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CHARACTERIZING MARINE SUBSURFACE FUNGI from OLIGOTROPHIC SOUTH PACIFIC GYRE SEDIMENTS a Thesis by MORGAN STARR SOBOL BS, Texas A

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CHARACTERIZING MARINE SUBSURFACE FUNGI from OLIGOTROPHIC SOUTH PACIFIC GYRE SEDIMENTS a Thesis by MORGAN STARR SOBOL BS, Texas A CHARACTERIZING MARINE SUBSURFACE FUNGI FROM OLIGOTROPHIC SOUTH PACIFIC GYRE SEDIMENTS A Thesis by MORGAN STARR SOBOL BS, Texas A&M University-Corpus Christi, 2016 Submitted in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE in MARINE BIOLOGY Texas A&M University-Corpus Christi Corpus Christi, Texas August 2018 © Morgan Starr Sobol All Rights Reserved August 2018 CHARACTERIZING MARINE SUBSURFACE FUNGI FROM OLIGOTROPHIC SOUTH PACIFIC GYRE SEDIMENTS A Thesis by MORGAN STARR SOBOL This thesis meets the standards for scope and quality of Texas A&M University-Corpus Christi and is hereby approved. Brandi Kiel Reese, PhD Jeffrey W. Turner, PhD Chair Committee Member Xavier Fonz Gonzales, PhD Committee Member August 2018 ABSTRACT Fungal communities from the deep marine subsurface may be important in global biogeochemical cycles through remineralization of sedimentary organic matter, but this has not yet been thoroughly observed. This study analyzes the fungal role in subsurface biogeochemical cycles and understands how these organisms have adapted to extreme environments, such as the nutrient and organic matter depleted sediments of the South Pacific Gyre. Sediment cores were collected during the Integrated Ocean Drilling Program Expedition 329 to the South Pacific Gyre on board the D/V JOIDES Resolution in the Fall of 2010. Two fungal isolates were cultured from 70 million year old sediments. Previous analysis found that the two isolates were closely related to Penicillium species. To fully characterize the isolates and test their physiological boundaries, we grew them at different temperatures, salinities and pH. Whole genomic analysis was used to understand the fungi’s physiology and metabolism on a molecular level. The fungi were found to prefer growth at mesophilic temperatures and low NaCl concentrations. Growth occurred between pH 3 and pH 8. The isolate from 12 mbsf grew optimally from pH 3 to pH 8 and the isolate from 124 mbsf grew optimally from pH 3 to pH 6. Fermentation of lactose and sucrose was confirmed, but not nitrate and sulfate reduction. The fungal isolates from the South Pacific Gyre sediment had physiological capabilities that were consistent with the in situ subsurface conditions and contained genes that were capable of utilizing the recalcitrant carbon sources found in situ. The results from this study expand on the fungal limits of life and highlight their important role global carbon cycle. v DEDICATION I would like to dedicate this work to everyone who has encouraged and supported my one dream to become a marine biologist. vi ACKNOWLEDGEMENTS First, I would like to thank to my advisor, Dr. Brandi Kiel Reese, for her immense support and guidance during my three years as her student. She introduced me to the small, close knit community of marine subsurface scientists and helped fueled my love for studying life in extreme environments. I am also grateful for the opportunities she has provided me with and for the future collaborations that will forever support my career as a scientist. To Dr. Jeffrey Turner, Dr. Xavier Gonzales, Dr. Blair Sterba-Boatwright, and other Texas A&M University – Corpus Christi professors, thank you for your support, mentorship, and kind use of equipment and supplies. Many thanks to my lab mates in both the Reese and Turner labs for assisting me with my research both in the lab and on the command line, for constantly reminding me of things I had forgotten, but most importantly, for your friendship. Thank you to Dr. Fumio Inagaki and Tatsuhiko Hoshino for providing the DNA extraction method and sequencing for the fungal genomes and to Dr. Martha Ariza and Dr. Heath Mills for the early analyses of the fungal isolates. Also, thank you to the science party and crew of IODP Expedition 329 for sample collection and geochemical analysis. I would like to thank the College of Science and Engineering for the graduate student scholarship which supported my first year of graduate school and the Ruth A. Campbell Endowed scholarship for supporting my second year. I also appreciate the travel awards from the Parent’s Council and the Marine Biology Program at Texas A&M University – Corpus Christi. To my funding sources, the National Science Foundation, the NASA Astrobiology Institute, the Center for Dark Energy Biosphere Investigations, and the Consortium of Ocean Leadership, thank you very much for your support. vii Lastly, I would like to give special thanks to closest family and friends. Thank you, mom and dad for originally inspiring my love for the ocean and never letting me forget my true passion. I want to thank my sisters, other family, and friends for their unconditional love and support. Finally, I am greatly appreciative of Taylor for his continued understanding and moral support during this journey. Without you all, reaching my goal of becoming a marine biologist would not have been possible. Thank you! viii TABLE OF CONTENTS CONTENTS PAGE ABSTRACT .................................................................................................................................... v DEDICATION ............................................................................................................................... vi ACKNOWLEDGEMENTS .......................................................................................................... vii TABLE OF CONTENTS ............................................................................................................... ix LIST OF FIGURES ..................................................................................................................... xiii LIST OF TABLES ....................................................................................................................... xiv INTRODUCTION ........................................................................................................................ xv Discovery of Fungi in Marine Subsurface ............................................................................... xvi Energy Limitation .................................................................................................................... xvi Role of Fungi in Carbon Cycle .............................................................................................. xviii Ecological Importance ........................................................................................................... xxiii Summary ................................................................................................................................ xxiv CHAPTER I: ECOPHYSIOLOGY OF SOUTH PACIFIC GYRE FUNGI .................................. 1 Abstract ....................................................................................................................................... 1 Introduction ................................................................................................................................. 2 Materials and Methods ................................................................................................................ 3 Results ......................................................................................................................................... 9 Discussion ................................................................................................................................. 18 ix Summary ................................................................................................................................... 26 CHAPTER II: MOLECULAR CHARACTERIZATION OF FUNGI IN THE OLIGOTROPHIC MARINE SUBSURFACE ............................................................................................................ 27 Abstract ..................................................................................................................................... 27 Introduction ............................................................................................................................... 28 Materials and Methods .............................................................................................................. 30 Results ....................................................................................................................................... 34 Discussion ................................................................................................................................. 43 Summary ................................................................................................................................... 50 REFERENCES ............................................................................................................................. 51 LIST OF APPENDICES ............................................................................................................... 68 Table A.1. NCBI accession numbers used. ............................................................................... 68 Table A.2. SPG-F1 media control pH and weights for 4ºC. ..................................................... 70 Table A.3. SPG-F1 culture pH and weights for 4ºC. ................................................................ 71 Table A.4. SPG-F15 media control pH and weights 4ºC. ........................................................ 72 Table A.5. SPG-F15 culture pH and weights for 4ºC. .............................................................. 73 Table A.6. Media control pH and weights for 10ºC. ...............................................................
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