Publication Date

April 2018

Advisor(s)

Frederick Cohan

Major

Biology (BIOL)

Language

English (United States)

Abstract

Bacterial species have traditionally been broadly defined to contain large amounts of ecological and genetic diversity, making it difficult to identify the most newly divergent products of ecological diversification (or speciation). The ability to determine the most newly divergent phylogenetic groups is necessary for discovering and describing the ecological dimensions on which bacteria diverge and become able to coexist indefinitely. This study uses the ecotype as the unit of demarcation for bacteria, which encompasses sequence clusters of bacteria who share a genetic history, are each ecologically distinct, and whose members are ecologically homogenous, to explore the ecological dimensions of early divergence among close relatives of Bacillus subtilis in Death Valley National Park. We collected rhizosphere soil samples of Artemisia tridentata, Juniperus osteosperma, Opuntia basillaris, and O. polyacantha, as well as free soil samples along a transect stretching from 900 to 3000 meters in Death Valley. This study focuses on a subset of the soil samples: Opuntia rhizosphere and associated free soil samples from 900 m, 1200 m, 1500 m, 1770 m, 1980 m, 2130 m, and 3000 m. We isolated candidates of the B. subtilis-B. licheniformis clade from our samples using a series of metabolic tests, and sequenced the gyrA gene. I then reconstructed the phylogenetic relationships among the isolates, with the addition of reference strains, to identify the most recent products of speciation. Lastly, I ran Ecotype Simulation 2 to detect putative ecotypes from the sequence clustering of the gyrA gene. ES2 detected a total of 81 putative ecotypes among all isolates. Of these, ES2 detected a total of 41 putative ecotypes containing strains isolated from the elevation gradient in Death Valley, with 29 of them not previously identified or published. These putative ecotypes fell within the three major clades most closely related to B. atrophaeus, B. subtilis subsp. spizizenii, and B. subtilis subsp. inaquosorum. I confirmed the hypothesized ecotypes to be ecologically distinct by showing that the putative ecotypes were differentially associated with rhizosphere or free soil, as well as with elevation. Elevation appears to be associated with early divergence, but does not play a predominant role in driving early diversification. Furthermore, ecotypes tend to quantitatively differ in their rhizosphere associations, suggesting some level of speciation occurring regarding changes in preference to rhizosphere or free soil. This study adds elevation and rhizosphere association as ecological dimensions of speciation in B. subtilis and close relatives. There are many future directions in which to expand and supplement this work. There is opportunity to use next-generation sequencing technologies to further investigate the ecological dimensions of diversification and the rate of ecotype formation. Comparing genome content of strains of different ecotypes could allow for the identification of strains that possess unique sets of genes that code for different ecological functions, enhancing knowledge of ecotype differences. Whole genome analysis can also offer further insight into discovering whether individual ecotypes are each ecologically homogeneous. This work contributes to the broader goals of microbial ecologists to identify the ecological dimensions upon which bacteria diversify, and challenges researchers to continue to discover more of the ecological dimensions driving early diversification among B. subtilis and close relatives.

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