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Phylogenetic-Based Characterization of Microbial Eukaryote Community Structure and Diversity of an Estuary in the Salish Sea

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Phylogenetic-Based Characterization of Microbial Eukaryote Community Structure and Diversity of an Estuary in the Salish Sea Western Washington University Western CEDAR WWU Graduate School Collection WWU Graduate and Undergraduate Scholarship Spring 2017 Phylogenetic-Based Characterization of Microbial Eukaryote Community Structure and Diversity of an Estuary in the Salish Sea Taylor L. Clement Western Washington University, [email protected] Follow this and additional works at: https://cedar.wwu.edu/wwuet Part of the Biology Commons Recommended Citation Clement, Taylor L., "Phylogenetic-Based Characterization of Microbial Eukaryote Community Structure and Diversity of an Estuary in the Salish Sea" (2017). WWU Graduate School Collection. 581. https://cedar.wwu.edu/wwuet/581 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]. PHYLOGENETIC-BASED CHARACTERIZATION OF MICROBIAL EUKARYOTE COMMUNITY STRUCTURE AND DIVERSITY OF AN ESTUARY IN THE SALISH SEA By Taylor L. Clement Accepted in Partial Completion of the Degree requirements for the Degree Master of Science Kathleen L. Kitto, Dean of the Graduate School ADVISORY COMMITTEE Chair, Dr. Robin Kodner Dr. Heather Fullerton Dr. Craig Moyer Dr. Suzanne Strom 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. I acknowledge that I retain ownership rights to the copyright of this work, including but not limited to the right to use all or part of this work in future works, such as articles or books. Library users are granted permission for individual, research and non-commercial reproduction of this work for educational purposes only. Any further digital posting of this document requires specific permission from the author. Any copying or publication of this thesis for commercial purposes, or for financial gain, is not allowed without my written permission. Taylor Clement April 27, 2017 PHYLOGENETIC-BASED CHARACTERIZATION OF MICROBIAL EUKARYOTE COMMUNITY STRUCTURE AND DIVERSITY OF AN ESTUARY IN THE SALISH SEA A Thesis Presented to The Faculty of Western Washington University In Partial Fulfillment Of the Requirements for the Degree Master of Science By Taylor Clement April, 2017 ABSTRACT Coastal estuaries are highly dynamic environments. Due to a multitude of environmental variables, microbial communities in these systems are generally complex and difficult to predict. A majority of studies in estuaries have focused on relating environmental factors to changes in community response through indirect measurements such as biomass or chlorophyll. Though these studies are important for our understanding of these systems, they treat community members as a “black box” by focusing on the environmental input and biological output of the system on a broad scale. However, community composition and diversity dictates how cohabitants respond to both environmental stimuli and each other, which in turn impacts their community-level response. We utilized high throughput sequencing of the small subunit ribosomal RNA gene to phylogenetically characterize the diversity of microbial eukaryotes in a coastal estuary located in the Northeastern Salish Sea, and explore spatial and temporal patterns in community structure. Sampling was carried out daily to biweekly for four years during the summer seasons of 2013 to 2016. While diatoms and dinoflagellates were found to be the most abundant amplicon sequences in our samples, there was a diverse assemblage of less represented amplicon sequences that showed spatial and temporal patterns. We also found that our assessment of stability in this system varied based on timescale. Our results highlight the importance of repeated sampling to characterize microbial eukaryotes in dynamic environments, and the importance of including small and/or rare taxa in future ecological studies of these systems. We also discuss current challenges and limitations of this methodological approach to evaluate microbial eukaryotes. iv ACKNOWLEDGEMENTS First and foremost, my warmest thanks go out to my advisor, Dr. Robin Kodner, for her patience, support, and guidance, and for the opportunities she provided throughout the course of my thesis research. Additionally, thanks to Dr. Craig Moyer for his expertise in microbial genomics and for graciously allowing me to utilize some of his lab equipment. Thanks to Dr. Heather Fullerton for her invaluable support and wisdom during troubleshooting and lab setbacks, as well as manuscript editing. Also, thanks to Dr. Suzanne Strom, for her help in analyzing the community composition in my dataset. This thesis would not have been accomplished without the collaboration of multiple undergraduate assistants, who completed the cell counts and assisted with data collection, including: Ciara Asomato, Rachel Lekanoff, and Staci Nazario. Thank you to the “carpet scientists” of the Kodner Lab, Eric Hervol and Ryan McLaughlin, for providing endless guidance and support during my data analysis, and Kevin Hager, for helping me work through the lab protocols. Big thanks to the captains during research cruises: Nate Schwarck and Jay Dimond, Marco Hatch, and Andrés Quesada. I would also like to express my gratitude toward my family, fellow graduate students, and the faculty and staff at WWU as well as the Salish Sea Research Center at Northwest Indian College (NWIC). This work would not have been possible without the support of WWU’s Office of Research and Sponsored Programs providing a Project Development Award to Dr. Kodner and a Graduate Research Award to me in 2015. I also received generous funding from Rosemary Flora, through the Flora Scholarship and summer support through the Biology Department’s Fraser Endowment. v TABLE OF CONTENTS ABSTRACT.............................................................................................................................iv ACKNOWLEDGEMENTS.......................................................................................................v LIST OF FIGURES.................................................................................................................vii LIST OF TABLES.................................................................................................................viii INTRODUCTION.....................................................................................................................1 METHODS................................................................................................................................7 RESULTS................................................................................................................................13 DISCUSSION..........................................................................................................................33 CONCLUSION........................................................................................................................52 LITERATURE CITED............................................................................................................53 SUPPLEMENTAL FIGURES AND TABLES.......................................................................70 vi LIST OF FIGURES Figure 1: Map of Bellingham Bay………………...…..………………....……………..…..…5 Figure 2: Stacked bar graph of sequences annotated by lineage each year……………….…14 Figure 3: Heatmap of annotated sequence counts each year at each depth.……...….......…..16 Figure 4: Stacked bar graphs of highly-represented amplicon sequences………………...…17 Figure 5: Bubble plot of less-represented amplicon sequences…………..….....………....…19 Figure 6: ePCAs by year, week and depth…...........................................................................22 Figure 7: Phylogenetic trees from ePCA of less-represented amplicon sequences……….…23 Figure 8: BWPD by week and depth each year……………………………………………...25 Figure 9: Pairwise weighted Unifrac comparisons by year and depth………….....…......…..27 Figure 10: BWPD and proportions of microeukaryote sequences at different timescales…..29 Figure 11: Proportions of less represented amplicon sequences at different timescales….....31 Figure 12: Heatmaps of Intensive Sampling Week……...…………........……………..……32 Supplemental Figure S1: Surface vs CM cell count data……….………..……….........……66 Supplemental Figure S2: diatom:dinoflagellate ratios in cell counts vs sequences……...….67 Supplemental Figure S1: Rarefaction Curves of low-read samples………..………......……68 vii LIST OF TABLES Supplemental Table S1: Cruise and sample metadata………………..……..……….....……69 Supplemental Table S2: Sequence count and sequences placed by pplacer……..….....….…71 Supplemental Table S3: unknown taxa……..……………………….....……..…...........……73 Supplemental Table S4: Diatom cell counts……..………………..……...…..…...........……74 Supplemental Table S5: Dinoflagellate cell counts……..….……….....…………………….75 viii INTRODUCTION Marine microbial eukaryotes are incredibly diverse groups of
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