MICROBIAL DIVERSITY AND EVOLUTION IN GULF OF MEXICO HYPERSALINE ENVIRONMENTS Lisa Marie Nigro A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in particial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Marine Sciences. Chapel Hill 2015 Approved by: Andreas Teske Samantha Joye Barbara MacGregor Carol Arnosti Brian White © 2015 Lisa Marie Nigro ALL RIGHTS RESERVED ii ABSTRACT Lisa Marie Nigro: MICROBIAL DIVERSITY AND EVOLUTION IN GULF OF MEXICO HYPERSALINE ENVIRONMENTS (Under the direction of Andreas Teske) My thesis investigated the microbial diversity and taxonomic composition of hypersaline and non-extreme sediment environments in the Gulf of Mexico to explore environmental controls on microbial community structure and function. Environmental conditions and resource availability are considered key drivers of microbial diversity and evolution. Extreme environments are traditionally thought to select for more specialized individuals and lower overall diversity, while non-extreme environments foster diverse communities of generalists that are capable of occupying a wider niche. However, extreme environments can also be heterogeneous and complex systems, with a wide variety of energetic resources, providing both challenges and opportunities for diversification. The sediment microbiomes of this study represented natural hydrocarbon seeps, a deep- sea hypersaline anoxic basin (Orca Basin, 26% salinity), Deepwater Horizon (DWH) oil spill- impacted sediments, and Continental Slope background sediments, all collected in November 2010. These samples were analyzed with ~5 million 16S rRNA gene amplicon sequences. Shannon diversity estimates indicated that Orca Basin hypersaline sediments, Continental Slope sediments, and DWH-contaminated surface sediments had similar species diversity, while hydrocarbon seep sediment diversity was significantly lower. UNIFRAC beta diversity analysis indicated that microbial communities inhabiting Orca Basin hypersaline sediments and iii hydrocarbon seep sediments had taxa unique to each of these site types, while sediments from the Continental Slope and DWH area were not statistically more similar to each other than other sediments. Taxonomic analysis showed that seep cores contained higher abundances of ANME-1 and Candidate Division JS1, while Orca Basin hypersaline sediment-associated sequences were dominated by Marine Group I Archaea, Bacteroidetes and halotolerant Deltaproteobacteria. The microbial composition of the Orca Basin brine was also investigated with 16S rRNA Sanger sequencing, which indicated that Candidate Division KB1-related sequences were the most abundant. A partial KB1 genome sequenced from a single sorted cell revealed that its proteome is dominated by acidic amino acids; therefore, KB1 likely incorporates potassium ions for osmoregulation. KB1 also contains complete enzymes for the acetyl-CoA pathway, and has genes consistent with a glycine betaine transporter cassette as well as genes that may allow for use of glycine betaine as an energy source. iv I dedicate this work to my parents, Linda and Dominic Nigro. v ACKNOWLEDGMENTS I would first like to thank my thesis advisory committee for providing excellent guidance on this project. To my primary advisor, Andreas Teske for introducing me to the project as well providing funding, space and resources in his lab to conduct experiments. He also gave me guidance and suggestions for the thesis and manuscripts. I am incredibly grateful to Mandy Joye. She provided everything from enthusiasm and project guidance to temporary employment when funding was scarce. Mandy also introduced me to the experience of collecting samples with a submarine and provided me with space and resources in her lab to conduct some of the geochemical measurements. She was also an amazing mentor and provided the encouragement and resources to develop my career. To Barbara MacGregor for being there, even at 1:00 am, to help with my thesis and share stories of life in New England. She also provided detailed feedback and pushed for high standards. To Carol Arnosti for always being a constant voice of reason when things seemed out of hand or unclear, as they often are in graduate school. Lastly, to Brian White who served on my committee as a member that had a very different expertise, yet always attended my committee meetings and encouraged discussion. I’d also like to thank my “lab family” that was always there for me, both professionally and personally: Tingting Yang, JP Balmonte, Zena Cardman, Luke McKay, Cassandre Lazar, Lindsay D’Ambrosio and Andrew Hyde. Also to the other MASC students in the program, especially Natalie Cohen, Carly Moreno, Jill Arriola, Isaac Westfield and Anna Jalowska. vi I also received support from other laboratories that helped on my project. While I was at the University of Georgia, Kim Hunter trained me on several of the geochemical experiments and provided all around experimental support. Kimi Takagi provided great discussions about life, both professional and otherwise, and was instantly there for me when I was a guest in the lab. Matt Saxton provided a great working atmosphere in the molecular group at UGA and was always there to give advice. Also thanks to Siobain Duffy at Rutgers University, who is both an amazing friend and someone I could always rely on to help me find my way. Most importantly, I’d like to thank my family. My parents, Linda Nigro and Dominic Nigro for not pushing me to go to college, but also encouraging me to do so once I wanted to persue an advanced degree. You were there supporting me the whole way. Also to my brother William Nigro for being a great friend as well as a great sibling. I would also like to thank the crew and scientists on the R/V Atlantis (leg AT18-2) and R/V Pelican (leg PE22-12) as well as Chief Scientists Mandy Joye (Atlantis) and Mark Pagani and Courtney Warren (Pelican). UNC Research Computing allowed access to their servers, and UNC’s bioinformatics provided excellent support, especially by Jeff Roach. Sean Gibbons at University of Chicago gave me initial bioinformatics advice. This project was funded by NSF Award EF-0801741. The UNC Off-Campus Dissertation Fellowship and UNC Dissertation Completion Fellowship provided tuition and stipend assistance. vii TABLE OF CONTENTS LIST OF TABLES ..........................................................................................................................x LIST OF TABLES ........................................................................................................................ xi LIST OF ABBREVIATIONS ..................................................................................................... xiv CHAPTER 1: INTRODUCTION ....................................................................................................1 CHAPTER 2: MICROBIAL DIVERSITY OF DIVERSE GULF OF MEXICO SEDIMENTS ..................................................................................................................................6 2.1 Introduction ....................................................................................................................6 2.2 Site Descriptions ............................................................................................................7 2.3 Materials and Methods .................................................................................................11 2.3.1 Sample collection .............................................................................................11 2.3.2 Nucleic acid extraction and sequencing ...........................................................11 2.3.3 Data analysis ....................................................................................................11 2.4 Results and Discussion ................................................................................................12 2.4.1 Hypersaline environment associated taxa ........................................................13 2.4.2 Methane cycle-associated taxa .........................................................................14 2.4.3 Japan Sea Group 1 (JS1) .................................................................................15 2.4.4 Patterns of microbial taxa in seep-associated cores .........................................21 2.4.5 Deepwater Horizon area associated taxa .........................................................22 2.4.6 Beta diversity analysis .....................................................................................27 2.5 Conclusions ..................................................................................................................31 viii CHAPTER 3: MICROBIAL DIVERSITY OF HYPERSALINE SEDIMENTS ASSOCIATED WITH ORCA BASIN ..........................................................................................33 3.1 Introduction ..................................................................................................................33 3.2 Materials and Methods .................................................................................................36 3.2.1 Sample collection and processing ....................................................................36 3.2.2 Geochemical analysis preparation ...................................................................37 3.2.3 Geochemical measurements.............................................................................38 3.2.4 DNA extraction and sequencing