1 RICE UNIVERSITY Mechanisms underlying the costs and benefits in grass-fungal endophyte symbioses by Andrew James Davitt A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE Master of Arts APPROVED, THESIS COMMITTEE: lefinji£r A. Rudgers distant Professor & Committee Chair Ecology and Evolutionary Biology Evan Siemann Professor Ecology and Evolutionary Biology VolkerH.WTRudolf Assistant Professor Ecology and Evolutionary Biology HOUSTON, TEXAS APRIL, 2010 UMI Number: 1486071 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. UMT Dissertation Publishing UMI 1486071 Copyright 2010 by ProQuest LLC. All rights reserved. This edition of the work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Mechanisms underlying the costs and benefits in grass-fungal endophyte symbioses by Andrew James Davitt Nearly all plants have developed symbiotic associations with microbes above- and belowground. These symbionts often alter the ecology of their hosts by enhancing nutrient uptake, increasing stress tolerance, or providing protection from host enemies. Understanding the dynamics of symbiosis requires testing how ecological factors alter not only the fitness consequences of the symbiosis, but also the rate of symbiont transmission. Here we asked how changes in the biotic and abiotic context alter both the costs and benefits of interactions between grass hosts and symbiotic fungal endophytes and rates of symbiont transmission. First, we assessed how shade and the presence of endophyte symbiosis affected host plant growth across six grass species. Our results demonstrate a novel benefit of endophyte symbiosis via the amelioration of shade stress. Second, we examine how interactions between a fungal endophyte and its grass host change along a gradient of water availability and in the presence versus absence of soil microbes. We show that benefits of the symbiosis were strongest when water was limiting. Together, our results highlight the context dependent nature of grass endophyte symbioses. ACKNOWLEDGEMENTS First and foremost, I would like to thank Jennifer Rudgers for her unwavering support, wisdom, and guidance. I also extend my gratitude to my committee members Evan Siemann and Volker Rudolf for numerous discussions, comments, and advice. I would like to thank Ken Whitney, Sami Hammer, Kerri Crawford, Liz Seifert, Loren Albert, Chris Chen, Robert Jakubowski, and Marcus Stansberry for field and laboratory assistance; my lab mates and friends Jeff Ahern, Amy Savage, and Scott Chamberlain for creating a friendly working environment; Laura Johnson, who was very helpful in many regards; Kelly Lyons for inspiring my love of ecology; and of course, my family for their unconditional love and emotional support. Last, but definitely not least, I thank Kerri Crawford for her love, friendship, and encouragement throughout the process. TABLE OF CONTENTS 1. List of tables v 2. List of figures vi 3. List of appendices vii 4. Chapter 1: Do the costs and benefits of fungal endophyte symbiosis vary with light availability? 1 5. Chapter 2: Understanding context dependency in plant microbe symbioses: the influence of abiotic and biotic contexts on host fitness and the rate of symbiont transmission 44 LIST OF TABLES Table 1.1. List of grass and endophyte species used for the study, including information on original source population location, natural endophyte frequencies, treatment for endophyte removal, and the range of light habitats in which they occurred. 39 Table 1.2. Statistical results from ANOVA analyzing effects of endophyte treatment, shade treatment and species on total biomass, root:shoot ratio, SLA, and endophyte density. 40 Table 2.1. Statistical results from M/ANOVA examining the effects of the endophyte treatment, water treatment, and soil treatment on plant reproductive responses: days to flowering, the number of inflorescences, inflorescences per tiller, seed weight, and reproductive effort. 77 Table 2.2. Statistical results from M/ANOVA examining the effects of the endophyte treatment, water treatment, and soil treatment on plant growth responses: total biomass, belowground biomass, aboveground biomass, root:shoot ratio, and the final number of tillers. 78 LIST OF FIGURES Figure 1.1. Differences in the frequency of endophyte symbiosis for grass species from shaded habitats vs. not restricted to shaded habitats. 35 Figure 1.2. Effects of the shade treatment on total biomass across all species (a) and effect of the shade and endophyte treatment in P. alsodes (POAL) (b). 36 Figure 1.3. Effects of the shade and endophyte treatment on the number of inflorescences in A. perennans (AGPE). 36 Figure 1.4. Effects of the shade and endophyte treatment on specific leaf area. 37 Figure 1.5. Effects of the shade treatment on root:shoot ratio across all species (a) and effects of the endophyte treatment on species grouped by habitat type (b). 38 Figure 1.6. Effects of the shade treatment on endophyte density. 38 Figure 2.1. Effects of the fungal endophyte and water availability on days to flowering in Agrostis hyemalis. 12» Figure 2.2. Effects of the fungal endophyte and water availability on plant reproductive responses. 74 Figure 2.3. Effects of the fungal endophyte and soil type (live or sterile) on total plant biomass. 75 LIST OF APPENDICES Appendix 1. Phylogenetic trees used for examining relationships between endophyte frequency and habitat type. 41 1 CHAPTER 1: DO THE COSTS AND BENEFITS OF FUNGAL ENDOPHYTE SYMBIOSIS VARY WITH LIGHT AVAILABILITY? 2 Summary We examined whether fungal endophytes modulated host plant responses to light availability, a potentially novel benefit of endophyte symbiosis. To look for general patterns across host plant species, we conducted a review of the literature to evaluate whether natural frequencies of endophyte symbiosis in grass species from shaded habitats were higher than frequencies in grass species spanning more diverse habitats. Then, in a greenhouse experiment, we assessed how four levels of shade and the presence of endophyte symbioses affected host plant growth across six grass species, and we compared species restricted to shaded environments to species that occupied a broader range of light environments. In our literature survey, endophytes were more common in grasses from shaded habitats than those that were not. In the greenhouse, we found large benefits of endophyte symbiosis for one species {Poa abodes), and strong negative effects of shade for all but one species (Festuca subverticillata). However, only one species showed evidence of an endophyte-mediated growth response to shade. In Agrostis perennans symbiotic plants produced 22% fewer inflorescences than non-symbiotic plants at the lowest level of shade, but made 53% more inflorescences at the highest level of shade, showing greater benefits of symbiosis under light limitation. Some species exhibited endophyte-mediated allocation to traits in response to shade. Under high shade, symbiotic Poa autumnalis invested in greater specific leaf area than symbiont-free plants, but this effect did not occur under high light. Furthermore, in grass species from broad habitat ranges, symbiosis increased the rootrshoot ratio, whereas species from shade-restricted habitats 3 showed no symbiont-mediated response. Finally, shade increased endophyte density in host leaf tissues across all species tested. Our results demonstrate a novel benefit of endophyte symbiosis via the amelioration of shade stress for one grass host and highlight the potential for symbiosis to alter the plasticity of hosts. Keywords: mutualism, context-dependency, phylogenetically independent contrasts, Epichloe, Neotyphodium, tall fescue 4 Introduction Nearly all plants have developed symbiotic associations with endophytic or mycorrhizal fungi (Petrini, 1986). The fossil record suggests that some of these interactions are older than 400 million years (Redecker et al, 2000), suggesting that fungal associations have played a long and important role in the evolution of life on land. These symbionts have been important in plant evolution because they alter the ecology of their hosts, often by enhancing nutrient uptake, increasing stress tolerance, or providing protection from host enemies (Clay & Schardl, 2002; Smith & Read, 1997; Hartley & Gange, 2009). Furthermore, both mycorrhizal fungi, which occur belowground, and aboveground fungal endophytes can have strong impacts on community composition (Clay & Holah, 1999; Hartnett & Wilson, 1999), succession (Janos, 1980; Rudgers et al, 2007), and nutrient cycling (Franzluebbers et al, 1999; van der Heijden et al, 2008). Given the ecological and evolutionary importance of plant-fungal symbioses, there is great interest in understanding mechanisms through which these symbioses are maintained at high frequencies in plant populations (Rudgers et al, 2009). Isolating these mechanisms necessitates identifying the ecological factors that generate variation in the relative costs and benefits of symbiosis. Positive, negative and neutral effects of symbionts on plant fitness are expected to arise from variation in the relative costs and benefits