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Bacillus globisporus and Bacillus psychrophilus are one among many pairs of ecologically distinct taxa that are distinguished by very few nucleotide differences in 16S rRNA gene sequence. This study has investigated whether the lack of divergence in 16S rRNA between such species stems from the unusually slow rate of evolution of this molecule, or whether other factors might be preventing neutral sequence divergence at 16S rRNA as well as every other gene. B. globisporus and B. psychrophilus were each surveyed for restriction-site variation in two protein-coding genes. These species were easily distinguished as separate DNA sequence clusters for each gene. The limited ability of 16S rRNA to distinguish these species is therefore a consequence of the extremely slow rate of 16S rRNA evolution. The present results, and previous results involving two Mycobacterium species, demonstrate that there exist closely related species which have diverged long enough to have formed clearly separate sequence clusters for protein-coding genes, but not for 16S rRNA. These results support an earlier argument that sequence clustering in protein-coding genes could be a primary criterion for discovering and identifying ecologically distinct groups, and classifying them as separate species.

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