Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat

Abstract

The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119,000 nearly full-length sequences and 28,000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Figure 1

The GN microbial mat (a) photograph and (b) taxonomic composition by depth. Taxonomic assignments were made with tax2tree (Materials and methods). (a) and (b) are consistent in scale. Numeric values on top and bottom of (b) correspond to the phylum legend to aid orientation. As discussed in the text, the dashed white lines indicate approximate depths of the photic/oxic (0–3 mm depth, zone A), low sulfide (3–6 mm depth, zone B) and high sulfide (6 mm and deeper, zone C) zones seen with light intensity and microelectrode measurements (Jorgensen and Des Marais, 1986; Jorgensen, 1992; Ley et al., 2006).

Figure 2

Abundance of some prominent phyla and taxonomic subgroups by depth in the mat. (a) Cyanobacteria, (b) Bacteroidetes, (c) Proteobacteria, (d) Chloroflexi, (e) Planctomycetes, (f) Spirochetes, (g) and (h) other low-abundance phyla.

Figure 3

(a) Procrustes plot comparing PCoA analysis results from unweighted UniFrac distances for 454 and Sanger sequences. (b) Detrended Procrustes plot (Methods and materials) illustrates the correlation between PC1 and depth in the microbial mat. Points are colored by depth in the microbial mat with red indicating the top layers, yellow indicating the middle layers and blue indicating the bottom layers.

Figure 4

(a) Phylogenetic gain (new phylogenetic tree branch length, right axis) and OTU gain (left axis) by layer in the GN mat for Sanger reads with respect to the Greengenes database. (b) OTU counts from 97% identity to 35% identity for Sanger sequences. The red line shows the number of OTUs in Greengenes at a given OTU identity threshold; the blue line shows the number of OTUs in the GN data set at a given OTU identity threshold; and the green line shows the number of OTUs in the GN but not Greengenes data base, that is, the number of novel OTUs in the GN data set, at a given identity threshold. The y axis is log scale, so lines are not present when y=0. (c) Alpha rarefaction plots for Sanger reads. Plots are color-coded by depth in the mat according to the inset: red indicates top layers, yellow and green indicate middle layers and blue indicates bottom layers.