A robust phylogenetic framework for ophiostomatoid fungi: orders Ophiostomatales and Microascales (Sordariomycetes, Ascomycota)

Ophiostomatoids are an ecological group of microfungi that commonly associate with bark and ambrosia beetles. As well as being insect symbionts, they play significant ecological roles as plant pathogens, and include species responsible for major forest tree diseases. Despite their ecological similarities, ophiostomatoids are distributed across two quite distantly related orders, the Microascales and Ophiostomatales. Historically, these fungi were considered a single natural group; however, molecular studies have revealed their independent origins and convergent ecological strategies. Previous phylogenetic studies of these fungi have typically focused on resolving taxonomic issues or understanding individual lifestyles, such as beetle-cultivated ambrosia lineages or vascular wilt pathogens. As a result, we lack a comprehensive phylogenetic framework that integrates dense species-level sampling with ecological data across both orders. Such frameworks are essential for understanding the broader phylogenetic and ecological context in which key fungal lifestyles have evolved. Here, we assembled and analysed all available sequence data for the Microascales and Ophiostomatales from seven widely used fungal marker loci to reconstruct a densely sampled phylogeny for each order. We evaluated locus performance and showed that whilst individual loci fail to resolve many taxa, concatenated datasets produce robust, well-supported topologies consistent with published genomic studies. By mapping ecological traits onto these trees, we show that lifestyle diversity and beetle associations are much more variable in the Microascales than in the Ophiostomatales, despite comparable species richness. Presenting both orders together provides a unique comparative perspective on the ecology and evolution of ophiostomatoids. As metabarcoding datasets of ophiostomatoids become increasingly common, this integrative framework can offer a valuable resource for environmental sequence identification and investigating fungal lifestyle switches, which in turn can support future biodiversity and ecology studies.