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Using PCR Amplification and Genetic Sequence Analysis of 18S Rrna University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses 8-6-2009 Using PCR Amplification and Genetic Sequence Analysis of 18S rRNA Genes to Survey the Microbial Diversity and Distribution of Eukaryotic Microbes Inhabiting Two Thermo-acidic Streams in Yellowstone National Park, Wyoming Robert Harvey Jr. University of New Orleans Follow this and additional works at: https://scholarworks.uno.edu/td Recommended Citation Harvey, Robert Jr., "Using PCR Amplification and Genetic Sequence Analysis of 18S rRNA Genes to Survey the Microbial Diversity and Distribution of Eukaryotic Microbes Inhabiting Two Thermo-acidic Streams in Yellowstone National Park, Wyoming" (2009). University of New Orleans Theses and Dissertations. 978. https://scholarworks.uno.edu/td/978 This Thesis is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO 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 University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Using PCR Amplification and Genetic Sequence Analysis of 18S rRNA Genes to Survey the Microbial Diversity and Distribution of Eukaryotic Microbes Inhabiting Two Thermo-acidic Streams in Yellowstone National Park, Wyoming A Thesis Submitted to the Graduate Faculty of the University of New Orleans In partial fulfillment of the Requirements for the degree of Master of Science In Biological Sciences By Robert C. Harvey Jr. B.S. Our Lady of Holy Cross College, 1994 M.S. Louisiana State University, 1998 August, 2009 Table of Contents List of Figures....................................................................................................... iii List of Tables ......................................................................................................... v Abstract ................................................................................................................ vi Introduction ........................................................................................................... 1 Extremophiles: Practical Significance ........................................................ 1 Acidic Geothermal Habitats: Yellowstone Park, a Natural Laboratory for Microbial Ecology .................................................................................. 3 Microbiological and Molecular Methods for Studying Microbes in situ ........ 4 Algal Mat Communities in Acidic Geothermal Streams .............................. 6 Taxonomic Overview of the Thermophilic Red Algae (Cyanidiales) ........... 7 Cyanidiales in Nature ............................................................................... 12 Other Organisms Associated with Thermoacidophilic Algal Mat Communities ............................................................................................ 16 Genomic Analysis of the Cyanidiales: Evolutionary Significance ............ 18 Research Objectives ................................................................................ 20 Methods .............................................................................................................. 21 Study Sites ............................................................................................... 21 Sample Collection .................................................................................... 24 DNA Extraction ......................................................................................... 27 PCR Amplification .................................................................................... 28 Cloning ..................................................................................................... 28 Sequence Analysis ................................................................................... 29 Results ................................................................................................................ 31 Detection and Identification of Eukaryotic Microbes ................................. 31 Temperature Gradient .............................................................................. 38 Seasonal Analysis .................................................................................... 41 Alluvium Creek Substrate Study ............................................................... 42 Discussion ........................................................................................................... 45 Characterization of Nymph Creek’s Algal Community .............................. 45 Analysis of Nymph Creek Algal Mat Populations, Along a Temperature Gradient .............................................................................. 47 Seasonal Analysis .................................................................................... 50 Alluvium Creek Comparison ..................................................................... 51 References .......................................................................................................... 55 Vita ...................................................................................................................... 62 ii List of Figures Figure 1. Cyanidiales dominated algae mat. ........................................................ 5 Figure 2. Map of Yellowstone Park. The location of study sites at Nymph Creek (Latitude 44.3844 and Longitude -110.2416) and Alluvium Creek (Latitude 44.75130 and Longitude -110.72694) are indicated .............................................................................................. 22 Figure 3A. Upstream high temperature region of Nymph Creek ........................ 25 Figure 3B. Downstream cooler region (T<38°C) of Nymph Creek ..................... 25 Figure 3C. Alluvium Creek flocculent site ........................................................... 26 Figure 3D. Alluvium Creek log site ..................................................................... 26 Figure 3E. Alluvium Creek rock site ................................................................... 26 Figure 4. Dendrogram based of ~680 nucleotides of 18S rRNA genes relating the predominant Nymph Creek thermoacidophilic red alga (ANC1) to other red algal sequences. Bootstrap values (>50%) are indicated at the relevant node. GenBank reference numbers are listed next to species ........................................................................... 35 Figure 5. Dendrogram based of ~680 nucleotides of 18S rRNA genes relating green algae detected in Nymph Creek (ANC2 and ANC3) to other green algal sequences. Bootstrap values (>50%) are indicated at the relevant node. GenBank reference numbers are listed next to species ................................................................................................ 36 Figure 6. Pie chart showing relative abundance of sequences of each phylotype obtained from cloned PCR products from Nymph and Alluvium Creek .................................................................................... 39 Figure 7. Relative abundance of phylotypes detected in a warm ~50°C upstream site and a cool ~30°C downstream site in Nymph Creek. Includes samples from different seasons. .............................. 40 Figure 8. Algae detected along a thermal gradient in Nymph Creek .................. 41 Figure 9. Algae detected at seasonal intervals in Nymph Creek at a warm upstream site and a cool downstream site ................................ 42 iii Figure 10. Relative abundance of phylotypes detected on two different substrates, a log and a rock, in Alluvium Creek. Data is from a single time point sample collected 08/18/01 .......................... 44 iv List of Tables Table 1. Currently recognized species of Cyanidiales and identifying characteristics ..................................................................................... 12 Table 2. Genomic DNA extraction methods for Nymph and Alluvium Creek sequence data: + successful attempt, - unsuccessful attempt ........... 32 Table 3. Phylotypes with their nearest GenBank relatives and the percent of similarity between sequences, the Rio Tinto phylotypes are prefaced by the text string “uncultured clone RT” in the second column ................................................................................................ 34 v Abstract A cultivation-independent approach, sequence analysis of 18S rRNA genes PCR-amplified from environmental DNA, was used to explore the diversity and distribution of eukaryotic microbes inhabiting algal mats in two acidic geothermal streams in Yellowstone National Park. The objectives were to: (1) clarify the identity of mat forming algae in Nymph Creek (2) survey microbial species in the Nymph Creek mat over seasonal intervals along a thermal gradient (3) compare microbial species in the Nymph Creek mat with those in Alluvium Creek mats (4) evaluate microbial species in algal mats formed on different substrates in Alluvium Creek. The results show that a novel red alga dominates high temperature regions (~50ºC) of Nymph Creek and two “Chlorella-like” algae predominate the cooler regions (<38ºC). The
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