Abstract
Long-term symbiotic associations often lead to reciprocal adaptation between the involved entities. One of the main challenges for studies of such symbioses is differentiating adaptation from neutral processes and phylogenetic background. Ambrosia fungi, cultivated by ambrosia beetles as their sole food source, provide an excellent model to study evolutionary adaptation in a comparative framework because they evolved many times, and each origin bears features seemingly convergently adapted to the symbiosis. We tested whether the symbiotic lifestyle of unrelated ambrosia fungi has led to convergence in the key feature of the symbiotic phenotype-nutrition provisioning to the vector beetles. We compared conidia and mycelium content in three phylogenetic pairs of ambrosia fungi and their closely related nonambrosia relatives using an untargeted metabolomic assay. Multivariate analysis of 311 polar metabolites and 14063 lipid features revealed no convergence of nutrient content across ambrosia lineages. Instead, most variation of the metabolome composition was explained by phylogenetic relationships among the fungi. Thus the overall metabolome evolution of each ambrosia fungus is mostly driven by its inherited metabolism rather than the transition toward symbiosis. We identified eight candidate lipid compounds with expression levels different between the swollen ambrosia spores and other tissues, but they were not consistently elevated across ambrosia fungi. We conclude that ambrosia provisions consist either of nonspecific nutrients in elevated amounts, or of metabolites that are specific to each of the ambrosia symbioses.