Fine-scale distribution patterns of Synechococcus ecological diversity in microbial mats of Mushroom Spring, Yellowstone National Park

Abstract

Past analyses of sequence diversity in high-resolution protein-encoding genes have identified putative ecological species of unicellular cyanobacteria in the genus Synechococcus, which are specialized to 60°C but not 65°C in Mushroom Spring microbial mats. Because these studies were limited to only two habitats, we studied the distribution of Synechococcus sequence variants at 1°C intervals along the effluent flow channel and at 80-μm vertical-depth intervals throughout the upper photic layer of the microbial mat. Diversity at the psaA locus, which encodes a photosynthetic reaction center protein (PsaA), was sampled by PCR amplification, cloning, and sequencing methods at 60, 63, and 65°C sites. The evolutionary simulation programs Ecotype Simulation and AdaptML were used to identify putative ecologically distinct populations (ecotypes). Ecotype Simulation predicted a higher number of putative ecotypes in cases where habitat variation was limited, while AdaptML predicted a higher number of ecologically distinct phylogenetic clades in cases where habitat variation was high. Denaturing gradient gel electrophoresis was used to track the distribution of dominant sequence variants of ecotype populations relative to temperature variation and to O₂, pH, and spectral irradiance variation, as measured using microsensors. Different distributions along effluent channel flow and vertical gradients, where temperature, light, and O₂ concentrations are known to vary, confirmed the ecological distinctness of putative ecotypes.

Fig. 1.

Neighbor-joining phylogenetic tree based on B′-like Synechococcus psaA sequences recovered from a 60°C site in the Mushroom Spring mat. Brackets show clusters of sequences demarcated as PEs by ES or as an EC by AdaptML; subclades with dominant variants within PEs also are indicated. The ratio of the number of dominant sequence variants to the total number of variants in a PE or subclade is indicated within parentheses to the right of the PE designation. The triangle orientation indicates association with upper (▴) and lower (▾) mat layers based on DGGE results. The location of the Synechococcus sp. strain B′ genomic homolog is shown by the arrow. Single sequences demarcated as PEs are indicated with asterisks. The scale bar indicates the number of fixed point mutations per nucleotide position.

Fig. 2.

Neighbor-joining phylogenetic tree based on A-like Synechococcus psaA sequences recovered from 60, 63, and 65°C sites in the Mushroom Spring mat. Brackets show clusters of sequences demarcated as PEs by ES or as ECs by AdaptML; subclades with dominant variants within PEs also are indicated. The ratio of the number of dominant sequence variants to the total number of variants in the PE, subclade, or EC is indicated within parentheses to the right of the PE designation. Colored symbols indicate the temperature at which the majority of sequences within a putative ecotype were collected (blue, 60°C; purple, 63°C; red, 65°C); the triangle orientation indicates association with upper (▲) and lower (▼) mat layers based on DGGE results. Circles indicate temperature distribution only. Numbers separated by colons to the right of some symbols indicate the number of sequences retrieved from each site (60°C:63°C:65°C). The location of the Synechococcus sp. strain A genomic homolog is shown by the arrow. Single sequences demarcated as PEs are indicated with asterisks. The scale bar indicates the number of fixed-point mutations per nucleotide position.

Fig. 3.

Denaturing gradient gel electrophoresis analysis of PCR-amplified psaA gene segments obtained from Mushroom Spring microbial mat samples collected at different temperature-defined sites (59 to 65°C) along the main flow path in the effluent channel. Labeled bands were purified, sequenced, and found to correspond to the dominant variants of putative ecotypes B′7, B′9, A1, A7, and A9 shown in Fig. 1 and 2. The direction of electrophoresis is top to bottom.

Fig. 4.

Vertical distribution of psaA variants, light, O₂, and pH at 60°C (A) and 63°C (B) sites in the microbial mat inhabiting the effluent channel of Mushroom Spring. The column on the left shows denaturing gradient gel electrophoresis analyses of PCR-amplified psaA gene segments obtained from 80-μm-thick vertical sections of the top green layers. Arrows indicate the different positions of closely migrating dominant alleles, which appear as one band in whole-mat samples and comigrate with the PE A1 and A7 band that was purified and sequenced (Fig. 3). The direction of electrophoresis is left (top) to right (bottom). The middle column shows spectral scalar irradiance (400 to 700 nm). Data were normalized to the downwelling irradiance at the mat surface. The spectral coverage in deeper layers was narrowed due to spectrometer stray light at wavelengths of <500 nm. The column on the right shows in situ depth profiles of O₂ concentration (solid symbols) and pH (open symbols) measured at noon (between 12:00 p.m. and 1:00 p.m. at an irradiance of >1,600 mmol photons m⁻² s⁻¹).

Fig. 5.

Denaturing gradient gel electrophoresis analyses of PCR-amplified psaA gene segments obtained from clones containing dominant alleles of closely related A-like putative ecotypes and mat DNA from 65 and 60°C samples (MS65 and MS60, respectively).