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A Biodiversity Study of High Temperature Mud Pool Microbial Communities: Implications of Regional/Geographical Isolation and Endemism

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A Biodiversity Study of High Temperature Mud Pool Microbial Communities: Implications of Regional/Geographical Isolation and Endemism A BIODIVERSITY STUDY OF HIGH TEMPERATURE MUD POOL MICROBIAL COMMUNITIES: IMPLICATIONS OF REGIONAL/GEOGRAPHICAL ISOLATION AND ENDEMISM by Benjamin R. Wheeler II A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Marine Studies Winter 2006 Copyright 2006 Benjamin R. Wheeler II All Rights Reserved UMI Number: 1432290 Copyright 2006 by Wheeler, Benjamin R., II All rights reserved. UMI Microform 1432290 Copyright 2006 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 A BIODIVERSITY STUDY OF HIGH TEMPERATURE MUD POOL MICROBIAL COMMUNITIES: IMPLICATIONS OF REGIONAL/GEOGRAPHICAL ISOLATION AND ENDEMISM by Benjamin R. Wheeler II Approved: __________________________________________________________ S. Craig Cary, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: __________________________________________________________ Nancy M. Targett, Ph.D. Dean of the Graduate College of Marine Studies Approved: __________________________________________________________ Conrado M. Gempesaw II, Ph.D. Vice Provost for Academic and International Programs ii ACKNOWLEDGMENTS The love and support of my family will always be my inspiration through everything that I do in life. Thank you Mom, Dad, Jim, and Mudder for always believing in me and for your overwhelming encouragement. A special thanks to Dr. Craig Cary for the opportunity to branch out into the realm of molecular biology and conduct my research in some of the most beautiful places on Earth. For those experiences, I will be forever grateful. Thank you to Dr. William Ullman and Dr. David Kirchman for their continued enthusiasm, support, guidance, and patience throughout my studies and work at CMS. For expert guidance and encouragement in learning the ins and outs of molecular biology, I would like to thank Dr. Kathy Coyne, Dr. Barbara Campbell, and Dr. Carol DiMeo. I would also like to thank the faculty, staff, and students of the Thermophile Research Unit, University of Waikato, New Zealand for their support and hospitality, especially Dr. Roy Daniel, Dr. Hugh Morgan, Tom Neiderberger, and Lee- Anne Rawlinson for their assistance in field sampling. Thank you to Karen Savidge for her time and patience in assisting me with nutrient analyses and Ken Voglesonger and Alf Harris for their assistance with ion, elemental, and SEM analysis. My years at CMS were blessed with the encouragement and support of a fantastic group of friends. My sincerest thanks go to Elizabeth and Sarah McCliment for their friendship and encouragement through both my graduate work and in life. Thanks to Justin Ossolinski, Tommy More, Ryan Dale, and Mia Steinberg for their friendship and countless hours of skimboarding. Thanks to Brandon Jones for his iii friendship and comic relief throughout the trying moments of proposal writing. A special thanks goes to Steve and Becky Thur, Robert Trouwborst, Lindsay Kendall, and Mike League for being great friends and fantastic housemates. Thanks to Kevin Portune for countless hours of musical entertainment and to Karen Pelletreau for hours of conversation and encouragement through late nights of lab work. I am surrounded by a fantastic group of friends in Hawaii that have been a constant source of encouragement and make Hawaii truly an island paradise. Thanks to Paul Lethaby, Susan Curless, David Nichols, Tara Clemente, Rita Steyn, Christine Tallamy, Brian Glazer, Christine Pequignet, Michael Stat, and Jamie Becker. I would also like to thank Dr. Ruth Gates and the entire Gates Lab for their patience, encouragement, and support through my thesis writing and defense preparation. Thank you to Peggy Conlon, Debbie Booth, Connie Edwards, Julie Tigue, Doris Manship, Lisa Perelli, and the entire CMS support staff for always having the time to answer a thousand questions and for making sure I was always prepared for the next step. Throughout my life I have been blessed with the greatest friends anyone could ever wish for…thank you to Brantley Cauley, Jeff Waters, Saundra Butcher, Pedro de Cardenas, Matthew Bell, Cecile Deen, Mike Deen, Becky Holyoke, Amanda Whitmire, and Kitty Fielding for your unwavering love and friendship over the years. This work was supported by the National Geographic Society and the National Science Foundation. Student support provided by the Delaware Biotechnology Institute and the Graduate Research Trainee program. iv TABLE OF CONTENTS LIST OF TABLES.........................................................................................................vii LIST OF FIGURES......................................................................................................viii ABSTRACT.................................................................................................................... x Chapter 1 - INTRODUCTION .................................................................................................. 12 Background....................................................................................................... 12 Biodiversity Studies of High Temperature Ecosystems..................................... 13 Origins of Life................................................................................................... 15 High Temperature Mud Pools........................................................................... 16 Geography and Hydrothermal Systems............................................................. 17 Molecular Methodologies for Determination of Biodiversity ............................ 19 Objectives.......................................................................................................... 22 References......................................................................................................... 25 2 - ACCESSING THE BACTERIAL COMMUNITIES OF HIGH TEMPERATURE MUD POOLS: A COMPARATIVE STUDY OF REGIONAL AND GEOGRAPHICAL ISOLATION...................................... 30 Abstract ............................................................................................................. 30 Introduction....................................................................................................... 31 Materials and Methods...................................................................................... 37 Sample collection, preparation and field measurements ............................ 37 Nucleic acid extraction optimization and recovery of extracellular DNA............................................................................................. 38 Geochemistry (New Zealand regional survey) and water content determination................................................................................ 40 Bacterial community fingerprinting analysis............................................. 40 Sequence analysis..................................................................................... 42 Results............................................................................................................... 43 Field sampling and geochemistry ............................................................. 43 Nucleic acid extraction optimization ......................................................... 46 Bacterial community fingerprinting analysis............................................. 50 Discussion......................................................................................................... 59 Conclusion ........................................................................................................ 71 References......................................................................................................... 73 3 - PHYLOGENETIC ANALYSIS OF THE MICROBIAL COMMUNITY INHABITING A HIGH TEMPERATURE MUD POOL FROM THE TAUPO VOLCANIC ZONE, NEW ZEALAND.............................................. 83 Abstract ............................................................................................................. 83 Introduction....................................................................................................... 84 Materials and Methods...................................................................................... 87 Sample collection, preparation and field measurements ............................ 87 Geochemistry ........................................................................................... 88 Water content, elemental and particle size analyses................................... 89 v Nucleic acid extraction.............................................................................. 89 Community fingerprinting analyses.......................................................... 90 Clone library construction ........................................................................ 93 Results............................................................................................................... 95 Chemical, elemental, and particle size analysis.......................................... 95 PCR-DGGE Bacterial and Archaeal community characterization............. 99 Bacterial and Archaeal community characterization of the Tokaanu mud pool .................................................................................... 102 Discussion......................................................................................................
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