CHEMICALLY-MEDIATED INTERACTIONS IN SALT MARSHES: MECHANISMS THAT PLANT COMMUNITIES USE TO DETER CLOSELY ASSOCIATED HERBIVORES AND PATHOGENS A Dissertation Presented to The Academic Faculty by Robert Drew Sieg In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the School of Biology Georgia Institute of Technology May 2013 CHEMICALLY-MEDIATED INTERACTIONS IN SALT MARSHES: MECHANISMS THAT PLANT COMMUNITIES USE TO DETER CLOSELY ASSOCIATED HERBIVORES AND PATHOGENS Approved by: Dr. Julia Kubanek, Advisor Dr. Meghan Duffy School of Biology Department of Ecology and School of Chemistry and Biochemistry Evolutionary Biology Georgia Institute of Technology University of Michigan Dr. Mark Hay Dr. Jeremy Long School of Biology Department of Biology Georgia Institute of Technology San Diego State University Dr. Joseph Montoya School of Biology Georgia Institute of Technology Date Approved: February 25, 2013 To my parents, for being a source of encouragement, to my wife, for reminding me to eat, and to Harold, it’s been a blast. ACKNOWLEDGEMENTS It’s surreal to be writing an acknowledgements section for my dissertation. My first year of graduate school seems so distant, as does my progression from taking classes, to qualification exams, to two years worth of research on red tides, to developing my own project in the salt marsh. I could not have gotten to this point on my own. First, I would like to thank my parents for giving me such an encouraging environment to grow up in, and prevent me from losing focus on my long term goals. I would also like to thank my wife Tracey, who somehow agreed to have a wedding in the midst of writing this dissertation and applying for jobs. Her tolerance as I became progressively more buried in my writing and lost track of reality was key towards getting through the last year of graduate school. A never-ending supply of freshly brewed tea didn’t hurt either. I thank my advisor, Julia Kubanek, for understanding my desire to switch projects halfway through graduate school, for giving me space when I needed it, and advice when I was struggling. Furthermore, without Julia I would not have participated in the Georgia Tech REU program as a liaison between students and faculty, which gave me my first exposure to Sapelo Island, expanded my interests in undergraduate education, and ultimately helped me secure a job after graduation. I also wish to thank those faculty whom I worked with as a teaching assistant or through CETL, namely Linda Green, Marc Weissburg, Cara Gormally, Lydia Soleil, and Lin Jiang. You showed me what it means to be an effective educator, and helped me to develop my own pedagogy. Over my six years in graduate school, I’ve seen many people come and go through the Kubanek lab, but the one constant in the lab was Kelsey Poulson. We shared iv an academic brain during our first two years, and were always willing to grab a coffee to hash out experimental designs, understand backwards results, and discuss life in general. Thanks for being a pleasure to work around and for being a friend. I was lucky enough to have two wonderful undergraduates to work with on my marsh project for over two years, and I’m happy to see Kevin Wolfe and Drew Willey pursuing their academic careers beyond Georgia Tech. Thanks to Clare Redshaw, Emily Prince, Paige Stout, and Amy Lane for advice during my early years, Troy Alexander, Jessie Roy, Remington Poulin, and Margi Teasdale for countless comments as I prepared these manuscripts, and the great undergraduates that have passed through our lab. The administrative staff and faculty on Sapelo Island at the University of Georgia Marine Institute were very accommodating and knowledgeable while I conducted my field research. In particular, thanks to Gracie Townsend for day-to-day troubleshooting, Mary Price for help with facilities and permitting, and Lloyd Dunn and Steve Pennings for advice about the ecology of the island. There are so many friends and colleagues that provided me with support and laughter during my time at Tech, and in the interest of time (and space) I cannot go into the details about how you helped me individually. Nevertheless, thanks to Stu and Anna Auld, Doug and Rachel Rasher, Rachel and Jackson Potterkowski, Melanie Heckman, Tim Ellestad, Nick Goodyear, Dylan Grippi, Cat and Mark Christie, Hilary Smith, Kathryn Pigg, Seb Engel, Zach Marion, Nick Parnell, and those who I’ve forgotten. Finally, I would like to thank my committee members: Mark Hay, Joe Montoya, Meghan Duffy, and Jeremy Long. Your advice and criticism throughout the years made this dissertation possible. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES viii LIST OF FIGURES ix SUMMARY xii CHAPTER 1 INTRODUCTION 1 2 CHEMICAL ECOLOGY OF MARINE ANGIOSPERMS: STATE OF THE FIELD AND OPPORTUNITES AT THE INTERFACE OF MARINE AND TERRESTRIAL SYSTEMS 5 Abstract 5 Introduction 6 Interactions between Marine Angiosperms and Herbivores 9 Allelopathic Interactions between Marine Angiosperms 28 Interactions of Marine Angiosperms with Pathogens and Fouling Organisms 32 Settlement and Metamorphic Cues from Marine Angiosperms 40 Community and Ecosystem Effects 44 Conclusions 51 3 MULTIPLE CHEMICAL DEFENSES PRODUCED BY SPARTINA ALTERNIFLORA DETER FARMING SNAILS AND THEIR FUNGAL CROP 53 Abstract 53 Introduction 55 Methods 59 vi Results 66 Discussion 76 4 CHEMICAL DEFENSES AGAINST HERBIVORES AND FUNGI LIMIT ESTABLISHMENT OF FUNGAL FARMS ON SALT MARSH ANGIOSPERMS 84 Abstract 84 Introduction 86 Methods 89 Results 97 Discussion 107 5 CONCLUSIONS AND FUTURE DIRECTIONS 117 APPENDIX A: Variation in S. alterniflora chemical defenses among growth forms and populations 131 APPENDIX B: Cage design for induction experiment in Chapter 3 133 APPENDIX C: Spectroscopic data for structure elucidation of α-dimorphecolic acid and orientin 134 REFERENCES 146 vii LIST OF TABLES Page Table 3.1: Two-factor ANOVA from month-long field caging experiment to test potency of S. alterniflora chemical defenses after altering exposure to herbivores or fungi. 74 Table 4.1: Selected salt marsh plant traits. Expressed values represent mean percent content by dry mass ± 1 S.E for all traits. Superscript letters within a column represent significant differences among species for a given trait (one-way ANOVA with Tukey’s HSD test, P < 0.05, n = 5-11). 101 Table C.1 Comparison of carbon and hydrogen chemical shifts for α- dimorphecolic acid collected by RDS to literature values. 139 Table C.2. Comparison of carbon and hydrogen chemical shifts for orientin collected by RDS to literature values. 145 viii LIST OF FIGURES Page Figure 2.1: Secondary metabolites involved in chemically-mediated interactions between marine angiosperms and organisms in their surrounding environment. 26 Figure 3.1: Isolation of antifungal and antigrazer compounds from short form S. alterniflora. Bars represent inhibition of the fungus Mycosphaerella sp. (A-D, black) or feeding by the snail L. irrorata (A, E-G, white) relative to paired controls. Separation methods include (A) liquid-liquid partitioning, (B) silica gel column chromatography or (E) C18 silica gel column chromatography, (C, F) Sephadex LH-20 size-exclusion column chromatography, and (D, G) C18 silica HPLC. Asterisks (*) represent significant differences between treatments and paired controls (1-tail t-test, n - = 3-5 (vs. Mycosphaerella sp.), n = 16-20 (vs. L. irrorata)), while error bars represent 1 S.E. Tested concentrations relative to natural isolated yields are denoted in the upper right hand corner of each graph in square brackets. 67 Figure 3.2: Chemical defenses produced by S. alterniflora to prevent fungal farming. (A) α-dimorphecolic acid, a fatty acid that inhibits growth of the fungus Mycosphaerella sp. and (B) orientin, which reduces grazing by the snail L. irrorata in conjunction with other phenolic compounds. 68 Figure 3.3: Growth inhibition of α-dimorphecolic acid against the fungi Mycosphaerella sp. (black) and P. spartinicola (grey) when embedded in agar representing whole tissue (A) or surface (B) concentrations. Significant differences in growth between paired treatments and controls determined by 1-tail t-test (P < 0.05, n = 4) and denoted by asterisks. Bars represent 1 S.E. 70 Figure 3.4: Chemical defenses in short form S. alterniflora after cage manipulations in the field against the fungi Mycosphaerella sp. (black bars) and P. spartinicola (grey bars) or the snail L. irrorata (white bars). Caging treatments denoted on the x-axis. Extracts were added to agar at (A) natural or (B) half natural isolated concentrations. Significant differences between paired treatments and controls determined by 1-tail t-test (P < 0.05, n = 9-15) and denoted by asterisks (*, P < 0.05), (**, P < 0.01), (***, P < 0.001). 75 ix Figure 4.1: (A) L. irrorata densities relative to the abundance of five plant species among three middle elevation marshes on Sapelo Island, GA surveyed in July 2011; n = 150 quadrats site-1 to determine plant abundance, n = 100 plants site-1 surveyed to measure L. irrorata densities on each species. (B) Average L. irrorata densities across all sites; letters represent significant differences in snail densities among plant species (one-way ANOVA with Tukey’s HSD test, P < 0.01, n = 300 plants species-1). Error bars represent 1 S.E. in this and all later figures. 98 Figure 4.2: L. irrorata distributions in multi-choice mesocosm experiments measuring snail habitat preferences. Letters denote significant differences in snail distributions among plant species. Statistical differences among treatments determined by Friedman’s test with multiple comparisons (P < 0.001, n = 20). 100 Figure 4.3: Feeding preferences of L. irrorata when offered plant constituents as either (A) ground, reconstituted tissue (to eliminate structural differences, all plants offered simultaneously) or (B) as crude extracts embedded in an artificial diet of palatable ground alga (to directly test chemical defenses, each extract offered with a paired control).