The epsomitic phototrophic microbial mat of Hot Lake, Washington: community structural responses to seasonal cycling

Abstract

Phototrophic microbial mats are compact ecosystems composed of highly interactive organisms in which energy and element cycling take place over millimeter-to-centimeter-scale distances. Although microbial mats are common in hypersaline environments, they have not been extensively characterized in systems dominated by divalent ions. Hot Lake is a meromictic, epsomitic lake that occupies a small, endorheic basin in north-central Washington. The lake harbors a benthic, phototrophic mat that assembles each spring, disassembles each fall, and is subject to greater than tenfold variation in salinity (primarily Mg(2+) and SO(2-) 4) and irradiation over the annual cycle. We examined spatiotemporal variation in the mat community at five time points throughout the annual cycle with respect to prevailing physicochemical parameters by amplicon sequencing of the V4 region of the 16S rRNA gene coupled to near-full-length 16S RNA clone sequences. The composition of these microbial communities was relatively stable over the seasonal cycle and included dominant populations of Cyanobacteria, primarily a group IV cyanobacterium (Leptolyngbya), and Alphaproteobacteria (specifically, members of Rhodobacteraceae and Geminicoccus). Members of Gammaproteobacteria (e.g., Thioalkalivibrio and Halochromatium) and Deltaproteobacteria (e.g., Desulfofustis) that are likely to be involved in sulfur cycling peaked in summer and declined significantly by mid-fall, mirroring larger trends in mat community richness and evenness. Phylogenetic turnover analysis of abundant phylotypes employing environmental metadata suggests that seasonal shifts in light variability exert a dominant influence on the composition of Hot Lake microbial mat communities. The seasonal development and organization of these structured microbial mats provide opportunities for analysis of the temporal and physical dynamics that feed back to community function.

Keywords: 16S tag sequencing; Hot Lake; community assembly; magnesium sulfate; microbial diversity; phototrophic microbial mats; phylogenetic turnover; seasonal cycling.

Figure 1

Physical characteristics of Hot Lake. (A) Aerial photograph of Hot Lake on August 6, 2011 showing the surrounding mixed grass and pine communities common within its endorheic basin and the gypsum flats flanking the lake. Mat was sampled at the location indicated by the yellow arrowhead. On the inset map of the state of Washington, the location of Hot Lake is represented by a white star. QuickBird imagery was provided by DigtalGlobe and Land Info Worldwide Mapping, inset map from the National Atlas of the United States. Seasonal changes in water level can be clearly seen from photographs of the north-easternmost basin of Hot Lake taken on July 7, 2011 (B) and October 20, 2011 (C).

Figure 2

Seasonal variation in the environmental conditions experienced by the mat community in Hot Lake. (A) Variation in salinity (as represented by total dissolved solids) and temperature in water proximal to sampled mat. December values are from water immediately below ice cover. (B) Variation in irradiance throughout 2011 as recorded by remote automated weather station OVLW1. Maximal recorded daily irradiance near Hot Lake was 9574 W/m² on June 26, while just 160 W/m² was recorded at minimum on January 7.

Figure 3

Ultrastructure of the Hot Lake mat sampled on September 1, 2011. (A) Cross-section of the Hot Lake mat at the millimeter scale. Orange (O), green (G), and pink (P) lamina are readily apparent along with interspersed carbonate minerals (C). (B) Ultrastructure of a 50 μm-thick section of the Hot Lake mat (100X magnification).

Figure 4

Light penetration into the Hot Lake mat. (A) Spectrally-resolved transmission of light through the mat as measured by fiber-optic microprobe. Numbers above the curve represent the depth the probe was inserted into the mat in millimeters. (B) Attenuation of wavelengths representing local maxima in absorbance. Values denote wavelength in nanometers.

Figure 5

Inter-sample variability in community structure. (A) Random sampling strategy. A grid comprising nine subsamples, each 5 mm on a side and encompassing the entire depth of the mat (usually 3–5 mm), was cut into the center of each mat sample, three of which were selected for sequencing per plate using a random number generator. Two plates containing independent mat samples were subsampled at each time point. (B) Mean Bray-Curtis distance as a function of the relationship between two samples. No significant difference in mean Bray-Curtis distance, as determined by unpaired Student's t-test assuming unequal variance, was detected between samples that share an edge (e.g., sample 2 and 5 in panel A) or corner (e.g., sample 5 and 7), or non-contiguous samples from the same plate (e.g., sample 2 and 7) or on different plates collected at the same sampling time point. A significant difference was observed between samples collected at the same time point and those collected at other time points (as denoted by the asterisk, p < 1 × 10⁻²⁶). (C) Neighbor-joining tree of Bray-Curtis β-diversity by sample.

Figure 6

Seasonal cycling of phylotypes within the Hot Lake mat community. (A) Variation in major phyla of the mat community. Phyla representing > 0.5% of the reads for at least one time point were included. (B) Variation in classes Cyanobacteria and Chloroplast. Family IV, Family XIII, and Cyanobacteria incertae ordinis represent subordinate taxa of class Cyanobacteria. (C) Variation in class Alphaproteobacteria and subordinate families Rhodobacteraceae, Rhodospirillaceae, and Alphaproteobacteria incertae familiaris. (D) Variation in Gammaproteobacteria and Deltaproteobacteria. Families Chromatiaceae and Ectothiorhodospiraceae are subordinate families of class Gammaproteobacteria and, like Deltaproteobacteria, contain many members involved in dissimilatory sulfur cycling.

Figure 7

Depth-resolved and seasonal abundance of major operational taxonomic units in the mat community. Intensity of color depicts log₂ transformed relative abundance data. (A) Seasonal cycling of major mat OTUs. After processing, reads were clustered at 97% identity using the average neighbor algorithm as implemented in mothur v. 1.29 and classified by kmer analysis using the Ribosomal Database Project training set 9 (released 3/20/2012). Each unique sequence was also mapped to the corresponding regions of the near full-length 16S sequences using the nucmer algorithm (as implemented in MUMmer 3.23). Short reads were considered to match full-length sequences if they were >99% identical across the entire amplified region. As near full-length sequences were also classified using the same protocol as the short reads, the classification with the best taxonomic resolution or bootstrap value was reported for an OTU as long as > 50% of its reads mapped to the corresponding near full-length sequence. ^*HL7711_P3D1 shares its V4 region with HL7711_P3F7 and HL7711_P3G11. (B) Depth-resolved abundance of major OTUs within mat sampled on July 7, 2011 and cryosectioned. OTUs are identical to those in panel A with the exception of OTU 273, which is omitted due to a lack of reads in the depth-resolved samples. Depths reported are the maxima for each sample and represent a 0.5 mm-thick laminar section.

Figure 8

Alpha diversity of the Hot Lake mat community. (A) Alpha diversity, richness, and evenness around the seasonal cycle. Unitless Simpson values are plotted on the left axis. Error bars represent standard error of the mean. Statistically significant differences (p < 0.05) are labeled above the point with the same letter. (B) Depth gradient in alpha diversity, richness, and evenness. Unitless Simpson values are plotted on the left axis. Depths are reported as the maximum for each sample (i.e., 0.5 mm denotes 0–0.5 mm).

Figure 9

Phylogenetic reconstruction of near full-length 16S sequences from the Hot Lake mat representing major OTUs. Clones were generated from mat sampled on July 7, 2011 and are in bold. Clusters of sequences with >99% identity are represented by a single sequence; the number of sequences represented by each is noted parenthetically. While a neighbor-joining tree is depicted above, nodes duplicated using a maximum-likelihood algorithm employing the general time-reversible model are notated with a diamond. Values near nodes represent neighbor-joining bootstrap values greater than 80. Terminal node colors denote phyla according to the same scheme used in Figure 6A. Classes Alphaproteobacteria and Gammaproteobacteria are enclosed in brackets.