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University of Florida Thesis Or Dissertation Formatting SPATIAL DISTRIBUTION OF MICROBIAL COMMUNITIES ACROSS COLONIES AND GENOTYPES IN NURSERY-REARED ACROPORA CERVICORNIS By NICOLE MILLER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2019 © 2019 Nicole Miller To my father ACKNOWLEDGMENTS First and foremost, I would like to thank Dr. Julie Meyer for her continuous guidance and support. Her invaluable mentoring has been foundational to my accomplishments as a graduate student. I would also like to thank Dr. Tom Frazer for the opportunity to pursue this graduate degree and for his contributions to ensuring a successful project. I sincerely thank my committee for providing me with the skill sets necessary to start a budding successful career. I would also like to express my gratitude to the personnel of the Central Caribbean Marine Institute of Little Cayman. I would like to thank Paul Maneval and Dr. Carrie Manfrino for providing the resources and capabilities to conduct project sampling. Finally, I would like to acknowledge the relentless support and encouragement of my family and friends. I am deeply grateful to my father for his inspiration to explore the natural world through science, as well as, my grandmother for giving me the strength to endeavor through hardships. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 6 LIST OF FIGURES .......................................................................................................... 7 ABSTRACT ..................................................................................................................... 8 CHAPTER 1 INTRODUCTION .................................................................................................... 10 2 MATERIALS AND METHODS ................................................................................ 13 Site Description ....................................................................................................... 13 Sample Collection ................................................................................................... 15 Sequencing ............................................................................................................. 16 Raw Read Quality Control ...................................................................................... 17 Analysis .................................................................................................................. 20 3 RESULTS ............................................................................................................... 21 Summary of Read Counts ....................................................................................... 21 Differentiation of Community Composition .............................................................. 24 Differential Abundance Among Communities ......................................................... 26 4 DISCUSSION ......................................................................................................... 28 Microbial Community Composition ......................................................................... 28 Community Heterogeneity ...................................................................................... 30 LIST OF REFERENCES ............................................................................................... 32 BIOGRAPHICAL SKETCH ............................................................................................ 38 5 LIST OF TABLES Table page 2-1 Sample metadata and read numbers before and after quality control. ............... 18 6 LIST OF FIGURES Figure page 2-1 Location of the ICON Reef Nursery relative to the Island of Little Cayman, Cayman Islands. ................................................................................................. 14 2-2 Photograph of the coral colonies used in this study, in situ, suspended from a PVC frame at the ICON Reef 60ft depth nursery.. .............................................. 15 2-3 Example of the three types of sample locations from a single A. cervicornis colony. ................................................................................................................ 16 3-1 Relative abundance of amplicon sequence variants of the V4 region of 16S rRNA genes in A. cervicornis nursery corals, colored by Order. ......................... 23 3-2 Principal Component Analysis of the Atchison distance among microbial communities from three coral genotypes (G, R, Y) and branch types from an A. cervicornis nursery. ........................................................................................ 24 3-3 Relative distances to the centroid across colonies, colored by genotype. .......... 25 3-4 Differential abundance of ASVs across coral genotypes in A. cervicornis. ......... 27 7 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SPATIAL DISTRIBUTION OF MICROBIAL COMMUNITIES ACROSS COLONIES AND GENOTYPES IN NURSERY-REARED ACROPORA CERVICORNIS By Nicole Miller May 2019 Chair: Julie Meyer Cochair: Tom Frazer Major: Interdisciplinary Ecology Coral reef degradation is a global concern and can be attributed to broad suite of anthropogenic stressors. Acropora cervicornis and A. palmata were historically the two primary reef building taxa of the Caribbean. Populations have declined approximately 95% since the early 1980s. These architecturally important coral species have been listed as Critically Endangered by the IUCN Red List and have been protected under the Endangered Species Act since 2006. The intention of this study is to define the microbiome associated with nursery-reared A. cervicornis. In characterizing the microbiome of A. cervicornis from a central Caribbean nursery, we were able to determine the spatial structure and heterogeneity of the microbiome within and between colonies. Samples were taken from three distinct locations on the colony (basal branch, intermediate branch, or branch tip) from each of three replicate colonies representing three unique genotypes. Bacterial and archaeal community composition were determined by sequencing 16S rRNA genes with Illumina Miseq. While Rickettsiales were the most abundant bacteria in all samples, microbiomes were significantly different 8 between coral genotypes. In addition, within colony microbiome variation was not statistically significant. These findings suggest that sampling from any one location on a host coral is likely to provide a representative microbial community for the entire colony. This study provides baseline data for future studies seeking to describe the A. cervicornis microbiome and its roles in coral health, adaptability, and resilience. 9 CHAPTER 1 INTRODUCTION Worldwide, the loss of tropical coral reefs is a growing concern because of the ecosystem services these keystone species provide. In the Caribbean, Acropora cervicornis and A. palmata were once the most abundant stony corals in the Caribbean and their branching morphologies are thought to provide “unparalleled ecosystem structure and function” (Precht et al., 2002) on Caribbean coral reefs. Ecosystem functions provided by Acropora corals include contributing to reef growth and biodiversity, island formation, and coastal buffering. Numerous efforts are being implemented to restore Acropora cervicornis populations in the wake of changing habitat and climate. Over 60 organizations in the Caribbean are working toward A. cervicornis restoration (Young et al., 2012). The development of coral gardening techniques, involving the fragmentation of corals and growth in ocean nurseries before outplanting back to reefs, has provided abundant coral biomass for restoration efforts (Lirman et al., 2014; Lirman and Schopmeyer, 2016). Since 1980, as much as 98% of native populations of Acropora corals have diminished throughout the Caribbean (Aronson and Precht, 2001).This decline has been recorded from Florida to Colombia, encompassing all of the natural range of A. cervicornis (Jackson et al., 2001; Patterson et al., 2002). Deterioration of A. cervicornis and its habitat has been linked to human disturbance factors such as overfishing and increased nutrient run-off onto reefs (Hughes, 1994). However, the most substantial losses to coral population in the Caribbean have been attributed to disease (Hughes et al., 2003; Lamb et al., 2016). The majority of A. cervicornis mortality has been due to White Band Disease (Aronson and Precht, 2001). White Band Disease is characterized 10 by the direct sloughing off of coral tissue from the skeleton (Kline and Vollmer, 2011) and has been attributed to potential bacterial pathogens identified as Vibrio (Ritchie and Smith, 1998) or Rickettsia-like organisms (Peters, Oprandy, and Yevich, 1983). Coral microbiomes have been proposed to influence host immunity and nutrient cycling as well as house potential pathogens that can cause disease (Bourne et al. 2009, Muller et al. 2012). To date, only a handful of studies have examined the microbiomes of Caribbean Acropora corals. In a study of several Caribbean coral species, including A. cervicornis, recent work has indicated that bacterial community structure of corals is consistent among coral
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