Masters Thesis

Prokaryotic diversity of Boiling Springs Lake, Lassen Volcanic National Park

The identification of organisms present in an environment is a crucial prerequisite for understanding the role of organisms in the environment and the processes influencing the diversity of those organisms. In general, the basic ecology of acidic thermal environments is poorly understood, partially due to the lack of cultivability of prokaryotes from those environments. The purpose of this project was to examine the prokaryotic community composition in Boiling Springs Lake (BSL), a hot, acidic lake in Lassen Volcanic National Park (LVNP). Culture-independent methods were used to identify the prokaryotes living in BSL and to compare the prokaryotic community composition at four sites around the lake, ranging in temperature from 52.2-82.3ºC. First, prokaryotes were identified using a 16S rRNA clone library constructed from water samples collected at the warmest of the four sites. Analysis of the clone library identified sequences from the domains Bacteria and Archaea. Approximately 75% of the clones sequenced and 27% of the identified phylotypes belonged to the domain Bacteria. Terminal Restriction Fragment Length Polymorphism (TRFLP), a molecular method that has been used to approximate diversity within microbial communities, was used to compare community composition at all four sampling sites. TRFLP diversity fingerprints were examined for intra-sample and inter-sample variation in an effort to evaluate the sensitivity and reproducibility of this method in detecting variation between and among prokaryotic communities in BSL. Some variation between independent extractions of the same sample was observed, but, in most cases, it was less than the variation observed between different samples. TRFLP was able to resolve community composition differences between samples where differences were expected based on temperature. All of the phylotypes identified in the clone library were detected by TRFLP in at least one of the sampling sites, and six of the phylotypes were detected in water samples from all four sites. Nonmetric multidimensional scaling ordination of TRFLP fingerprints generally resulted in clusters of extractions correlated with sampling site and temperature. TRFLP fingerprints from the warmest site were characterized by a Thermoplasmatales-like phylotype, an Ignicoccus-like phylotype, and Hydrogenobaculum. The results of this study provide an initial look at the bacterial diversity present in BSL and suggest that differences in community composition around the lake are correlated with temperature. They also indicate that TRFLP is a sensitive and repeatable enough method to detect variation in the prokaryotic community composition at various sites in BSL.

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