Molecular Phylogenetic Investigation of Microbial Diversity and Nitrogen Cycling in Lava Tubes Jennifer Jane Marshall Hathaway
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University of New Mexico UNM Digital Repository Biology ETDs Electronic Theses and Dissertations 5-1-2010 Molecular phylogenetic investigation of microbial diversity and nitrogen cycling in lava tubes Jennifer Jane Marshall Hathaway Follow this and additional works at: https://digitalrepository.unm.edu/biol_etds Recommended Citation Hathaway, Jennifer Jane Marshall. "Molecular phylogenetic investigation of microbial diversity and nitrogen cycling in lava tubes." (2010). https://digitalrepository.unm.edu/biol_etds/48 This Thesis is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Biology ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. MOLECULAR PHYLOGENETIC INVESTIGATION OF MICROBIAL DIVERSITY AND NITROGEN CYCLING IN LAVA TUBES BY JENNIFER J. MARSHALL HATHAWAY B.S., BIOLOGY, STANFORD UNIVERSITY 2001 THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science Biology The University of New Mexico Albuquerque, New Mexico May, 2010 © 2010, Jennifer Jane Marshall Hathaway iii DEDICATION This work is dedicated to my parents, Bill and Jane Marshall, who taught me at a very young age to see the wonder of the natural world, and to ask questions about the way it works. And to my husband, Simon Hathaway, who teaches me every day about finding joy in both the little and big moments in my life. Thank you for allowing me to have this adventure. iv ACKNOWLEDGEMENTS I would like to acknowledge first and foremost my advisor, Dr. Diana Northup. She has a love of science that is infectious, and I am grateful that I was able to work with such an extraordinary mentor. Without her guidance this work would have not been completed. I thank my committee members, Dr. Robert Sinsabaugh and Dr. Andrea Porras- Alfaro, for their suggestions and guidance in developing and completing this work, and their contributions to my professional development. I thank the members of the Northup Lab, past and present, for making it an enjoyable work atmosphere to come to every day. I especially thank Dr. Armand Dichosa for the countless hours of invaluable conversation about science and caves. Lurdes and Airidas Dapkevicius provided support and generous hospitality while I was in the Azores, and I thank them for their help, and their friendship. I want to thank my family and my husband’s family for encouraging me to undertake this endeavor from the start, and supporting me every step of the way. Finally, I want to thank my husband, Simon, for his never ending love and support in whatever project I am undertaking. He listened patiently to my explanations of my research, and helped me find ways to overcome any obstacle in my path. v MOLECULAR PHYLOGENETIC INVESTIGATION OF MICROBIAL DIVERSITY AND NITROGEN CYCLING IN LAVA TUBES BY JENNIFER J. MARSHALL HATHAWAY ABSTRACT OF THESIS Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science Biology The University of New Mexico Albuquerque, New Mexico May 2010 vi MOLECULAR AND PHYLOGENETIC INVESTIGATION OF MICROBIAL DIVERSITY AND NITROGEN CYCLING IN LAVA TUBES By Jennifer J. Marshall Hathaway B.S., BIOLOGY, STANFORD UNIVERSITY, 2001 MASTER OF SCIENCE, BIOLOGY, UNIVERSITY OF NEW MEXICO, 2010 ABSTRACT Worldwide, lava tubes host colorful microbial mats on their walls and ceilings. Little is known about the diversity and ecological roles of the bacteria communities in the subsurface ecosystem of lava tubes. White and yellow microbial mats were collected from four lava tubes from the Azorean island of Terceira and from four lava tubes on the Big Island of Hawai‘i, to compare and contrast the diversity of bacteria found in lava tubes. The 16S rRNA gene was sequenced in order to determine the diversity within these caves, and to begin to elucidate the environmental controls on microbial diversity in the subsurface by comparing community structure to environmental parameters. One hundred ninety two sequences from 16 samples were analyzed. Fifteen phyla were found across the samples. With more Actinobacteria clones retrieved from Hawaiian communities, while more Alphaproteobacteria clones were found in Azorean communities. The Actinobacteria exhibited considerable novel diversity, with several vii distinct novel clades that shared less than 94% sequence identity. Geographical location was the major contributor to differences in community structure. The diversity of ammonia oxidation (amoA) and nitrogen fixation (nifH) genes in bacterial mats from lava tube walls in the Azores was investigated using denaturing gradient gel electrophoresis (DGGE). The lava tubes were found under different land use categories, pasture, forested and sea/urban. Soil and water samples from each lava tube were analyzed for nutrient content. Nitrosospira-like sequences dominated the ammonia oxidizing bacteria (AOB) community, and the majority of the diversity was found in lava tubes under forested land. The nitrogen fixation community was dominated by Klebsiella pneumonia-like sequences, and diversity was evenly distributed between pasture and forested land. The results suggest that land use is impacting the AOB more than the nitrogen fixing bacteria. Furthermore, the results of these studies underscore the need for further investigation of these unique ecosystems. viii TABLE OF CONTENTS List of Figures…………………………………………………………………………xi List of Tables………………………………………………………………………….xii Chapter 1: Introduction……………………………………………………………………1 References……………………………………………………………………..5 Chapter 2: Investigation of Novel Microbial Diversity in Azorean and Hawaiian Lava Tubes Introduction……………………………………………………………………9 Results Analysis of Community Composition…………………………….….11 Analysis of Actinobacteria…………………………………………...16 Scanning Electron Microscopy………………………………………22 Discussion……………………………………………………………………22 Experimental Procedure Site Description and Sample Collection…………………….……….27 DNA Extraction, Amplification, and Sequencing…………………...28 Molecular Phylogenetics………………………………………...…..30 Scanning Electron Microscopy………………………………………31 Acknowledgements…………………………………………………………..31 References……………………………………………………………………32 Chapter 3: Diversity of Ammonia Oxidation (amoA) and Nitrogen Fixation (nifH) Genes in Lava Tubes of Terceira, Azores, Portugal Introduction…………………………………………………………………..38 Material and Methods Sample Site Description……………………………………………...41 Sample Collection……………………………………………………41 Water and Soil Chemistry………………………………...………….41 Molecular Methods…………………………………………………..42 Denaturing Gradient Gel Electrophoresis…………………….……...47 Results Water and Soil Chemistry……………………………………………48 Ammonia Oxidation ……………………………………….………...48 Nitrogen Fixation…………………………………………………….50 Discussion……………………………………………………………………64 Conclusions……….………………………………………………………….68 ix Acknowledgements………………………………………………………….68 References…………………………………………………………………...69 Chapter 4 Conclusions………………………………………………………………….74 References…………………………………………………………………...76 x List of Figures Chapter 2: Figure 1. Inside Azorean and Hawaiian Lava Tubes…………………………12 Figure 2A. Phyla Found by Lava Tube……………………………………….13 Figure 2B. Phyla Found in Lava Tubes by Location…………………………14 Figure 3. Chao 1 Estimate of Richness……………………………………….17 Figure 4. Principal Component Analysis of Hawai‘i and the Azores….….…..18 Figure 5. Sequence Identity of Actinobacteria………………………….….….19 Figure 6. Parsimony Tree of Actinobacteridae……………………….…….…20 Figure 7. Parsimony Tree of Acidimicrobiales and Rubrobacteridae……..….21 Figure 8. SEM Similarities Between Hawai‘i and the Azores………………..23 Chapter 3: Figure 1. Map of Terceira Island……………………………………………..44 Figure 2. Different Land Use Practices……………………………………….45 Figure 3. DGGE Image of amoA Gels…………………………………….…..59 Figure 4. DGGE Image of nifH Gels…………………………………….…....60 Figure 5. Venn Diagrams of OTUs by Land Use……………………….…….61 Figure 6. Parsimony Tree of amoA OTUs……………………………….……62 Figure 7. Parsimony Tree of nifH OTUs……………………………….……..63 xi List of Tables Chapter 2 Table 1. List of Samples and Abiotic Factors…………………………………29 Chapter 3 Table 1: Abiotic Factors Associated with Each Cave …………………………43 Table 2. Primers and Thermocycling Conditions……………………………...46 Table 3. Water and Soil Chemistry…………………………………………….49 Table 4. amoA DGGE Band Identification…………………………………....51 Table 5. nifH DGGE Band Identification……………………………………..54 xii Chapter 1 Introduction Extreme environments that harbor microbial life are found on the surface and in the subsurface across the planet, from sulfur hot springs to the salt flats to dry deserts to the hydrothermal vents at the bottom of the ocean (Rothschild and Mancinelli, 2001). These microbes play important roles in shaping the ecosystems they inhabit. Microbes are central in cycling nutrients through the precipitation and dissolution of minerals, both at the surface (Ehrlich, 1999) and in the subsurface (Northup and Lavoie, 2001; Barton and Northup, 2007). Caves and lava tubes often have moderate temperature and relatively stable humidity throughout the year, but are still considered extreme environments because of their low nutrient availability and productivity (Moore and Sullivan, 1997; Northup and Welbourn, 1997; Barton, 2006). The microorganisms of limestone caves have received greater scientific attention in the last 20 years, while lava tubes and their microbial communities have received far less attention (Northup and Welbourn, 1997). Parenthetically, the recent