Abstract: The availability of genome sequences for closely related microorganisms has at the same time clarified and complicated our view of species delineation. While 16S rRNA gene based classification generally corresponds to genomic and ecological differences, organisms grouped as one species often display both significant gene content variation as well as resource partitioning. In order to address the relationship between differences in gene content and sequence and ecological divergence in ecosystem context, I investigated links between genotype and ecology of two populations of Leptospirillum Group II bacteria in comprehensively characterized natural acidophilic biofilm communities. These populations share 99.7 percent 16S rRNA gene sequence identity and 95 percent average amino acid identity between their orthologs. One predominates during early colonization and the other typically proliferates in later successional stages, forming distinct tens to hundreds µm diameter patches. Absence of protein expression, measured via mass spectrometry-based community proteomics, of most population-specific gene content supports the argument that much of the laterally transferred gene pool found in closely related isolate genomes is of a transient, non-adaptive nature. Evolutionary signatures, and population-resolved expression patterns emphasize how sequence and expression variation of shared genes contributes to ecological divergence. As such, we highlight an interesting parallel to higher organisms, where evolution of gene expression has been suggested as an important factor in species differentiation. Ecological divergence between these two populations can be viewed as an example of r- vs. K-selection. The early colonizer proliferates optimally in the absence of competition from other organisms, thanks to adaptations that allow it to rapidly propagate in the AMD environment. Adaptations of the late colonizer allow its proliferation in conditions with high inter- and intra-specific competition for resources. Such distinct ecological strategies mediated by subtle genomic differences between closely related organisms, which are often found to co-exist in natural microbial systems, exemplify how fine-scale variation within ecological functional groups can have significant effects on community structure and functioning.