Hyperdiverse, bioactive, and interaction-specific metabolites produced only
in co-culture suggest diverse competitors may fuel secondary metabolism of
xylarialean fungi
Authors: Franco Mario E.E. , Nickerson Megan N. , Bowen Benjamin P. , Louie Katherine , Northen Trent R. , U’Ren Jana M. 
Published: January 2025

Abstract
Xylariales is one of the largest and most ecologically diverse fungal
orders. Xylarialean fungi are well-known for their chemical diversity,
reflecting a hyperdiversity of biosynthetic gene clusters (BCGs), even compared
to other bioactive fungi. Enhanced secondary metabolism appears linked to the
number of horizontal gene transfer (HGT) events and gene duplications, which is
highest in the clade that also has a greater ability to both degrade
lignocellulose as saprotrophs and interact with a wider variety of plant and
lichen hosts as symbiotic endophytes. Thus, one hypothesis for BGC
diversification in this clade is that diverse competitive interactions—in both
their free-living and symbiotic life stages—may exert selective pressure for HGT
and a diverse metabolic repertoire. Here, we tested this hypothesis using
untargeted metabolomics to examine how pairwise co-culture interactions between
seven xylarialean fungi influenced their metabolite production. Overall, we
detected >9,000 features, including putatively anti-microbial, insecticidal,
and medicinal compounds. In total, 6,115 features were over-represented in co-
cultures vs. 2,071 in monocultures. No features occurred in all 21 interactions,
and only 39% of features occurred in >10 different co-culture combinations.
Each additional co-culture interaction resulted in an 11 to 14-fold increase in
metabolite richness. Consistent with HGT, metabolite profiles did not reflect
phylogenetic relationships. Overall, the diversity and specificity of
metabolites support the role of widespread and diverse competitive interactions
to drive diversification of xylarialean metabolism. Additionally, as plant
hormones were only detected in co-culture, our results also reveal how
competition may influence the outcome of endophytic symbioses.

Importance
Saprotrophic and endophytic xylarialean fungi are among the most
prolific producers of bioactive secondary metabolites, which are often
encoded by sets of physically clustered genes (i.e., biosynthetic gene
clusters). Secondary metabolites have numerous industrial uses as
antibiotics, pharmaceuticals, and insecticidal toxins, but their ecological
importance for competitive and symbiotic interactions with other fungi,
bacteria, and plants is not fully understood. Here, we examined how
co-culture interactions with ecologically and genetically diverse
competitors influence the metabolite production of seven xylarialean
strains. Among 21 pairwise interactions, we detected >9,000 metabolite
features, 67% of which were produced only in specific co-cultures and not in
monocultures. Over 8,000 features were unclassified at any level and likely
represent novel bioactive compounds. Our data illustrate that competitor
identity and ecology significantly impact xylarialean fungal secondary
metabolite production, key information that will enable us to further
harness the immense biotechnological potential of these fungi.