Western Washington University Western CEDAR WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship Fall 2016 Bacterial Diversity and Community Structure of Lithotrophically- Driven Microbial Mats from the Mariana Arc and Back-Arc Kevin W. Hager Western Washington University, [email protected] Follow this and additional works at: https://cedar.wwu.edu/wwuet Part of the Biology Commons Recommended Citation Hager, Kevin W., "Bacterial Diversity and Community Structure of Lithotrophically-Driven Microbial Mats from the Mariana Arc and Back-Arc" (2016). WWU Graduate School Collection. 547. https://cedar.wwu.edu/wwuet/547 This Masters Thesis is brought to you for free and open access by the WWU Graduate and Undergraduate Scholarship at Western CEDAR. It has been accepted for inclusion in WWU Graduate School Collection by an authorized administrator of Western CEDAR. For more information, please contact [email protected]. BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE OF LITHOTROPHICALLY-DRIVEN MICROBIAL MATS FROM THE MARIANA ARC AND BACK-ARC By Kevin W. Hager Accepted in Partial Completion of the Requirements for the Degree Master of Science Kathleen L. Kitto, Dean of the Graduate School ADVISORY COMMITTEE Chair, Dr. Craig Moyer Dr. Heather Fullerton Dr. Robin Kodner Dr. Dietmar Schwarz MASTER’S THESIS In presenting this thesis in partial fulfillment of the requirements for a master’s degree at Western Washington University, I grant to Western Washington University the non-exclusive royalty-free right to archive, reproduce, distribute, and display the thesis in any and all forms, including electronic format, via any digital library mechanisms maintained by WWU. I represent and warrant this is my original work, and does not infringe or violate any rights of others. I warrant that I have obtained written permissions from the owner of any third party copyrighted material included in these files. 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Kevin Hager December 2, 2016 BACTERIAL DIVERSITY AND COMMUNITY STRUCTURE OF LITHOTROPHICALLY-DRIVEN MICROBIAL MATS FROM THE MARIANA ARC AND BACK-ARC A Thesis Presented to The Faculty of Western Washington University In Partial Fulfillment Of the Requirements for the Degree Master of Science By Kevin Hager December 2, 2016 ABSTRACT The Mariana region exhibits a rich array of hydrothermal venting conditions in a complex geological setting, which provides a natural laboratory to study the influence of local environmental conditions on microbial community structure as well large-scale patterns in microbial biogeography. We used high-throughput amplicon sequencing of the bacterial SSU rRNA gene from 22 microbial mats collected from four hydrothermally active locations along the Mariana Arc and back-arc to explore the structure of lithotrophically-based microbial mat communities in order to better assess the hypothesis that these communities represent hotspots of microbial diversity. The vent effluent was classified as iron- or sulfur-rich corresponding with two distinct community types, dominated by either Zetaproteobacteria or Epsilonproteobacteria, respectively. The Zetaproteobacterial-based communities had the highest richness and diversity, which supports the hypothesis that Zetaproteobacteria are ecosystem engineers. The Epsilonproteobacteria-dominated mats were less abundant and split into two groups based on the prevalence of the genera Sulfurovum/Sulfurimonas or Thioreductor/Lebetimonas, which oxidize or reduce sulfur compounds, respectively. In addition, we also compare two sampling techniques, showing that higher diversity in microbial mats is associated with bulk sampling compared to fine-scale sampling. Overall, we present a comprehensive analysis and new insights into community structure and diversity of the lithotrophically-driven microbial mats from a hydrothermal region associated with high microbial biodiversity. iv ACKNOWLEDGEMENTS Many thanks go out to my advisor, Dr. Craig Moyer, for all the opportunities, insights, and support he provided during my time as a part of his research group. Also, Dr. Heather Fullerton’s expertise, encouragement, and knowledge has been invaluable while working on this project. I am grateful for the guidance provided by my committee members, Dr. Dietmar Schwarz and Dr. Robin Kodner. A special thanks goes out to my brother, Kyle Hager, for the many discussions we have had about microbiology and the use of Illumina sequencing technology. I would like to also express my gratitude toward my family, fellow graduate students, and the faculty and staff of the biology department who I’ve worked closely with over the course of my studies here at WWU. This research would not have been possible without the crew of the R/V Roger Revelle and the skilled pilots that operated ROV Jason II on the 2015 Submarine Ring of Fire cruise. I would also like to acknowledge Dr. David Butterfield for his collaboration on this project and for sharing the geochemical data he processed. The funding for this work was provided by WWU’s Office of Research and Sponsored Programs, National Science Foundation, and Truc and Jerry Thon through the Thon family scholarship for graduate studies in biology. v TABLE OF CONTENTS ABSTRACT ...........................................................................................................................iv ACKNOWLEDGEMENTS ...................................................................................................v LIST OF FIGURES ...............................................................................................................vii LIST OF TABLES .................................................................................................................viii INTRODUCTION .................................................................................................................1 METHODS ............................................................................................................................5 RESULTS ..............................................................................................................................9 DISCUSSION ........................................................................................................................14 REFERENCES ......................................................................................................................22 FIGURES AND TABLES .....................................................................................................30 SUPPLEMENTAL FIGURES AND TABLES .....................................................................43 APPENDIX ............................................................................................................................46 APPENDIX FIGURES ..........................................................................................................49 vi LIST OF FIGURES Figure 1: Bathymetric map of the Mariana and photos of microbial mats ............................30 Figure 2: Community structure dendrogram and stacked bar graphs ....................................32 Figure 3: Heatmap of abundant Zeta-, Epsilon-, and Gamma-proteobacterial OTUs ...........34 Figure 4: CCA triplot of abundant Zeta-, Epsilon-, and Gamma-proteobacterial OTUs ......36 Figure 5: Rarefaction curves of mats collected with scoops vs. biomat samplers .................38 Supplemental Figure 1: Heatmap of abundant OTUs from scoops vs. biomat samplers ......43 Appendix Figure 1: T-RFLP community structure dendrogram ...........................................49 Appendix Figure 2: T-RFLP and sequencing dendrogram comparison (tanglegram) ..........51 vii LIST OF TABLES Table 1: Sample locations and descriptions ...........................................................................40 Table 2: Geochemistry data ...................................................................................................41 Table 3: Sequencing and diversity metrics ............................................................................42 Supplemental Table 1: Top BLAST hits of unclassified Gammaproteobacteria ..................45 viii INTRODUCTION Hydrothermal vent systems allow for investigations into the fundamentals of microbial ecology and biogeography as well as planetary processes such as global nutrient and carbon cycling (Dick et al., 2013; Nakagawa and Takai, 2008; Resing et al., 2015). The geochemistry of hydrothermal vents make them compelling and pragmatic systems for the study of early life on Earth or other potentially habitable zones such as Saturn’s moon, Enceladus (Martin et al., 2008; McKay et al., 2008). The steep redox gradients and high concentration of reduced substrates (e.g., Fe(II), H2S, and H2) in hydrothermal vent habitats provide energetically favorable conditions that support luxuriant microbial mats with phylogenetically diverse lithoautotrophic microbes (Emerson and Moyer, 2010; Amend et al., 2011). Iron is the second most abundant metal in Earth’s crust (Kappler