Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes

TLDR

ANI is a robust genomic relatedness metric that can replace labor‑intensive DNA‑DNA hybridization, with a 95‑96% threshold previously suggested but not yet validated across all prokaryotic lineages. The authors examined ANI distributions across 6,787 genomes from 22 phyla to determine whether the 95‑96% cutoff applies universally and to identify the 16S rRNA similarity that corresponds to this ANI threshold. They performed pairwise ANI calculations on 6,787 genomes and over one million 16S rRNA similarity comparisons, using two‑fold cross‑validation to link ANI thresholds to 16S rRNA similarity. The study found a distinct intra‑ versus inter‑species ANI split at 95‑96% and established 98.65% 16S rRNA similarity as the species threshold, corroborating earlier estimates and accelerating genomic taxonomy.

Abstract

Among available genome relatedness indices, average nucleotide identity (ANI) is one of the most robust measurements of genomic relatedness between strains, and has great potential in the taxonomy of bacteria and archaea as a substitute for the labour-intensive DNA-DNA hybridization (DDH) technique. An ANI threshold range (95-96%) for species demarcation had previously been suggested based on comparative investigation between DDH and ANI values, albeit with rather limited datasets. Furthermore, its generality was not tested on all lineages of prokaryotes. Here, we investigated the overall distribution of ANI values generated by pairwise comparison of 6787 genomes of prokaryotes belonging to 22 phyla to see whether the suggested range can be applied to all species. There was an apparent distinction in the overall ANI distribution between intra- and interspecies relationships at around 95-96% ANI. We went on to determine which level of 16S rRNA gene sequence similarity corresponds to the currently accepted ANI threshold for species demarcation using over one million comparisons. A twofold cross-validation statistical test revealed that 98.65% 16S rRNA gene sequence similarity can be used as the threshold for differentiating two species, which is consistent with previous suggestions (98.2-99.0%) derived from comparative studies between DDH and 16S rRNA gene sequence similarity. Our findings should be useful in accelerating the use of genomic sequence data in the taxonomy of bacteria and archaea.