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Prokaryotic Diversity of Boiling Springs Lake, Lassen Volcanic National Park

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Prokaryotic Diversity of Boiling Springs Lake, Lassen Volcanic National Park PROKARYOTIC DIVERSITY OF BOILING SPRINGS LAKE, LASSEN VOLCANIC NATIONAL PARK HUMBOLDT STATE UNIVERSITY By Andrea Bartles A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment Of the Requirements for the Degree Master of Art In Biology (May, 2007) PROKARYOTIC DIVERSITY OF BOILING SPRINGS LAKE, LASSEN VOLCANIC NATIONAL PARK HUMBOLDT STATE UNIVERSITY By Andrea Bartles We certify that we have read this study and that it conforms to acceptable standards of scholarly presentation and is fully acceptable, in scope and quality, as a thesis for the degree of Master of Arts. Approved by the Master’s Thesis Committee: ________________________________________________________________________ Patricia Siering, Major Professor Date ________________________________________________________________________ Mark Wilson, Committee Member Date ________________________________________________________________________ Michael Camann, Committee Member Date ________________________________________________________________________ Brian Arbogast, Committee Member Date ________________________________________________________________________ Michael Mesler, Graduate Coordinator Date ________________________________________________________________________ Chris A. Hopper, Interim Dean Date Research, Graduate Studies & International Programs ABSTRACT Prokaryotic Diversity of Boiling Springs Lake, Lassen Volcanic National Park Andrea Bartles The identification of organisms present in an environment is a crucial prerequisite for understanding the role of organisms in the environment and the processes influencing the diversity of those organisms. In general, the basic ecology of acidic thermal environments is poorly understood, partially due to the lack of cultivability of prokaryotes from those environments. The purpose of this project was to examine the prokaryotic community composition in Boiling Springs Lake (BSL), a hot, acidic lake in Lassen Volcanic National Park (LVNP). Culture-independent methods were used to identify the prokaryotes living in BSL and to compare the prokaryotic community composition at four sites around the lake, ranging in temperature from 52.2-82.3ºC. First, prokaryotes were identified using a 16S rRNA clone library constructed from water samples collected at the warmest of the four sites. Analysis of the clone library identified sequences from the domains Bacteria and Archaea. Approximately 75% of the clones sequenced and 27% of the identified phylotypes belonged to the domain Bacteria. Terminal Restriction Fragment Length Polymorphism (TRFLP), a molecular method that has been used to approximate diversity within microbial communities, was used to compare community composition at all four sampling sites. TRFLP diversity fingerprints were examined for intra-sample and inter-sample variation in an effort to evaluate the sensitivity and reproducibility of this method in detecting variation between and among iii prokaryotic communities in BSL. Some variation between independent extractions of the same sample was observed, but, in most cases, it was less than the variation observed between different samples. TRFLP was able to resolve community composition differences between samples where differences were expected based on temperature. All of the phylotypes identified in the clone library were detected by TRFLP in at least one of the sampling sites, and six of the phylotypes were detected in water samples from all four sites. Nonmetric multidimensional scaling ordination of TRFLP fingerprints generally resulted in clusters of extractions correlated with sampling site and temperature. TRFLP fingerprints from the warmest site were characterized by a Thermoplasmatales-like phylotype, an Ignicoccus -like phylotype, and Hydrogenobaculum . The results of this study provide an initial look at the bacterial diversity present in BSL and suggest that differences in community composition around the lake are correlated with temperature. They also indicate that TRFLP is a sensitive and repeatable enough method to detect variation in the prokaryotic community composition at various sites in BSL. iv ACKNOWLEDGEMENTS I would sincerely like to thank the people who made it possible for me to complete this project. Most of all, I would like to thank Patty Siering and Mark Wilson for their guidance and support on every aspect of this project. Over the past four years, they have been invaluable as both teachers and friends. Thanks to Mike Camann and Brian Arbogast for their assistance in developing this project and Mike Camann for his assistance with the statistical analyses. Thanks to Anthony Baker for sharing his laboratory knowledge and space. Thanks to Michelle Hauser, Luke Hamm, and Christy Whitehouse for their assistance in collecting and processing samples. Michelle also did extremely helpful preliminary work on TRFLP. Thanks to Michelle Ansell for helping me complete plasmid preps. Thanks to Ryan Brodie and Jocelyn Jones for collecting AODC data. Thanks to Chris White for working side by side with me on TRFLP, sharing his data, and providing encouragement along the way. Thanks to Donnie Carter for his suggestions in the lab. I would also like to thank Casey Lu for giving me guidance and providing me with opportunities to help me become a better teacher. Finally, I would like to thank Marty Yip for encouraging me to accomplish my goals, providing emotional support when I needed it, and helping me to keep balance in my life along the way. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………………...iii ACKNOWLEDGEMENTS……………………………………………………………….v TABLE OF CONTENTS…………………………………………………………………vi LIST OF TABLES………………………………………………………………………..ix LIST OF FIGURES……………………………………………………………………….x CHAPTER 1: BACKGROUND AND OBJECTIVES……………………………………1 Overview…………………………………………………………………………..1 Site Description……………………………………………………………………2 Thermoacidophilic Life…………………………………………………………...6 Cultivation vs. Culture Independent Investigation………………………………10 Molecular Estimates of Diversity………………………………………………..12 CHAPTER 2: CLONE LIBRARY AND PHYLOGENY……………………………….22 Introduction………………………………………………………………………22 Methods…………………………………………………………………………..24 Sample Collection………………………………………………………..24 Acridine Orange Direct Counts (AODC)………………………………..26 Nucleic Acid Extractions………………………………………………..26 Creation of Site D rRNA Gene Clone Library………………………….28 Sequence Analysis and Phylogeny Estimation…………………………29 Results…………………………………………………………………………..31 vi Site Characterization……………………………………………………..31 Community Analysis…………………………………………………….32 Discussion………………………………………………………………………..39 CHAPTER 3: TERMINAL-RESTRICTION FRAGMENT LENGTH POLYMORPHISM (TRFLP)……………………………………………………………45 Introduction………………………………………………………………………45 Methods…………………………………………………………………………..49 Sample Collection and Nucleic Acid Extraction………………………...49 PCR Amplification………………………………………………………49 Mung Bean Nuclease Digestion…………………………………………51 Restriction Enzyme Digestion…………………………………………...51 Polyacrylamide Gel Electrophoresis…………………………………….52 TRFLP Analysis…………………………………………………………54 Results……………………………………………………………………………54 Intra-sample Variation…………………………………………………...54 Inter-sample Variation…………………………………………………...57 Inter-site Variation……………………………...………………………..60 Comparison of TRFLP with Clone Library……………………………...66 Statistical Analysis……………………………………………………….66 Discussion………………………………………………………………………..69 Assessment of the Sensitivity, Repeatability and Resolution of TRFLP..69 Sources of variation……………………………………………...69 vii Intra-sample variation……………………………………………70 Inter-sample variation……………………………………………71 Assessment of Diversity in BSL…………………………………………73 Inter-site variation…………………...…………………………...73 Pooled vs. Averaged Extractions………………………………...76 Factors influencing microbial communities……………………...76 Factors influencing TRFLP results………………………………77 Suggestions for Future Studies…………………………………………..79 CHAPTER 4: CONCLUSION…………………………………………………………..81 REFERENCES…………………………………………………………………………..84 viii LIST OF TABLES Table Page 1 Common known thermoacidophiles………………………………………………9 2 Common phylotypes in BSL site A sediment clone libraries……………………23 3 Summary of BSL site D clone library phylotypes……………………………….33 4 Summary of nucleic acid extractions from BSL water samples collected July 19, 2004…………………………………………………………..50 5 Bray-Curtis similarity indices showing variation between multiple extractions from each BSL sample (higher values indicate greater similarity).………….….56 6 Bray-Curtis and Jaccard similarity indices showing variation between water samples from BSL (higher values indicate greater similarity)……………58 7 Bray-Curtis similarity indices showing variation between sampling sites in BSL (higher values indicate greater similarity)……………………………….64 8 Bray-Curtis similarity indices comparing TRFLP profiles of pooled samples with averaged TRFLP profiles from sampling sites in BSL (higher values indicate greater similarity). For each site, pooled samples are compared with averaged results for all extractions and with averaged results for only those extractions included in the pooled sample. All results are from the primer set U341F/U1406R……………………………….…65 9 Comparison of averaged TRFLP results for sites A, B, C, and D with phylotypes detected in site D clone library………………………………………67 ix LIST OF FIGURES Figure Page 1 Maps of Lassen Volcanic National Park. (a) LVNP is located at the southern end of the Cascade Range in northern California. (image taken from www.lib.utexas.edu ) (b) Shaded area indicates LVNP boundaries. BSL (indicated
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