1 AN ABSTRACT OF THE DISSERTATION OF Lu Wang for the degree of Doctor of Philosophy in Microbiology presented on September 8, 2020. Title: Effects of Anthropogenic Stressors on Seagrass and Coral Microbiomes Abstract approved: ______________________________________________________ Ryan S. Mueller Seagrasses and coral reefs play important roles in nutrient cycling, coastal protection, and maintaining marine biodiversity. However, these coastal marine organisms are declining globally due to anthropogenic stressors, such as rising ocean temperatures, ocean acidification, and eutrophication. These organisms live in close association with their microbiomes, which can be beneficial or detrimental to the host organism, depending upon environmental conditions. This body of work utilized 16S rRNA amplicon and metagenome sequencing to characterize changes in the taxonomic composition and functional potential of seagrass and coral microbiomes under varying environmental stressors. This work was done to gain fuller understanding of seagrass and coral holobiont responses to the changing oceans. Chapter 2 describes the increased prevalence of Dark Spot Syndrome (DSS) in the coral Siderastrea siderea associated with thermal stress, and changes in the microbiome associated with diseased corals, exhibiting characteristics of dysbiosis (increased compositional variation amongst diseased samples). Importantly, we found no microbial taxa associated with DSS, and concluded DSS is a general stress response and not microbe mediated. Chapter 3 presents the effects of eutrophication on Zostera marina, or eelgrass. Using a mesocosm experiment, we characterized the microbiome and plant host morphology and physiology responses to nutrient enrichment. Fertilization led to increased plant size and enriched nitrogen and sulfur cycling bacteria in root-associated samples. This study contributes both eelgrass physiology and microbiome responses to eutrophication to the breadth of seagrass literature. Chapter 4 examines changes to the leaf microbiome of the seagrass Posidonia oceanica under acidified conditions. Samples were collected from naturally occurring CO2 vents in Ischia, Italy, which simulate future ocean acidification scenarios. In acidified samples, we identified decreases in relative abundance of microbial carbon fixation genes, and enrichment for heterotrophic bacteria and genes involved in biofilm production. Seagrass transplantation is a major component of restoration efforts after population declines, and Chapter 5 examines the eelgrass rhizobiome response to transplantation. We transplanted eelgrass individuals with and without intact rhizosphere sediment and characterized plant morphology and belowground microbiome succession over 4 weeks. Eelgrass transplanted without intact rhizospheres exhibited declines in root biomass and dysbiosis at the start of the experiment, but after one week, eelgrass plants and their belowground microbiomes demonstrated resilience to transplantation in our mesocosm environment. As a whole, these studies described demonstrable changes to host-associated microbiomes under future ocean conditions. In particular, this work presents a significant contribution to the nascent field of seagrass microbiome research. These studies have identified key members of healthy and disturbed seagrass microbiomes; this knowledge can be used to generate hypotheses and future experiments targeting specific host-microbe interactions. This work shows the importance of interdisciplinary collaboration and integration of microbiome data with plant and environmental metrics to gain a full appreciation of the system. Lastly, though these valuable coastal organisms are currently on the decline, the integration of microbiome data may lead to successful management and restoration efforts. ©Copyright by Lu Wang September 8, 2020 All Rights Reserved Effects of Anthropogenic Stressors on Seagrass and Coral Microbiomes by Lu Wang A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented September 8, 2020 Commencement June 2021 Doctor of Philosophy dissertation of Lu Wang presented on September 8, 2020 APPROVED: Major Professor, representing Microbiology Head of the Department of Microbiology Dean of the Graduate School I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request. Lu Wang, Author ACKNOWLEDGEMENTS I express my deepest and most sincere gratitude to everyone who has supported and believed in me during this journey. Thank you to my committee members for their expertise, patience, and wisdom. I would particularly like to thank and acknowledge my advisor, Dr. Ryan S. Mueller, an all-star scientist and mentor, for guiding me on my scientific journey. I feel extremely lucky to have landed in the Mueller Lab. Thank you to friends near and far, and to the friends I made along the way, especially fellow microbiology graduate students with whom I could celebrate and commiserate. Special thanks to past and present members of the Mueller Lab, particularly Hanna Delgado, MK English, and Dr. Brandon Kieft. Thank you to Cinamon Moffett at HMSC, who was vital in the success of chapters 3 and 5. Thank you to Dr. James Kaldy, who passed on seagrass research wisdom. Thank you to everyone who helped me in the field – 100-pound buckets of mud are not easy to carry, and I literally could not have done it alone. I appreciate all the funding sources who took a chance on me – the Department of Microbiology at OSU (Nicholas L. Tartar Graduate Student Fellowship, Middlekauf Graduate Research Assistant Award, Joan Countryman Suit Scholarship, Teaching Assistantships), OSU Graduate School (Dissertation Completion Award), Hatfield Marine Science Center (Mamie Markham Research Award), South Slough National Estuarine Research Reserve (Margaret Davidson Fellowship FY2019 Spring Quarter), and the Oregon Shell Club. Lastly, thank you to my loved ones – my mom and dad for their support and encouragement. Thank you to Stephen Black for everything, even coming out to the field with me and inadvertently becoming an expert in seagrass epiphyte removal. And of course, thanks to my cat Magus for keeping me grounded. CONTRIBUTION OF AUTHORS Lu Wang: First author in the dissertation and chapters therein, study design and conception, performed experiments, data analysis, manuscript preparation and edits Andrew A. Shantz: Fieldwork, data analysis, manuscript preparation and edits Jérôme P. Payet: Field work, sample preparation, manuscript preparation and edits Thomas J. Sharpton: Data analysis Amelia Foster: Sample preparation Deron E. Burkepile: Study design and conception, fieldwork, data analysis, manuscript edits Rebecca Vega Thurber: Study design and conception, data analysis, manuscript preparation and edits Gema Hernán: Sample collection Jorge Terrados: Sample collection Fiona Tomas: Study design and conception, manuscript preparation and edits MK English: Performed experiments, data analysis, manuscript preparation and edits Ryan S. Mueller: Study design and conception, performed experiments, data analysis, manuscript preparation and edits TABLE OF CONTENTS Page 1 Introduction …………………………………………………………………..….….1 2 Corals and their microbiomes are differentially affected by exposure to elevated nutrients and a natural thermal anomaly ……………………………………..……...14 2.1 Abstract ………………………………………………………….….…...15 2.2 Introduction ……………………………………………………..….……16 2.3 Methods ……………………………………………………….….….…..19 2.3.1 Nutrient Enrichment Experimental Design ………….….….….19 2.3.2 Disease and Bleaching Surveys …………………….….……....20 2.3.3 Coral Mucus and Seawater Sampling ………………..………..21 2.3.4 Microbial Metagenome Library Generation and Sequencing ....22 2.3.5 Bioinformatic Analyses of Metagenomic Data ………….….…23 2.3.6 Statistical Analyses for Environmental and Metagenomic Data ………………………………………………………………..………26 2.4 Results …………………………………………………………..……….28 2.4.1 Time and Treatment Variably Affect Agaricia sp. Bleaching ...28 2.4.2 Thermal Stress Associated with Dark Spot Syndrome in Siderastrea siderea …………………………………………,……….30 2.4.3 Siderastrea siderea Microbiome Community Structure Shifts ...32 2.4.4 Coral-Associated Viral Consortia Shift During Thermal Stress ………………………………………………………………….…….32 2.4.5 Coral-Associated Archaea Shift in Abundance During Warming ………………………………………………………………..………33 2.4.6 Coral-Associated Fungi Shift Across the Thermal Stress Event ……………………………………………………………….……….34 2.4.7 Indicator Species of Healthy Coral Microbiomes …….……….34 TABLE OF CONTENTS (Continued) Page 2.4.8 Coral Microbiome Function is Altered During Thermal Stress ………………………………………………………….…………….34 2.4.9 Thermal Stress Shifts the Microbiomes of DSS Afflicted Corals ………………………………………………………….…………….36 2.5 Discussion ……………………………………………….……………….37 2.5.1 Nutrient Exposure May Prolong Temperature-Mediated Bleaching in Agaricia Corals …………………………………….….37 2.5.2 Coral Disease Linked to Thermal Stress ………………………38 2.5.3 Siderastrea siderea Disease and Microbial Diversity ………….38 2.5.4 Thermal Anomaly Associated with Taxonomic Functional Microbiome Shifts …………………………………………………..40 2.6 Conclusions ……………………………………………………………...44 2.7 Experimental Design Considerations and Future Work ………………...44 2.8 Author Contributions ……………………………………………………46 2.9 Acknowledgments ……………………………………………………….46 2.10 Tables …………………………………………………………………..47