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

Abstract Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Here, we establish a robust genome-based framework for Aeromonas genomic species delineation. We analysed average nucleotide identity (ANI) across 3,782 genomes representing 33 currently recognized Aeromonas species and identified 95.2% ANI as a better threshold for the delineation of Aeromonas species. We defined the clusters at the 95.2% ANI cutoff as genomic species, and in the 3,782 genomes, identified 34 clusters. Of those, 30 correspond to existing Aeromonas species, whilst four are novel genomic species. The remaining three existing species were not supported as separate genomic species and merged into closely related species. The 34 genomic species were supported by genus core genome phylogenetic analysis and digital DNA-DNA hybridization (dDDH) values were less than 55%, between genomic species. We also found that eight existing Aeromonas species had intraspecies dDDH values below 70%, indicating that this conventional threshold does not consistently resolve species boundaries within Aeromonas. We further developed AeromonasGStyper, a genomic species typing tool that assigns query genomes based on ANI similarity to medoid genomes. Further testing of this tool on an additional 584 genomes validated its utility and identified four new genomic species. Thus, the Aeromonas genus currently comprises 38 genomic species. This framework standardized genome-based Aeromonas genomic species identification and supports future genomic surveillance. Competing Interest Statement The authors have declared no competing interest. Footnotes We have rewritten sections in the introduction, results, and discussion. We define delineation with the ANI threshold and clustering as "genomic species" instead of "ANI-defined species" and clearly distinguish the term from current established species definitions throughout the manuscript. Additionally, we have tested our AeromonasGStyper tool on an additional 584 genomes retrieved from the NCBI and SRA databases, representing the most recently available genomes in public databases.