University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 7-26-2016 Haloarchaeal Species Genesis: Diversity, Recombination, and Evolution Nikhil Ram Mohan University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Ram Mohan, Nikhil, "Haloarchaeal Species Genesis: Diversity, Recombination, and Evolution" (2016). Doctoral Dissertations. 1207. https://opencommons.uconn.edu/dissertations/1207 Haloarchaeal Species Genesis: Diversity, Recombination, and Evolution Nikhil Ram Mohan University of Connecticut, 2016 ABSTRACT The processes key to the generation of new species in eukaryotes is well understood but the same is not true for the prokaryotic world. Allopatric speciation is widely accepted as the driving force in eukaryotic species genesis. However, little is known about prokaryotic speciation primarily due to the abundance of prokaryotes in this world. They inhabit every fathomable niche, present high cell densities, complex community structures, and are easily dispersed between large distances. For example, a gram of soil or a milliliter of seawater house millions and millions of a vast variety of microorganisms. This complexity in the prokaryotic world makes inferring an overall evolutionary processes extremely difficult. To reduce the complexity, the system studied needs to be narrowed down. This thesis focusses on one environment that is so extreme that it is conducive for the growth of only certain groups of organisms. The system is characterized by high salinity, much greater than that of seawater, which is a prerequisite for the survival of an entire class of Archaea call the Halobacteria (Haloarchaea). Studying these environments reduces the microbial complexity in comparison to some of the more easily habitable areas like soil or water. Haloarchaea are the dominant group of i organisms in hypersaline environments. The dissertation describes a multiscale approach studying haloarchaeal communities to determine the evolutionary forces influencing species genesis in the haloarchaea. First, a spatial distribution analysis of the haloarchaeal communities using both PCR amplified environmental gene marker as well as metagenomic comparisons reveal unique haloarchaeal communities in geographically distant hypersaline environments. Similar to eukaryotes, endemism is apparent in haloarchaea. However, unlike the eukaryotes, there is neither a distance-decay relationship nor is there a similarity in haloarchaeal communities based on whether they are from lakes or salterns. Second, temporal analysis on one haloarchaeal community reveals stability in the community at the genus level. Stable environmental conditions provided by the hypersaline environments ensure stability in the community with only fluctuations in the relative abundances with respect to salinity. Third, comparing individuals within a community showed widespread genomic variations between isolates. Comparing a multi gene concatenated phylogeny and whole genome fingerprinting exposed that even isolates that were identical at each locus tested had varying genome patterns. This happens at a rate much greater than the accumulation of third codon substitutions. Finally, assaying two highly conserved genes from 109 haloarchaeal genomes evidenced the existence of extensive recombination at predicted rates far greater than the rate of mutation in haloarchaea. This dissertation discusses a working model for species genesis in haloarchaea based on the obtained evidence. It involves frequent recombination within the community members at each geographically distant site, homogenizing them, and maintaining endemic populations while often forming stable chimaeras that could become new species. These findings in haloarchaea offer a peek into the mysteries of the prokaryotic world. ii Haloarchaeal Species Genesis: Diversity, Recombination, and Evolution Nikhil Ram Mohan B.Tech, Dr.M.G.R. Educational and Research Institute, 2007 India A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2016 iii Copyright by, Nikhil Ram Mohan 2016 iv APPROVAL PAGE Doctor of Philosophy Dissertation Haloarchaeal Species Genesis: Diversity, Recombination, and Evolution Presented by Nikhil Ram Mohan, B.Tech. Major Advisor _______________________________________________________________________ R. Thane Papke Associate Advisor _______________________________________________________________________ Johann Peter Gogarten Associate Advisor _______________________________________________________________________ Spencer Nyholm Associate Advisor _______________________________________________________________________ Daniel Gage Associate Advisor _______________________________________________________________________ Kent Holsinger University of Connecticut 2016 v ACKNOWLEDGEMENTS I take this opportunity to express my deepest gratitude to my advisor, Dr. R. Thane Papke, without whose support and invaluable guidance I would not be where I am this day. We have had many exciting discussions through the years that fueled my curiosity and helped develop not only my skills but also mold me into the scientist I am. Thank you for being a great advisor. I would also like to thank my advisory committee, Drs. Peter Gogarten, Spencer Nyholm, Kent Holsinger, and Dan Gage for taking time away from their busy schedules to always help me out with my problems and answer my queries. Having been in the Papke lab as long as I have, I was lucky to know some very nice people as they made their way through the lab. I was and am fortunate to have had a lab full of friendly people who made the lab a stimulating place to work at. I would also like to thank all of the friends I have made outside of the lab, graduate school would not have been the same without all of you. None of this would have been possible if not for the everlasting support of my family. My parents, uncle, aunt, brother, and sister have been the foundation that I stand on. I am deeply indebted to them. Though infrequently, visiting my brother and his family would be my only consolation for spending all this time away from my family in India. Finally, I would like to thank my wife, Kunica, for she is my rock, my biggest cheerleader, my best friend. The past five years have been the best of my life and I cannot imagine having gotten this far without you. The future holds a different field in science for me but the haloarchaea will always have a special place in my heart since this small group of microorganisms have taught me all I know! vi Table of Contents Chapter 1 : Introduction .............................................................................................................. 1 1.1 Modes of Speciation ............................................................................................................ 1 1.2 Challenges in the Prokaryotic World ................................................................................ 3 1.3 Hypersaline environments – the ideal ‘Island’ for Prokaryotic Biogeography ............. 5 1.4 The Haloarchaea ................................................................................................................. 6 1.4.1 Frequent gene transfer and recombination in Haloarchaea ........................................ 7 1.5 Choice of molecular marker ............................................................................................. 10 1.5.1 16S rRNA and rpoB genes ........................................................................................... 10 1.5.2. The bacteriorhodopsin gene ....................................................................................... 13 1.6 Overarching goals of this thesis. ...................................................................................... 14 1.6.1 Biogeography of the Haloarchaea ................................................................................ 14 1.6.2 Temporal analysis of one Haloarchaeal community .................................................... 15 1.6.3 Dynamics of Individual Haloarchaea in a population .................................................. 16 1.6.4 Assay the extent of recombination in the Haloarchaea ................................................ 17 Chapter 2 : Cell sorting analysis of geographically separated hypersaline environments .. 18 Chapter 3 : Analysis of geographically separated hypersaline environments reveals uniquely constituted haloarchaeal communities. ..................................................................... 30 3.1 Abstract .............................................................................................................................. 30 3.2 Materials and Methods ..................................................................................................... 31 3.2.1 Sequence acquisition from environmental DNA .......................................................... 31 3.2.1.1 DNA isolation and PCR amplification of bop gene .............................................. 31 3.2.1.2 Cloning, Plasmid Isolation and Sequence acquisition ........................................... 32 3.2.2 Sequence acquisition from available metagenomes ..................................................... 32 3.2.2.1 Extraction of bop, 16S rRNA, and rpoB genes from metagenomes ..................... 32 3.2.3 Sequence analysis ........................................................................................................