Ecological interactions drive metabolomic diversification in Amazonian <i>Pseudonocardia</i> symbionts

Fungus-growing ants engage in a multipartite symbiosis, including <i>Pseudonocardia</i> bacteria that produce antifungal metabolites to protect their fungal cultivar from the specialized pathogen <i>Escovopsis</i>. While different bioactive metabolites have been reported from ant-associated <i>Pseudonocardia</i>, most studies have focused on a limited number of strains, leaving the extent of chemical diversity across broader ecological contexts less resolved. Here, we investigated the antagonistic potential and metabolomic repertoires of 36 <i>Pseudonocardia</i> strains isolated from Amazonian <i>Paratrachymyrmex</i> ants. Pairwise bioactivity assays against two <i>Escovopsis</i> isolates revealed striking variability, with inhibition generally stronger and more diverse against the pathogenic fungus originating from the same ant genus. Untargeted liquid chromatography-tandem mass spectrometry metabolomics coupled with 16S rRNA-based phylogenetic analyses showed that closely related strains harbored highly divergent chemical profiles, underscoring a decoupling between taxonomy and metabolite output. Detailed analyses of selected isolates revealed the production of structurally diverse metabolites, including dentigerumycin analogs, provipeptide A, β-carbolines, and tetracycline-related compounds. Co-culture analysis uncovered metabolites absent in monocultures, including lichenysins, pepstatins, and hallobacillins, as well as conserved attinimicin, whose production was enhanced under pathogen challenge. These results highlight that both strain-specific metabolic repertoires and interaction-induced chemistry contribute to the defensive arsenal of <i>Pseudonocardia</i>. Together, our findings likely demonstrate that ecological pressures and local adaptation, rather than phylogeny alone, drive metabolomic diversification in this defensive symbiosis. Beyond their potential for novel bioactive compound discovery, these results provide understanding into the chemical basis of multipartite symbioses, the dynamics of defensive mutualisms, and the ecological forces shaping microbial diversity in underexplored environments such as the Amazon.IMPORTANCEMicrobial symbionts are central to host defense and natural product discovery, yet the factors driving their chemical diversification remain unclear. The fungus-growing ant-<i>Pseudonocardia-Escovopsis</i> system offers a powerful model to study how ecological context shapes microbial metabolism. By systematically characterizing multiple Amazonian <i>Pseudonocardia</i> strains, we show that antagonistic capacity and metabolomic repertoires vary widely, even among strains with highly similar 16S rRNA gene sequences, revealing a pronounced discordance between 16S-based phylogenetic relatedness and specialized metabolite production. These findings suggest the likely importance of ecological pressures and local adaptation in shaping metabolomic output, emphasizing symbiotic actinobacteria as both key ecological players and promising sources of antifungal natural products.