Ecology of speciation in the genus Bacillus

Abstract

Microbial ecologists and systematists are challenged to discover the early ecological changes that drive the splitting of one bacterial population into two ecologically distinct populations. We have aimed to identify newly divergent lineages ("ecotypes") bearing the dynamic properties attributed to species, with the rationale that discovering their ecological differences would reveal the ecological dimensions of speciation. To this end, we have sampled bacteria from the Bacillus subtilis-Bacillus licheniformis clade from sites differing in solar exposure and soil texture within a Death Valley canyon. Within this clade, we hypothesized ecotype demarcations based on DNA sequence diversity, through analysis of the clade's evolutionary history by Ecotype Simulation (ES) and AdaptML. Ecotypes so demarcated were found to be significantly different in their associations with solar exposure and soil texture, suggesting that these and covarying environmental parameters are among the dimensions of ecological divergence for newly divergent Bacillus ecotypes. Fatty acid composition appeared to contribute to ecotype differences in temperature adaptation, since those ecotypes with more warm-adapting fatty acids were isolated more frequently from sites with greater solar exposure. The recognized species and subspecies of the B. subtilis-B. licheniformis clade were found to be nearly identical to the ecotypes demarcated by ES, with a few exceptions where a recognized taxon is split at most into three putative ecotypes. Nevertheless, the taxa recognized do not appear to encompass the full ecological diversity of the B. subtilis-B. licheniformis clade: ES and AdaptML identified several newly discovered clades as ecotypes that are distinct from any recognized taxon.

FIG. 1.

Collection sites within Radio Facility Wash. Next to each site is the number of strains (if not zero) that were isolated from the clade most closely associated with silty soil (i.e., that containing putative ecotypes 12 and 19). This silt-associated clade was isolated from silty and sandy sites throughout the study area, and from both slopes.

FIG. 2.

Phylogenetic, Ecotype Simulation, and AdaptML analyses of the B. subtilis-B. licheniformis clade. The phylogeny is based on a maximum-parsimony analysis of the recombination-free concatenation of dnaJ, gyrA, and rpoB, rooted by strain C-125 of B. halodurans. The B. subtilis and B. licheniformis subclades are defined as the descendants of ancestors A and B, respectively, in the figure. Bootstrap support at >50% is indicated, except for nodes within putative ecotypes. The bracketed ecotype demarcations indicate the largest clades consistent with containing a single ecotype (in ES) and containing strains of a single habitat spectrum (in AdaptML). The habitat sources of strains from RFW are indicated in Table S1 in the supplemental material. For ecotypes with ≥5 RFW strains, the percentages of isolates from different habitats are indicated. Strains not isolated from RFW are indicated by asterisks, except that type strains are indicated by a “T.” Individuals that had recombined in one of the three protein-coding genes are indicated by an “R”; their positions in the tree are based on the remaining two genes that had not recombined. Putative ecotypes 1 to 17 have been demarcated previously (29). The strains previously assigned to PE 6 were found in the present analysis to fall within PE 12; those previously assigned to PE 9 are designated here as PE 9A and 9B.

FIG. 3.

Clade sequence diversity pattern for the B. subtilis-B. licheniformis clade. For both the observed and model curves, sequences for the concatenation of genes were binned by complete linkage clustering into clusters with different levels of minimum pairwise sequence identity, to yield the history of lineage splitting (29). The observed curve was based on the actual sequences from our survey of diversity, and the model curve was based on the average number of bins at each criterion level over 1,000 replicate runs of ES, using the parameter trio representing the maximum-likelihood solution (i.e., n = 28, Ω = 0.019, σ = 1.1).

FIG. 4.

Relationship between the mean heat adaptation index of a putative ecotype and the fraction of strains isolated from the south-facing slope. The heat adaptation index of each strain was quantified as the sum of the isomethyl-branched fatty acid levels divided by the sum of the anteisomethyl-branched and unsaturated fatty acid levels (Table 3).