Integrating ANI and phylogenies for re-evaluation of Fusobacterium taxonomy and disease associations.

The genus Fusobacterium encompasses significant pathogens implicated in diseases spanning from infections to cancer. However, taxonomic ambiguities persist within the genus, particularly concerning Fusobacterium nucleatum (sensu lato). Through genus-wide average nucleotide identity (ANI) and phylogenetic analyses of 540 Fusobacterium genomes, we identify an ANI gap (93.38%-93.89%) for species delineation, leading to comprehensive taxonomic revisions that resolve these ambiguities. We further establish gyrB and rpoB as high-resolution taxonomic markers with phylogenies consistently supporting the revised taxonomy. Leveraging these markers, we develop B&B, a general strategy for precise species identification without whole-genome sequencing, and validate its accuracy in clinically relevant strains. Integrating the revised taxonomy with genomic/metagenomic toolkits demonstrate broad utilities, reinterpreting key colorectal cancer-associated species. This work establishes a unified taxonomic framework and enables standardised species classification for Fusobacterium isolates and microbiomes, highlighting the genetic divergence among Fusobacterium species and providing the taxonomic precision essential for advancing Fusobacterium-related research.