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Characterization of the Marine Sponge Amphimedon Compressa Microbiome Across a Spatial Gradient Renee Michelle Potens Nova Southeastern University, [email protected]

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Characterization of the Marine Sponge Amphimedon Compressa Microbiome Across a Spatial Gradient Renee Michelle Potens Nova Southeastern University, Rp641@Nova.Edu Nova Southeastern University NSUWorks HCNSO Student Theses and Dissertations HCNSO Student Work 5-20-2016 Characterization of the Marine Sponge Amphimedon compressa Microbiome Across a Spatial Gradient Renee Michelle Potens Nova Southeastern University, [email protected] Follow this and additional works at: https://nsuworks.nova.edu/occ_stuetd Part of the Biodiversity Commons, Environmental Microbiology and Microbial Ecology Commons, Genetics Commons, Laboratory and Basic Science Research Commons, Marine Biology Commons, Molecular Genetics Commons, and the Oceanography and Atmospheric Sciences and Meteorology Commons Share Feedback About This Item NSUWorks Citation Renee Michelle Potens. 2016. Characterization of the Marine Sponge Amphimedon compressa Microbiome Across a Spatial Gradient. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, . (413) https://nsuworks.nova.edu/occ_stuetd/413. This Thesis is brought to you by the HCNSO Student Work at NSUWorks. It has been accepted for inclusion in HCNSO Student Theses and Dissertations by an authorized administrator of NSUWorks. For more information, please contact [email protected]. NOVA SOUTHEASTERN UNIVERSITY HALMOS COLLEGE OF NATURAL SCIENCE AND OCEANOGRAPHY Characterization of the Marine Sponge Amphimedon compressa Microbiome Across a Spatial Gradient By Renee Michelle Potens Submitted to the Faculty of Nova Southeastern University Halmos College of Natural Science and Oceanography in partial fulfillment of the requirements for the degree of Master of Science with a specialty in: Biological Science Nova Southeastern University April 2016 1 Thesis of RENEE MICHELLE POTENS Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science: Biological Science Nova Southeastern University Oceanographic Center Halmos College of Natural Science and Oceanography April 2016 Approved: Thesis Committee Major Professor :______________________________ Jose Lopez, Ph.D. Committee Member :___________________________ Robert Smith, Ph.D. Committee Member :___________________________ George Duncan, Ph.D. i TABLE OF CONTENTS 1.0 ACKNOWLEGMENTS .......................................................................... iv 2.0 ABSTRACT ................................................................................................v 3.0 LISTS.........................................................................................................vi 3.1 List of Figures ...................................................................................... vi 3.2 List of Tables ....................................................................................... ix 3.3 List of Abbreviations .............................................................................x 4.0 INTRODUCTION......................................................................................1 4.1 Significance of Sponges .........................................................................1 4.2 High-Throughput Sequencing ................................................................5 4.3 16S rRNA...............................................................................................8 4.4 Metagenomics and Microbiomes ...........................................................9 4.5 Microbial Symbionts ............................................................................13 4.6 Amphimedon compressa Spicule Taxonomy .......................................15 4.7 Amphimedon compressa Duchassaing & Michelotti, 1864 .................16 5.0 OBJECTIVES AND HYPOTHESES ....................................................18 5.1 Objectives ............................................................................................19 5.2 Hypotheses ...........................................................................................19 6.0 MATERIALS AND METHODS ............................................................20 6.1 Sponge Sample Collection ...................................................................20 6.2 Amphimedon compressa Spicule Taxonomy .......................................22 6.2.1 Individual Spicule Taxonomy ........................................................22 6.2.2 Pseudoskelton Preparation .............................................................22 6.2.3 Microscopy ....................................................................................23 6.3 Microbial DNA Extraction/Isolation ...................................................23 6.4 Illumina High-Throughput Metagenomic Sequencing ........................24 6.5 Earth Microbiome Project Acquisition of Microbiome Data .............24 6.6 Data Analysis .......................................................................................26 6.6.1 Metadata .........................................................................................26 6.6.2 Analytical Platform ........................................................................27 6.6.3 Analysis Categories .......................................................................28 6.6.4 Analysis of Biotic Data ..................................................................29 6.6.5 Operational Taxonomic Units ........................................................29 6.6.6 Rarefaction Analysis ......................................................................30 6.6.7 ANOVA for OTU Richness ...........................................................31 6.6.8 Alpha Diversity ..............................................................................31 6.6.9 Inverse Simpson .............................................................................32 6.6.10 Tukey for Multiple Comparisons of Means ...................................32 6.6.11 Beta Diversity ................................................................................33 6.6.12 Bray-Curtis Dissimilarity ...............................................................33 6.6.13 Non-metric Multidimensional Scaling ...........................................34 6.6.14 Heatmap .........................................................................................34 6.6.15 Simper Similarities.........................................................................34 6.6.16 Analysis of Abiotic Factors ...........................................................35 7.0 RESULTS .................................................................................................36 ii 7.1 Sponge Taxonomy by Spicule Analysis ..............................................36 7.2 Data Analysis with R ...........................................................................38 7.2.1 Triple Site Analysis........................................................................38 7.2.1.1 Operational Taxonomic Units ..................................................38 7.2.1.2 Rarefaction Analysis ................................................................38 7.2.1.3 OTU Richness ..........................................................................40 7.2.1.4 Alpha Diversity - Inverse Simpson ..........................................45 7.2.1.5 Beta Diversity ..........................................................................50 7.2.1.5.1 Bray-Curtis Dissimilarity ...................................................50 7.2.1.5.2 ADONIS ............................................................................51 7.2.1.5.3 Non-metric Multidimensional Scaling ...............................51 7.2.1.5.4 Heatmap .............................................................................53 7.2.1.5.5 Simper Similarity Percentages ...........................................56 7.2.1.6 BLAST Taxonomic Investigation ............................................60 7.2.2 Dual Site Analysis, Pairwise ..........................................................63 7.2.2.1 OTU Richness ..........................................................................64 7.2.2.2 Alpha Diversity - Inverse Simpson ..........................................66 7.2.2.3 Beta Diversity ..........................................................................74 7.2.2.3.1 Bray-Curtis Dissimilarity ...................................................74 7.2.2.3.2 Pair-wise NMDS ................................................................79 7.2.3 Single Site Analysis; Broward County, Florida, USA .............80 7.2.3.1 OTU Richness ....................................................................83 7.2.3.2 Alpha Diversity – Inverse Simpson ...................................84 7.2.3.3 Beta Diversity ....................................................................85 7.2.3.3.1 Bray-Curtis Dissimilarity .............................................85 7.2.3.3.2 Non-metric Multidimensional Scaling .........................86 8.0 DISCUSSION ...........................................................................................88 9.0 CONCLUSIONS ......................................................................................96 10.0 APPENDICES ..........................................................................................98 11.0 SUPPLIMENTARIES ...........................................................................121 12.0 REFERENCES .......................................................................................126 iii 1.0 ACKNOWLEDGEMENTS Thanks to my major professor Joe Lopez who first worked with me as an undergraduate and then took me on as his graduate student. Completion of this thesis was possible through his guidance and mentorship. My other
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