table 1. Insect visitors observed visiting experimental plants. 17 Table 2. Effects of stamen treatment and pollinator visitation rate on male and female fitness. 18 Table 3. Effects of stamen treatment and overall pollinator visitation rate on visits to individual plants. 22 Table 4. Arabidopsis lyrata populations and sampling scheme 45 Table 5. Models of trait differences between populations and mating systems 54 Table 6. Models testing for increased variance in short stamen length with shift to self-pollination 60 Table 7. Accessions included in the study of geographic variation in short stamen production 70 Table 8. Testing function of short stamens 75 Table 9. Plants, lines, and flowers sampled for QTL analysis 78 Table 10. Locations and 95% credible intervals for main-effect QTL peaks 86 Table 11. Significance of main effects and interactions 87 Table 12. Number that fall within QTL for stamen loss in Arabidopsis thaliana 88 Table 13. Details for candidate genes 89 Table 14. Results of three models in R/qtl 90 ix LIST OF FIGURES Figure 1. Experimental stamen removal treatments applied to flowers. 10 Figure 2. Male fitness of 2 vs. 4-staminate treatments over varying pollinator visitation rates. 16 Figure 3. Male fitness of with different stamen treatments over varying pollinator visitation rates. 19 Figure 4. Female fitness by treatment 20 Figure 5. Square-root transformed pollinator visitation rates of different treatments over varying overall pollinator visitation 24 Figure 6. Female fitness of with different stamen treatments over varying pollinator visitation rates 26 Figure 7. Effect of stamen removal on per-flower seed set 31 Figure 8. Geographic trends in short stamen number 34 Figure 9. Main effects of QTL 36 Figure 10. Epistasis imposes evolutionary constraint on short stamen loss 37 Figure 11. Arabidopsis lyrata flower preserved in alcohol, with linear measurements marked 44 Figure 12.