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. In addition to being insect symbionts, they play significant ecological roles as plant pathogens and include species responsible for major forest tree diseases. Despite their ecological similarities, ophiostomatoid fungi are distributed across two distantly related orders, Microascales and Ophiostomatales , which include many additional fungi with diverse ecological lifestyles. Previous studies have typically focused on localised taxonomic issues or specific fungal ecologies. As a result, public repositories suffer from fragmented locus coverage, sequence misidentifications, and uneven taxon sampling, leaving the community without a standardised, comprehensive dataset to study these fungi at scale. To address this, all available ophiostomatoid fungal sequence data across seven widely used marker loci were curated and analysed to reconstruct densely sampled alignments and trees covering 918 concatenated Microascales sequences and 859 Ophiostomatales sequences. Evaluation of locus performance showed that whilst individual loci exhibit extreme state frequency heterogeneity, composition bias, and topological discordance, concatenated datasets produce robust, well-supported topologies consistent with published genomic studies. Integrating phylogenies with broad ecological trait data reveals a clear contrast in data distribution between the two orders, with ecological lifestyles and beetle associations being broadly uniform and stable across the Ophiostomatales tree but highly heterogeneous in the Microascales , despite comparable species richness. These macroecological data distributions are also shaped by taxonomic philosophies, whereby the fragmentation of Microascales into small, restricted genera tends to visually amplify trait variation, whereas the large, heterogeneous genera of Ophiostomatales can obscure it. This unified, fully reproducible framework establishes the stable topology and alignments necessary for future molecular taxonomy and trait evolution analyses, while providing a high-quality reference for environmental sequence identification, which in turn can support future biodiversity and biosecurity research.