Gene presence–absence and evolutionary signals of positive selection associated with virulence divergence in Beauveria fungi

Beauveria bassiana is widely used as a mycoinsecticide and studied for its pathogenic mechanisms. However, the virulence biology of other Beauveria species remains poorly understood. In this study, we conducted comparative pathological and genomic analyses from seven species representing the B. bassiana and Beauveria asiatica species complexes. Virulence assays on Spodoptera exigua revealed substantial intra- and interspecific variability, with Beauveria neobassiana emerging as the most virulent. Notably, insect mortality does not correlate with ‘mycelia on cadavers’, highlighting the need for multiple virulence proxies. Draft genomes of the Beauveria species included in the virulence assays were assembled and annotated. Gene presence–absence analyses identified 195 orthogroups potentially related to virulence, including genes encoding proteases, transporters and lipid-modifying enzymes. Over 70% of these orthogroups lacked functional annotations, consistent with lineage-specific or rapidly evolving virulence factors. Genome-wide scans for positive selection occurring in high-virulence strains revealed enrichment in genes with signal peptides, suggesting roles as effectors. B. bassiana NHJ13051 and Beauveria thailandica MY4824 exhibited high numbers of gene gains and positively selected genes, suggesting independent evolution of virulence in different lineages. A secondary metabolite biosynthetic gene cluster containing terpene and non-ribosomal peptide synthase (NRPS) core genes is strongly associated with high-virulence strains. Targeted comparative analyses further showed that B. neobassiana harboured more NRPS core genes under positive selection than other Beauveria species included in this study. These findings reveal that virulence variation in Beauveria is shaped by lineage-specific gene gains, selection on secreted proteins and metabolic diversification. Our study provides a genomic framework for understanding entomopathogenicity beyond B. bassiana and highlights promising candidate genes for biocontrol enhancement.