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Diversity, Biogeography, and Geochemical Habitat of Korarchaeota in Continental Hot Springs UNLV Retrospective Theses & Dissertations 1-1-2008 Diversity, biogeography, and geochemical habitat of Korarchaeota in continental hot springs Robin Lea Miller University of Nevada, Las Vegas Follow this and additional works at: https://digitalscholarship.unlv.edu/rtds Repository Citation Miller, Robin Lea, "Diversity, biogeography, and geochemical habitat of Korarchaeota in continental hot springs" (2008). UNLV Retrospective Theses & Dissertations. 2414. http://dx.doi.org/10.25669/6h98-vit6 This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Retrospective Theses & Dissertations by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. DIVERSITY, BIOGEOGRAPHY, AND GEOCHEMICAL HABITAT OF KORARCHAEOTA IN CONTINENTAL HOT SPRINGS by Robin Lea Miller Bachelor of Science in Biological Sciences University of Nevada, Las Vegas 2006 A thesis submitted in partial fulfillment of the requirements for the Master of Science Degree in Biological Sciences School of Life Sciences College of Sciences Graduate College University of Nevada, Las Vegas December 2008 UMI Number: 1463517 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, If unauthorized copyright material had to be removed, a note will indicate the deletion. UMI UMI Microform 1463517 Copyright 2009 by ProQuest LLC. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 E. Eisenhower Parkway PC Box 1346 Ann Arbor, Ml 48106-1346 Thesis Approval w The Graduate College Mii^iraatKii*M »Ugi University of Nevada, Las Vegas November 19 ,20Qâ_ The Thesis prepared by Robin M iller Entitled Diversity, Biogeography, and Geochemical Habitat of Korarchaeota in Continental Hot Springs is approved in partial fulfillment of the requirements for the degree of Master of Science Examination Committee Chair Dean of the Graduate College Examination Cpmmittee Member Graduate (College Faculty Representative 11 ABSTRACT Diversity, Biogeography, and Geochemical Habitat of Korarchaeota in Continental Hot Springs by Robin Lea Miller Dr. Brian Hedlund, Committee Chair Professor of Biology University of Nevada, Las Vegas Microorganisms have traditionally been characterized based on their morphology and physiology. However, this formula does not account for the importance of a microorganism’s habitat, which is intimately linked to its physiology. The Korarchaeota comprise a deeply branching archaeal lineage that has proven to be difficult to isolate and, consequently, has remained uncharacterized until recently. The aim of this study was to evaluate the physicochemical and biogeographical distribution of Korarchaeota in hot springs of the Great Basin and Yellowstone National Park. Ninety-nine sediment samples were collected from hot springs with temperatures ranging from 40.7-94.3°C and pH ranging from 1.3-9.2, while recording as many coincident chemical properties as feasible. Polymerase chain reaction was used to screen samples for Korarchaeota 16S rRNA genes, and products were sequenced and phylogenetically analyzed. The resultant data set identified novel korarchaeal phylotypes and showed that korarchaeal phylotypes are not randomly distributed with respect to biogeography. Ill TABLE OF CONTENTS ABSTRACT..................................................................................................................................iii LIST OF FIGURES..................................................................................................................... vi LIST OF TABLES......................................................................................................................vii ACKNOWLEDGEMENTS .................................................................................................... viii CHAPTER 1 INTRODUCTION................................................................... 1 The archaea .......................................................... 1 Discovery and phylogeny of Korarchaeota..........................................................................3 Cultivation and quantitation of Korarchaeota..................................................................... 4 The distribution and phylogeny of Korarchaeota............................................................... 5 Morphology of Korarchaeota...............................................................................................11 Physiology of Korarchaeota................................................................................................. 14 Microbial biogeography and biodiversity ........................................................................... 15 Objectives ................................................................................................................................18 CHAPTER 2 MATERIALS AND METHODS................................................................. 20 Site description and sampling .............................................................................................. 20 DNA extraction and evaluation of quality of DNA ...........................................................25 Evaluation of possible Korarchaeota-spccific PCR primers ........................................... 26 Optimization of Korarchaeota-spcciïic PC R .................................................................... 27 Cloning and RFLP .................................................................................................................29 Phylogenetic analysis ............................. 30 Support vector statistics ........................................................................................................ 31 CHAPTER 3 RESULTS........................................................................................................ 33 Sample distribution ................................................................................................................33 Temperature and pH ..............................................................................................................35 Bulk water chemistry ............................................................................................................ 41 Support vector machine ........................................................................................................ 41 Phylogeny ............................................................................................................................... 44 CHAPTER 4 DISCUSSION..................................................................................................47 Discussion .............................................. 47 APPENDIX...................................................................................................................................53 IV REFERENCES............................................................................................................................80 VITA............................................................................................................................................. 85 LIST OF FIGURES Figure I Microscopy of “Candidatus Korarchaeum cryptofilum” ..................................13 Figure 2 Gel electrophoresis of PCR optimization assay .................................................28 Figure 3 Physical map of Great Basin and Yellowstone National Park geothermal regions .................................................................................................34 Figure 4 Temperature vs. pH plot of hot springs sampled in the Great Basin ............. 38 Figure 5 Temperature vs. pH plot of hot springs sampled in YNP ................................39 Figure 6 Temperature vs. pH plot of hot springs sampled in YNP with pH ranges ....40 Figure 7 Chemical analytes & Korarchaeota distribution in YNP & GB ....................43 Figure 8 Maximum likelihood phylogeny ..........................................................................46 VI LIST OF TABLES Table I Description of environments where Korarchaeota have been detected .............6 Table 2 Field kit method numbers and ranges ....................................................................22 Table 3 Primers used for PCR amplification and sequencing of I6S rRNA gene ....... 26 Table 4 Description of Great Basin and Yellowstone hot springs where Korarchaeota 16S rRNA genes were detected ................................................... 36 Vll ACKNOWLEDGEMENTS I would like to thank: The National Science Foundation for supporting this work under the CAREER grant MCB-0546865 to Brian P. Hedlund. INBRE grant P20 RR-016464 for supporting the SVM work by Cliris
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