© 2019 Molly R. Bindell ALL RIGHTS RESERVED
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© 2019 Molly R. Bindell ALL RIGHTS RESERVED DIVERSITY OF ARBUSCULAR MYCORRHIZAL FUNGI ASSOCIATED WITH SWITCHGRASS IN THE NEW JERSEY PINE BARRENS ECOSYSTEM By MOLLY R. BINDELL A dissertation submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Ecology and Evolution Written under the direction of Ning Zhang And approved by _____________________________________ _____________________________________ _____________________________________ _____________________________________ New Brunswick, New Jersey January, 2019 ABSTRACT OF THE DISSERTATION DIVERSITY OF ARBUSCULAR MYCORRHIZAL FUNGI ASSOCIATED WITH SWITCHGRASS IN THE NEW JERSEY PINE BARRENS ECOSYSTEM by MOLLY R. BINDELL Dissertation Director: Dr. Ning Zhang Arbuscular mycorrhizal fungi (AM fungi) are a group of mutualistic, root- colonizing, microorganisms that have enormous importance in ecosystem functioning, agricultural production, and habitat conservation. Despite their relatively small taxonomic diversity, they associate with over 80% of terrestrial plants. AM fungal species are often described through morphological differences, such as, spore wall features. However, next-generation sequencing (NGS) has become an increasingly popular method to expand our understanding of AM fungal global diversity patterns. The drivers of AM fungal diversity patterns remain poorly understood. This dissertation, therefore, combines Illumina MiSeq sequencing and microscopic observations to uncover the AM fungal communities inhabiting switchgrass roots in the Pine Barrens ecosystem, and compare those communities with those from agroecosystems. The AM fungal communities of the rare and unique Pine Barrens ecosystem were previously unknown. Illumina sequencing results uncovered several clades unique to the Pine Barrens ii ecosystem, as well as, clades unique to agricultural fields, with many potentially novel species. This dissertation also developed a fully annotated, bioinformatic workflow for the study of AM fungal diversity via Illumina sequencing. Developing this AM fungal Illumina workflow showed that certain bioinformatic decisions can alter downstream AM fungal diversity results dramatically. Reference database selection was found to be a key decision in the workflow process. A pot experiment was also conducted in order to explore whether or not the acidic soils of the Pine Barrens influence the extent of switchgrass root colonization by and diversity of AM fungi. Native Pine Barrens AM fungi and soil were either left alone or amended with calcium carbonate (lime) in order to test whether soil pH plays a role in shaping the AM fungal communities, as previous studies have shown mixed results. Although the experiment didn’t yield much significant data, it found that plants grown without AM fungi and those with acidic soil grew bigger than those with AM fungi and those with more neutral soil. Better control over inoculation procedures and abiotic factors may shed more light on these findings. While this dissertation research helps us better understand AM fungal global diversity patterns, lingering questions remain on how these fungi function in different environments, why species exist in certain places, and how these fungi can be realistically implemented in sustainable agricultural practices. iii Acknowledgements First and foremost, I must acknowledge my outstanding dissertation advisor, Dr. Ning Zhang. Not only did she have unwavering faith in my ability to work hard and succeed, but she gave me excellent research advice and guidance whenever it was needed. She afforded me the ability to make my own mistakes at times, in order to learn from them, and allowed me to use my creativity when designing experiments, all while sharing helpful tips along the way. I thank her for being supremely patient with me and for always believing in me. I would also like to thank my dissertation committee, Dr. James White, Dr. John Dighton, and Dr. Thomas Gianfagna, for giving me much necessary feedback on my experimental design and approaches. These extra perspectives were very much needed to successfully complete my research. Second, I must thank my fellow laboratory members. In particular, Emily Walsh and Dr. Jing Luo were both extraordinarily helpful and instrumental in helping me figure out how to get through these last 7+ years of research and writing. I could not have done it without their insights, direction, and words of encouragement. Third, I would like to thank all my sources of financial support throughout my time at Rutgers. Those include: Rutgers University awards, the National Military Family Association awards, Garden Club of America award, my GA support from Dr. Zhang, and my TA support from Rutgers University. In particular, I am very grateful to have had the opportunity to teach as a TA while a graduate student. I must thank Dr. Gregg Transue for giving me 5 years of amazing teaching and leadership opportunities within General Biology. Teaching was not what I anticipated it to be. I was surprised to discover that I could learn a thing or two from my students each semester. I was equally iv surprised to find that I loved teaching Biology. I hope I have the chance to dive into teaching more in the days and years to come. Last, I wish to give a massive thank you to all of my family and friends who have helped support me through these graduate school years. In particular, my parents, Anna and Alvin Silverstein, provided me with infinite babysitting help for my three kids, as well as, lots of love and emotional support along the way. My dad especially helped take care of my baby, Oren, in the last few months of my dissertation writing, making it possible for me to complete the writing phase. My in-laws, Paul and Laura Bindell, also helped care for my children when I needed to get some research or writing done. A special thank you, from the bottom of my heart, to my dedicated and patient husband, Yaakov (Jack). I could not have completed this huge undertaking without Jack’s constant support. He guided me through the toughest times even when I couldn’t see the light at the end of the tunnel. I certainly cannot thank everyone enough for all that they have done to guide me through this challenging obstacle course called graduate school. v DEDICATION This dissertation is dedicated to my precious children, Eliyahu, Chaim, and Oren, who, unbeknownst to them, put up with me and my late nights working on research, teaching, and writing for the last 7+ years. I love you boys to the moon and back! vi TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION............................................................................ii ACKNOWLEDGEMENTS................................................................................................iv DEDICATION....................................................................................................................vi TABLE OF CONTENTS..................................................................................................vii LIST OF TABLES...............................................................................................................x LIST OF FIGURES...........................................................................................................xii Chapter 1: Introduction........................................................................................................1 Arbuscular mycorrhizal fungi overview..................................................................2 Abiotic effects on arbuscular mycorrhizae..............................................................8 Host specificity of arbuscular mycorrhizae...........................................................13 Methods in AM fungal biodiversity and relevance of metagenomics...................14 Significance of switchgrass....................................................................................19 The Pine Barrens ecosystem..................................................................................23 Conclusions, main objectives, significance of study.............................................26 Chapter 2: Arbuscular mycorrhizal fungal communities associated with switchgrass roots are shaped by soil properties and land use.........................................................................31 Abstract..................................................................................................................31 vii Introduction............................................................................................................32 Materials and Methods...........................................................................................37 Results....................................................................................................................45 Discussion..............................................................................................................50 Chapter 3: Impacts of bioinformatic workflow decisions on the results of arbuscular mycorrhizal fungi diversity and community composition: an ‘Illumina’ting study..........66 Abstract..................................................................................................................66 Introduction............................................................................................................68 Materials and Methods...........................................................................................72