Temperature and Geographic Location Impact the Distribution and Diversity of Photoautotrophic Gene Variants in Alkaline Yellowstone Hot Springs

Abstract

Alkaline hot springs in Yellowstone National Park (YNP) provide a framework to study the relationship between photoautotrophs and temperature. Previous work has focused on studying how cyanobacteria (oxygenic phototrophs) vary with temperature, sulfide, and pH, but many questions remain regarding the ecophysiology of anoxygenic photosynthesis due to the taxonomic and metabolic diversity of these taxa. To this end, we examined the distribution of genes involved in phototrophy, carbon fixation, and nitrogen fixation in eight alkaline (pH 7.3-9.4) hot spring sites near the upper temperature limit of photosynthesis (71ºC) in YNP using metagenome sequencing. Based on genes encoding key reaction center proteins, geographic isolation plays a larger role than temperature in selecting for distinct phototrophic Chloroflexi, while genes typically associated with autotrophy in anoxygenic phototrophs, did not have distinct distributions with temperature. Additionally, we recovered Calvin cycle gene variants associated with Chloroflexi, an alternative carbon fixation pathway in anoxygenic photoautotrophs. Lastly, we recovered several abundant nitrogen fixation gene sequences associated with Roseiflexus, providing further evidence that genes involved in nitrogen fixation in Chloroflexi are more common than previously assumed. Together, our results add to the body of work on the distribution and functional potential of phototrophic bacteria in Yellowstone National Park hot springs and support the hypothesis that a combination of abiotic and biotic factors impact the distribution of phototrophic bacteria in hot springs. Future studies of isolates and metagenome assembled genomes (MAGs) from these data and others will further our understanding of the ecology and evolution of hot spring anoxygenic phototrophs. IMPORTANCE Photosynthetic bacteria in hot springs are of great importance to both microbial evolution and ecology. While a large body of work has focused on oxygenic photosynthesis in cyanobacteria in Mushroom and Octopus Springs in Yellowstone National Park, many questions remain regarding the metabolic potential and ecology of hot spring anoxygenic phototrophs. Anoxygenic phototrophs are metabolically and taxonomically diverse, and further investigations into their physiology will lead to a deeper understanding of microbial evolution and ecology of these taxa. Here, we have quantified the distribution of key genes involved in carbon and nitrogen metabolism in both oxygenic and anoxygenic phototrophs. Our results suggest that temperature >68ºC selects for distinct groups of cyanobacteria and that carbon fixation pathways associated with these taxa are likely subject to the same selective pressure. Additionally, our data suggest that phototrophic Chloroflexi genes and carbon fixation genes are largely influenced by local conditions as evidenced by our gene variant analysis. Lastly, we recovered several genes associated with potentially novel phototrophic Chloroflexi. Together, our results add to the body of work on hot springs in Yellowstone National Park and set the stage for future work on metagenome assembled genomes.

Keywords: Chloroflexi; anoxygenic photosynthesis; cyanobacteria; hot springs; metagenomics; photosynthesis; phototroph.

FIG 1

Principal component analysis of site meta-data. Principal components were calculated using the numeric data in Table S1A. Sites are labeled by site ID in corresponding Table S1 and shaded by Yellowstone National Park area.

FIG 2

Distribution of photosynthetic genes with temperature. The overall abundance (normalized ln(1 + reads mapped)) of genes that encode for Cyanobacterial photosystem II (psb) and type II anoxygenic photosynthesis reaction centers (puf) are shown as box plots for each site. Triangles represent the mean abundance for the gene set, and dots represent individual gene abundances, shaded and separated by the corresponding photosystem or reaction center gene (KEGG Orthology IDs are shown with gene name). Boxes represent the inter quartile range (Q1–Q3) and whiskers (lines) represent the maximum and minimum, with outliers removed (±2.5 standard deviations from the mean). A gray line divides the sites into high temperature and low temperature groups. Sites are ordered by increasing temperature.

FIG 3

Richness and distribution of psbA gene variants. Rank abundance plots for each site are displayed in increasing temperature order. Plots display abundances as normalized ln(1 + reads mapped) for each psbA OTU, and OTUs are ranked in order from most to least abundant. Bars are labeled with the OTU number. Striped bars represent OTUs that are present in more than one site.

FIG 4

Richness and distribution of pufLM gene variants. Rank abundance plots for each site are displayed in increasing temperature order. Plots display abundances as normalized ln(1 + reads mapped) for each pufLM OTU, and OTUs are ranked in order from most to least abundant. Bars are labeled with the OTU number. Striped bars represent OTUs that are present in more than one site.

FIG 5

Abundance and distribution of key genes in phototrophic carbon fixation pathways. The abundance (normalized ln(1 + reads mapped)) of key genes in the Calvin cycle (A) and the 3-hydroxypropionate bicycle (B) are shown as box plots for each site. Triangles represent the mean abundance for the gene set, and dots represent individual gene abundances, shaded by the genes. Boxes represent the inter quartile range (Q1–Q3), and whiskers (lines) represent the maximum and minimum, with outliers removed (±2.5 standard deviations from the mean). Sites are ordered by increasing temperature. A gray line divides the sites into high temperature and low temperature groups. Sites are ordered by increasing temperature. To determine significant differences in gene abundance in all sites, a Kruskal-Wallis H test followed by Dunn’s Multiple Comparison post hoc test for significant differences between sites. Only Bonferroni-adjusted P values < 0.05 are shown for brevity (all site comparison adjusted P values are shown in Table S3).

FIG 6

Richness and distribution of nifH gene variants. Rank abundance plots for each site are displayed in increasing temperature order. Plots display abundances as normalized ln(1 + reads mapped) for each nifH OTU, and OTUs are ranked in order from most to least abundant. Bars are labeled with the OTU number. Striped bars represent OTUs that are present in more than one site.