An ANI gap within bacterial species that advances the definitions of intra-species units

Large-scale surveys of prokaryotic communities (metagenomes) as well as isolate genomes have revealed that their diversity is predominantly organized in sequence-discrete units that may be equated to species. Specifically, genomes of the same species commonly show genome-aggregate average nucleotide identity (ANI) >95% among themselves and ANI <90% to members of other species, while genomes showing ANI 90-95% are comparatively rare. However, it remains unclear if such “discontinuities” or gaps in ANI values can be observed within species and thus, used to advance and standardize intra-species units such as strains and sequence types. By analyzing 18,123 complete isolate genomes from 330 bacterial species with at least ten genome representatives each as well as available long-read metagenomes, we show that such a discontinuity exists between 99.2-99.8% (mean 99.5%) ANI. The 99.5% ANI threshold is largely consistent with how sequence types have been defined in previous epidemiological studies but provides clusters with ∼20% higher accuracy in terms of evolutionary and gene-content relatedness of the grouped genomes, while strains should be consequently defined at higher ANI values (>99.99% proposed). Collectively, our results should facilitate future micro-diversity studies across clinical or environmental settings because they provide a more natural definition of intra-species units of diversity.