Defining Aeromonas species boundaries with large-scale average nucleotide identity analyses and genomic phylogeny

Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Here, we establish a robust genome-based framework for Aeromonas species delineation. We analyzed genome-wide average nucleotide identity (ANI) across 3,782 genomes representing the 33 currently recognized Aeromonas species and identified 95.2% ANI as the species boundary. This threshold defined 34 ANI-based species, comprising 30 corresponding to existing Aeromonas species and four potential novel species, while three existing species were merged into closely related species. A genus-level core genome of 673 loci was defined using 392 representative genomes, and core-genome phylogenetic analysis of 1,505 genomes showed consistency with ANI-based clustering. Digital DNA-DNA hybridization (dDDH) values between clusters were [≤] 55%, further supporting species separation. We also found that nine Aeromonas species had intraspecies dDDH values below 70%, cautioning the use of this cutoff for species delineation. We provide the tool Aeromonas ANI species typer (AeromonasANIStyper), an ANI-based species typing tool that assigns query genomes using ANI similarity to medoid genomes and achieved 100% accuracy. This framework supports standardized genome-based Aeromonas taxonomy and future surveillance. ImportanceAeromonas species have gained increased attention as emerging human enteric pathogens. Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Although an average nucleotide identity (ANI) threshold was proposed previously for the Aeromonas species delineation, the growing number and diversity of genomes requires a more rigorous reassessment of species boundaries. By analyzing 3,782 Aeromonas genomes, we identified 95.2 % ANI as an optimal cutoff for defining Aeromonas species. Using this threshold, we delineated 34 ANI-defined species, which were further validated by core genome phylogeny and digital DNA-DNA hybridization (dDDH). In addition, we clarified previously misidentified species. Importantly, we provide the Aeromonas ANI species typer (AeromonasANIStyper), a tool that accurately assigns Aeromonas genomes to ANI-defined species. These findings clarify Aeromonas species identification and support improved pathogen surveillance, evolutionary and epidemiological studies.