ABSTRACT EFFECT OF ALLIARIA PETIOLATA INVASION ON ECTOMYCORRHIZAL COLONIZATION OF QUERCUS RUBRA By Steven Michael Castellano Exotic plant invasion may result in the disruption of symbioses between plants and soil biota, potentially affecting plant fitness and community composition. I tested whether the invasive biennial Alliaria petiolata (garlic mustard) affects abundance and community composition of ectomycorrhizal fungi (EMF) associated with experimentally planted Quercus rubra (northern red oak) seedlings. Proportional EMF colonization, EMF species richness and diversity, and Q. rubra survival were lower for seedlings in a stand with high density A. petiolata than in a stand without A. petiolata. Community composition of EMF also differed between stands. A comparison of naturally occurring Q. rubra seedlings in a moderate vs. a low density site showed a trend toward less EMF with more A. petiolata, while richness and diversity did not differ. Though confounded by uncontrolled environmental variables, these results are consistent with A. petiolata negatively impacting ectomycorrhizal fungi, warranting more research on this indirect effect on native plants. EFFECT OF ALLIARIA PETIOLATA INVASION ON ECTOMYCORRHIZAL COLONIZATION OF QUERCUS RUBRA 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 Steven Michael Castellano Miami University Oxford, Ohio 2008 Advisor ___________________________ David L. Gorchov Reader ___________________________ Nicholas P. Money Reader ___________________________ Charles Kwit Table of Contents Introduction: 1 Methods: 5 Results: 13 Discussion: 15 Conclusions: 20 Literature Cited: 22 Appendices: Appendix A. 1938 aerial photograph of Tanglewood Preserve 46 Appendix B. Tree composition for all study sites 47 Appendix C. Aerial photograph of Reinhart Preserve and soil series description 49 Appendix D. Aerial photograph of Kramer Woods and soil series description 51 Appendix E. Aerial photograph of Tanglewood Preserve and soil series description 53 Appendiz F. Estimation of Alliaria petiolata cover at Tanglewood Preserve 55 Appendix G. Soil nutrient analysis of Tanglewood and Reinhart Preserves 56 Appendix H. Fungal morphotype description and total proportional abundance for 57 three sites Appendix I. Color plate of selected ECM morphotypes 58 Appendix J. Identities of successfully sequenced ITS samples of ECM morphotypes 59 ii List of Tables Table 1. Contingency table of germination success in high and no Alliaria sites 30 Table 2. Contingency table of seedling survival in high and no Alliaria sites- 30 Harvest 1 Table 3. Contingency table of over-winter seedling survival in high and no Alliaria 31 sites- Harvest 2 Table 4. Three-way ANOVA of mycorrhizal colonization of planted seedlings 31 Table 5. Weighted one-way ANOVA of mycorrhizal colonization, comparative study 31 Table 6. Ectomycorrhizal morphotypes found on Quercus rubra seedlings from 32 temperate forest sites Table 7. Comparison of fungal richness and Shannon-Weiner index between high 33 and no Alliaria sites Table 8. Comparison of fungal richness, Shannon-Weiner index between moderate 33 and no Alliaria sites Table 9. ECM community comparison (MRPP) between high and no Alliaria sites 34 Table 10. ECM community comparison (MRPP) between moderate and no 34 Alliaria sites List of Figures Figure 1. Survival of seedlings from planting date to harvest 1 35 Figure 2. Proportional mycorrhizal colonization of planted seedlings: Harvest 1 36 Figure 3. Proportional mycorrhizal colonization of planted seedlings: Harvest 2 37 Figure 4. Proportional ECM colonization of field germinated and bare root seedlings 38 Figure 5. Proportional mycorrhizal colonization of naturally growing seedlings 39 Figure 6. Rank abundance curves of ECM morphotypes in high and no Alliaria 40 density sites- Harvest 1 Figure 7. Rank abundance curves of ECM morphotypes in high and no Alliaria 41 density sites- Harvest 2 Figure 8. Rank abundance curves of ECM morphotypes in moderate and no Alliaria 42 density sites Figure 9. NMDS ordination of ECM community in high and no Alliaria density 43 sites-Harvest 1 Figure 10. NMDS ordination of ECM community in high and no Alliaria density 44 sites-Harvest 2 Figure 11. NMDS ordination of ECM community in moderate and no Alliaria density 45 sites iii In Memory Of John W. Thieret (1926-2006) Botanist, Teacher, and Friend iv Acknowledgments To the following people I am greatly indebted for their assistance on the project I present here. My advisor, Dr. David Gorchov, Professor of Botany, Miami University, who provided much needed guidance at all stages of this project, especially during the writing process as his editing skills greatly improved the quality of this thesis. I thank my other committee members: Nicholas P. Money, Professor of Botany, Miami University, whose delicate red pen danced the tango over every page of earlier drafts of the thesis, and to Charles Kwit, Visiting Professor, Miami University, for his ecological insights and his friendship, all of which improved the quality of my project. I am grateful for all the people who helped me with my field work: to Hayley Kilroy who braved the risk of frostbite with me to measure trees, to Jim Godby and Joe Fisher of the Cincinnati Park Board who helped plant acorns on a cold December morning, to my wife Meaghan and my daughters Savannah and Isabella who not only helped in the field but also in the home. Without my family’s support, love, and tolerance of me hiding in my office for days on end while I write, this project would have been doomed from the start. I thank everyone in the “Watson” lab, especially Aaron Kennedy, Pieter Pelser, Jenise Bauman, Susannah Fulton, and Melanie Link-Perez, for their help in learning molecular techniques and for providing reassurance when my reactions proved fruitless. I thank all my fellow graduate students for providing friendship and support during some of the more difficult stages of graduate education. I thank Hank Stevens, my “R Guru” for his help with some of the fancy code needed to produce the fancy figures in this thesis. I am grateful for the two pilots that drive our department, Barb Wilson and Vickie Sandlin, who do an amazing job at the helm and without whom, our department would not function. And finally, for my strength, my courage, and my perseverance even when my energy was low, and my burden great, I thank the wise and compassionate words of Jesus Christ. Thank you. Funding for this project was provided by MU Department of Botany Academic Challenge Grant, and the Summer Field Workshop in Botany. I thank Miami University Natural Areas and the Cincinnati Park Board for allowing me to conduct my field research on their property. v Introduction: Some species that are introduced into areas outside their native range become invasive, and spread very rapidly. Invasive species are reported to be a major cause of species decline and loss of biodiversity (Wilcove et al. 1998) and result in an estimated $120 billion in environmental damages and loses in the United States each year, of which non-native plants are a major contributor (Pimental et al. 2005). Plant invasions have the potential to impact native plant communities through niche displacement, resource competition, allelopathy, alteration of nutrient cycling, hydrology, and fire regimes, and disruption of mutualistic relationships (Vitousek et al. 1997, Gordon 1998, Mack et al. 2000, Mooney and Cleland 2001, Ehrenfeld 2001, Ehrenfeld 2003, Orr et al. 2005, Stinson et al 2006) A relationship understudied in the past is that between invasive plants and their effects on below-ground mutualisms between native plants and soil microbes. Soil microbes, especially fungi, are important for decomposition and nutrient cycling ultimately controlling the availability of some nutrients to plants and thus, play an integral role in ecosystem functioning (Chapin et al.1997). Recently this important area of research has been gaining more attention, especially concerning relationships between invasive plants and beneficial soil mycorrhizal fungi (Mooney and Cleland 2001, Roberts and Anderson 2001, Reinhart and Callaway 2004, Stinson et al. 2006). The association of plants and mycorrhizal fungi is thought to be one of the most ubiquitous mutualisms on earth, occurring widely (Lilleskov et al. 2004, Peterson et al. 2004, Reinhart and Callaway 2006), involving an estimated 95 % of all plants (Smith and Read 1997) including most woodland herbs (Whigham 2004). These fungi form mutualistic relationships with plants, in which they contribute to plant acquisition of water and nutrients while utilizing the plant as a carbon source. While most land plants benefit from endomycorrhizae, colonizing interior tissues of the plant root, about 8000 (3%) seed plant species form associations with ectomycorrhizal (ECM) fungi (Taylor and Alexander 2005). ECM host plants, mostly woody perennials, including trees such as Betula (birch), Fagus (beech), Pinus (pine), and Quercus (oak), are typically dominant components of woodlands (Smith and Read 1997, Taylor and Alexander 2005). The diversity of ECM fungi is quite high and while exact species numbers are unknown, the global diversity has been estimated to be 7000-10,000 species (Taylor and Alexander 2005). Plants forming ectomycorrhizal associations benefit by having greater access 1 to mineral nutrients, increased nutrient absorption, protection from pathogens,