Hyperdiverse, bioactive, and interaction-specific metabolites produced only in co-culture suggest diverse competitors may fuel secondary metabolism of xylarialean fungi.

Xylariales is one of the largest and most ecologically diverse fungal orders that is well-known for its chemical diversity. Enhanced secondary metabolism of Xylariales taxa is associated with increased gene duplication and horizontal gene transfer (HGT) of biosynthetic gene clusters (BGCs), especially in generalist taxa with both greater saprotrophic abilities and broader host ranges as foliar endophytic symbionts. Thus, one hypothesis for BGC diversification among more generalist fungi is that diverse competitive interactions-in both their free-living and symbiotic life stages with many hosts-may exert selective pressure for HGT and a diverse metabolic repertoire. Here, we used untargeted metabolomics to examine how competition (pairwise co-cultures) between seven xylarialean fungi influenced their metabolite production. Of the >9,000 total features detected, 6,115 and 2,071 were over-represented in co-cultures vs monocultures, respectively. For each strain, each additional co-culture interaction resulted in an 11- to 14-fold increase in metabolite richness compared to monocultures, reflecting the limited amount of metabolite overlap among different co-culture combinations. Phylogenetic relatedness and BGC content did not impact the diversity of metabolites produced in co-culture; however, co-cultures between more ecologically distinct fungi elicited the strongest metabolic response. Overall, the diversity, specificity, and putative bioactivity of metabolites over-represented in co-culture support the role of widespread and diverse competitive fungal interactions to drive xylarialean metabolic diversification. Additionally, as fungal-produced plant hormones were only detected in co-culture, our results reveal the potential for