Aspergillus flavus and Aspergillus oryzae are closely related fungal species with contrasting roles in food safety and fermentation. To comprehensively investigate their phylogenetic, genomic, and metabolic characteristics, we conducted an extensive comparative pangenome analysis using complete, dereplicated genome sets for both species. Phylogenetic analyses, employing both the entirety of the identified single-copy orthologous genes and six housekeeping genes commonly used for fungal classification, did not reveal clear differentiation between A. flavus and A. oryzae genomes. Upon analyzing the aflatoxin biosynthesis gene clusters within the genomes, we observed that non-aflatoxin-producing strains were dispersed throughout the phylogenetic tree, encompassing both A. flavus and A. oryzae strains. This suggests that aflatoxin production is not a distinguishing trait between the two species. Furthermore, A. oryzae and A. flavus strains displayed remarkably similar genomic attributes, including genome sizes, gene contents, and G + C contents, as well as metabolic features and pathways. The profiles of...

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