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Changes in Microbial Communities and Geochemical Energy Supplies Across The Changes in Microbial Communities and Geochemical Energy Supplies Across the Photosynthetic Fringe of Hot Spring Outflows in Yellowstone National Park by Joseph T. Romero A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science Approved November 2018 by the Graduate Supervisory Committee: Everett Shock, Chair Christy Till Hinsby Cadillo-Quiroz ARIZONA STATE UNIVERSITY December 2018 ABSTRACT Utilizing both 16S and 18S rRNA sequencing alongside energetic calculations from geochemical measurements offers a bridged perspective of prokaryotic and eukaryotic community diversities and their relationships to geochemical diversity. Yellowstone National Park hot spring outflows from varied geochemical compositions, ranging in pH from < 2 to > 9 and in temperature from < 30°C to > 90°C, were sampled across the photosynthetic fringe, a transition in these outflows from exclusively chemosynthetic microbial communities to those that include photosynthesis. Illumina sequencing was performed to document the diversity of both prokaryotes and eukaryotes above, at, and below the photosynthetic fringe of twelve hot spring systems. Additionally, field measurements of dissolved oxygen, ferrous iron, and total sulfide were combined with laboratory analyses of sulfate, nitrate, total ammonium, dissolved inorganic carbon, dissolved methane, dissolved hydrogen, and dissolved carbon monoxide were used to calculate the available energy from 58 potential metabolisms. Results were ranked to identify those that yield the most energy according to the geochemical conditions of each system. Of the 46 samples taken across twelve systems, all showed the greatest energy yields using oxygen as the main electron acceptor, followed by nitrate. On the other hand, ammonium or ammonia, depending on pH, showed the greatest energy yields as an - electron donor, followed by H2S or HS . While some sequenced taxa reflect potential biotic participants in the sulfur cycle of these hot spring systems, many sample locations that yield the most energy from ammonium/ammonia oxidation have low relative abundances of known ammonium/ammonia oxidizers, indicating potentially untapped I sources of chemotrophic energy or perhaps poorly understood metabolic capabilities of cultured chemotrophs. II TABLE OF CONTENTS Page LIST OF TABLES ............................................................................................................ iii LIST OF FIGURES ........................................................................................................... iv CHAPTER 1. INTRODUCTION ................................................................................................. 1 2. METHODS............................................................................................................ 7 Sample Collection – Linear Discriminate Analysis ..................................... 7 Primer Sets ................................................................................................. 10 DNA Extraction and Sequencing ................................................................ 10 Metabolic Energy Calculations ................................................................... 12 3. RESULTS ............................................................................................................. 17 Systems That Fell On or Near the Cox et al. (2011) Fringe Line ................. 19 Bison Pool ........................................................................................ 19 Rabbit’s Nest .................................................................................... 27 Old Blue Eyes................................................................................... 31 Peekaboo .......................................................................................... 35 Mound Spring ................................................................................... 40 Page Systems Exceeding the Cox et al. (2011) Fringe Line ................................. 46 Cyanidium Falls ............................................................................... 47 Mojito............................................................................................... 54 Goldilocks ........................................................................................ 62 Systems that Fall Below the Cox et al. (2011) Fringe Line ......................... 68 i CHAPTER Page Crater Hills Geyser ........................................................................... 71 Mutant Minnie .................................................................................. 76 Figure 8 ............................................................................................ 81 Empress ............................................................................................ 87 4. DISCUSSION........................................................................................................ 92 5. SUGGESTED FUTURE WORK ........................................................................... 96 WORKS CITED .................................................................................................... 99 APPENDIX A: SUPPLEMENTARY TABLES .................................................................... 106 B: SUPPLEMENTARY ENERGY FIGURES .................................................. 125 ii LIST OF TABLES Table Page 2.1. Linear discriminate analysis coefficients ..................................................................... 7 2.2. First step PCR protocol ............................................................................................... 11 2.3. Second step PCR protocol .......................................................................................... 12 2.4. 58 reactions list ........................................................................................................... 16 A.1. Geochemical meaurements ........................................................................................ 107-8 A.2. 16S relative abundances ............................................................................................. 109-16 A.3. 18S relative abundances ............................................................................................. 117-24 iii LIST OF FIGURES Figure Page 1.1. Temperature, pH, and sulfide effects on transition to photosynthesis .......................... 1 1.2. Photosynthetic fringes ................................................................................................ 2 1.3.Hot spring outflow schematics ..................................................................................... 5 1.4.Energetic trajectory schematics .................................................................................... 6 1.5. Concentration trajectory schematics ............................................................................ 6 2.1. Yellowstone sampling locations .................................................................................. 8 2.2. DNA extraction kit NanoDrop comparisons ................................................................ 11 3.1. 2014 Photosynthetic Fringe Sites ................................................................................ 17 4.1. Bison Pool 16S Taxonomic Analysis .......................................................................... 21 4.2. Bison Pool 18S Taxonomic Analysis .......................................................................... 23 4.3. Bison Pool Major Energetic Contributors.................................................................... 24 4.4. Bison Pool Oxidant and Reductant Energies ............................................................... 26 4.5. Rabbit’s Nest 16S Taxonomic Analysis ...................................................................... 28 4.6. Rabbit’s Nest 18S Taxonomic Analysis ...................................................................... 29 4.7. Rabbit’s Nest Major Energetic Contributors ............................................................... 30 4.8. Rabbit’s Nest Oxidant and Reductant Energies ........................................................... 31 4.9. Old Blue Eyes 16S Taxonomic Analysis ..................................................................... 32 4.10. Old Blue Eyes 18S Taxonomic Analysis ................................................................... 33 4.11. Old Blue Eyes Major Energetic Contributors ............................................................ 34 4.12. Old Blue Eyes Oxidant and Reductant Energies ........................................................ 35 iv Figure Page 4.13. Peekaboo 16S Taxonomic Analysis .......................................................................... 36 4.14. Peekaboo 18S Taxonomic Analysis .......................................................................... 38 4.15. Peekaboo Major Energetic Contributors .................................................................... 39 4.16. Peekaboo Oxidant and Reductant Energies ............................................................... 40 4.17. Mound Spring 16S Taxonomic Analysis ................................................................... 41 4.18. Mound Spring 18S Taxonomic Analysis ................................................................... 43 4.19. Mound Spring Major Energetic Contributors ............................................................ 45 4.20. Mound Spring Oxidant and Reductant Energies ........................................................ 46
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