EXAMINING THE LINK BETWEEN MACROPHYTE DIVERSITY, BACTERIAL DIVERSITY, AND DENITRIFICATION FUNCTION IN WETLANDS DISSERTATION Presented in Partial Fulfillment of the Requirements for The Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Janice M. Gilbert, B.E.S., B.Ed., M.E.S., M.S. ***** The Ohio State University 2004 Dissertation Committee: Professor Virginie Bouchard, Adviser Approved by Professor Serita D. Frey, Co-adviser Professor Olli H. Tuovinen Professor Frederick C. Michel, Jr. Adviser Environmental Science Graduate Program ABSTRACT The relationship between aquatic plant (macrophyte) diversity, bacterial diversity, and the biochemical reduction of nitrate (denitrification) within wetlands was examined. Denitrification occurs under anoxic conditions when nitrate is reduced to either nitrous oxide (N2O), or dinitrogen (N2). Although previous studies have identified physical and chemical factors regulating the production of either gas in wetlands, the role that macrophyte diversity plays in this process is not known. The central hypothesis, based on the niche-complimentarity mechanism, was that an increase in macrophyte diversity would lead to increased bacterial diversity, increased denitrification, and decreased N2O flux. This hypothesis was investigated in two mesocosm studies to control environmental conditions while altering macrophyte functional groups (FG) and functional group diversity. In Study #1, five macrophyte functional groups (clonal dominants, tussocks, reeds, facultative annuals, and obligate annuals) were each represented by two species. Fifty-five mesocosms with 5-6 replicates of 0, 1, 2, 3, 4, or 5 macrophyte FG (0-10 species) were established in the spring of 2001 and sampled in August 2001, September 2001, and April 2002. In Study #2, the clonal dominants were removed and forty-eight mesocosms with 6 replicates of 0, 1, 2, 3, or 4 macrophyte FG (0-8 species) were established in May 2002 and sampled in August and September 2002, and April 2003. In both studies, in situ denitrification, denitrification potential, sediment and interstitial ii water C pools, and bacterial biomass were measured. In Study #2, bacterial gDNA diversity using terminal restriction fragment length polymorphism (TRFLP) was also analyzed. Results showed no evidence of altered detrital C pools, denitrification flux, bacterial diversity or bacterial community composition due to macrophyte functional group diversity. However, distinct differences between individual macrophyte functional groups occurred. The tussocks and reeds exhibited higher denitrification function while the obligate annuals emitted significantly higher in situ N2O after nitrate addition. These results occurred despite no evidence for differences in bacterial diversity or bacterial community composition. These findings suggest that macrophyte community composition rather than diversity plays an important role in regulating denitrification and N2O emissions, and therefore has potentially important implications for a number of environmental issues pertaining to wetland mitigation, water quality, and global climate change. iii DEDICATION This dissertation is dedicated to my family who in numerous ways have made this long and important journey possible. Some are now with me only in spirit, Peter D. Fenton and Carl E. Stewart, but all have taken important turns as the wind beneath my wings. Thank you. iv ACKNOWLEDGMENTS I wish to thank my advisors Serita Frey and Virginie Bouchard for their time, guidance, encouragement, patience, and thought provoking discussions. I could not have found two more dedicated and interesting ecologists to be mentored by. You both taught me a great deal and I certainly grew under your tutelage. I also wish to thank my two committee members Olli Tuovinen and Frederick Michel who both contributed significant time, input, and encouragement which greatly enhanced this research project. I am indebted to numerous people for their technical support and expertise including Jerome Rigot, Bert Bishop, Carl Cooper, Doug Beak, Jeanne Durkalski, and Dedra Woner. Heartfelt thanks to my friends and fellow graduate students for many hours of field and laboratory help as well as contributions to the research through ideas and stimulating conversations: Dan Fink, Aaron Friend, Lisa Gardner, Tanna Holtz, Katie Hossler, Angelique Keppler, Mel Knorr, Rachel Lee, Becky Lippmann, Oor Nicomrat, Julie Pearson, Kelly Powell, Pascal Puget, Sharon Reed, Gregg Sablak, Rod Simpson, Alba Skorupa, Jamie Smialek, Sandrine Vandichele, Tonia White-Burford, the Waterman v Farm Crew: Mark, Darren, Ken and Dave, and my office mate and fellow molecular warrior, Wendy Gagliano. I would also like to thank 3 ladies in the School of Natural Resources office for their assistance, happy disposition, and encouragement throughout my time here, Mary Emmenegger, Pat Patterson, and Pat Polczynski. Many thanks to Rob McCartney and Mariana Asumendi at Kurtz Bros., Inc. for their help getting the mesocosm project started and with donations of soil. Thank you also to Andy at Savko, and Bob Webb at Columbus Irrigation for their interest in this project, great savings on supplies, and helpful information. Funding from the following sources was extremely appreciated: Environmental Science Graduate Program, School of Natural Resources, OARDC Research Enhancement Competitive Grants Program, Graduate School Research Alumni Award, and OSU Presidential Fellowship. And, last but certainly not least, I would like to thank profusely the following for their unending support, encouragement and help in ways too numerous to mention. Without you all, the completion of this dissertation would not have been possible: my fantastic family Enie, Greg, Sandy, Tom, Tanner, Nicole, Andrew, Nancy, Sam, Owen, Sharron, Allan, Shelley, Brian, Joanne, Rob,Vivian, Connie, Ruth, the Deys, and the Stewarts; my wonderful friends Lisa Mutchler, Frenchie, Dave Culver, and the ‘Lorimer Lake Gang’. vi VITA Citizenship……………………..Canadian 1993……………………………Honours B.E.S., University of Waterloo 1994……………………………B.Ed, Brock University 1997……………………………M.E.S. Environmental Studies, University of Waterloo 2000……………………………M.S. Environmental Science, The Ohio State University 1995-1997……………………..Graduate Teaching and Research Assistant, University of Waterloo 1998-present …………………..Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Gilbert, J.M., B.G. Warner, R. Aravena, J.C. Davies, and D. Brook. 1998. Mixing of floodwaters in a restored habitat wetland in Northeastern Ontario, Wetlands 9(1):106- 117. FIELDS OF STUDY Major Field: Environmental Science vii TABLE OF CONTENTS Page Abstract............................................................................................................................... ii Dedication.......................................................................................................................... iv Acknowledgments............................................................................................................. ..v Vita................................................................................................................................... vii List of Tables .......................................................................................................................x List of Figures................................................................................................................... xii Chapters: 1......Introduction.................................................................................................................1 2. Examining the relationships between macrophyte functional groups, macrophyte functional group diversity, and denitrification in wetlands ....................7 2.1 Abstract................................................................................................................7 2.2 Introduction........................................................................................................10 2.3 Methods..............................................................................................................15 2.3.1 Study site and experimental design......................................................15 2.3.2 Sediment and interstitial water C and N .............................................18 2.3.3 In situ denitrification............................................................................20 2.3.4 Denitrification potential ......................................................................22 2.3.5 Gas sample analysis .............................................................................23 2.3.6 Statistical analyses ...............................................................................23 2.4 Results................................................................................................................24 2.4.1 Macrophyte functional group comparisons ........................................24 2.4.1.1 Interstitial water and sediment characteristics .....................24 2.4.1.2 In situ denitrification............................................................27 2.4.1.3 In situ denitrification with added nitrate..............................29 2.4.1.4 Denitrification potential.......................................................30 2.4.1.5 Denitrification flux per C pools ...........................................31 2.4.2 Macrophyte functional group diversity comparisons ........................39