A Multi-Pronged Approach to Recover Diversity and Candidate Phyla from Hot Springs

Wilbur Hot Springs is a system of hot springs and a CO₂-driven geyser in Wilbur, California and serves as a natural source of mercury and reduced sulfur, which have important impacts on the water quality of the watershed. Hot spring ecosystems are also known to be rich sources of bacterial and archaeal candidate phyla, which can be challenging to recover and identify but likely play important ecological roles. Water and sediment was collected from 5 springs and the geyser in order to characterize the microbial community associated with each site and explore differences between sites, with a focus on candidate phyla. The composition of the microbial communities was assessed using 16S rRNA sequences from both Illumina-sequenced PCR amplicons (itags) and sequences extracted from shotgun metagenomic data using EMIRGE and an assembly-based pipeline. FOCUS was also used to generate a taxonomic profile of metagenomic reads based on k-mer composition relative to a reference database. Although many taxa were common to all springs, each spring had a distinct profile, with significant differences between sediment and water communities. Analysis methods differed in the amount of diversity they recovered, with EMIRGE yielding the fewest taxa and itags the most. Recovery of candidate phyla was greatest with the shotgun assembly-based method and reduced in itag datasets, likely due to PCR primer bias against these taxa, and also in FOCUS results, due to poor representation of candidate phyla in the reference database. These results highlight the utility of multi-pronged approaches to microbial community profiling, particularly when candidate phyla are targeted, and provide a foundation for future study of environmentally important transformations of mercury and sulfur in this ecosystem.