Setting new boundaries of 16S rRNA gene identity for prokaryotic taxonomy

The 16S ribosomal RNA (rRNA) gene is frequently sequenced to classify prokaryotes and identify new taxa. If sequences from two strains share less than ∼99% identity, the strains are usually classified as different species. Classification thresholds for genera and other ranks have also been proposed, but they are based on dated datasets. Here we update these thresholds by determining sequence identity of the 16S rRNA gene for nearly 18,000 type strains. This represents 86.5% of all strains validly published, and it involved making more than 161 million pairwise sequence alignments. In 90% of all cases, sequences from the same species shared a minimum of 97.2 to 100% identity. The corresponding values for genus were 90.3 to 99.0%, 79.6 to 94.1% for family, 73.1 to 90.1% for order, 72.2 to 86.3% for class, and 69.7 to 83.6% for phylum. We propose these values serve as thresholds for classifying new prokaryotic taxa. A major change from previous guidelines is recognizing that these boundaries overlap. This overlap has already been observed for relative evolutionary divergence, a metric correlated with 16S rRNA gene identity. Together with other metrics, 16S rRNA gene identity allows classification of prokaryotes from species to phylum.