Curated multi-marker analysis resolves fungal phylogeny and ecological patterns in two ophiostomatoid orders

A computational multi-marker analysis recovered Ophiostomatales and Microascales as monophyletic fungal orders and produced densely sampled reference trees connecting named, vouchered specimens with ecological lifestyles and beetle associations. The study assessed phylogenetic performance across seven widely used nuclear markers rather than conducting a field, animal or clinical experiment.

Researchers compiled publicly available sequences for the two orders and their closest outgroups. The loci included SSU, ITS, LSU, β-tubulin, EF1α, RPB1 and RPB2. Separate β-tubulin copies expanded the Microascales analysis to eight loci. Alignments were manually curated, suspected misidentifications and contaminants were checked with BLAST, and maximum-likelihood trees were inferred with IQ-TREE using partitioned models, SH-aLRT tests and ultrafast bootstrap analyses.

Of 18,121 downloaded Microascales sequences, 1,716 remained after curation. The final alignment contained 918 concatenated sequences and 15,995 sites, including 5,453 informative sites. For Ophiostomatales, 1,947 of 17,147 downloaded sequences remained, yielding 859 concatenated sequences across 21,603 sites, of which 4,363 were informative. These reductions largely reflected the removal of misidentified sequences and extensive redundancy among heavily sampled species.

Concatenating markers generally produced stronger support than analyses based on individual loci. ITS had the most available sequences in both orders, but it also showed high sequence variation and extreme nucleotide-frequency heterogeneity. These properties may complicate reconstruction of deeper relationships. The analysis also found widespread disagreement among genes and alignment sites, with low site-concordance values across most nodes and gene-concordance values ranging from complete agreement to little or no support.

In Microascales, the tree supported the previously proposed subdivision among Ceratocystidaceae, Gondwanamycetaceae, Microascaceae and Halosphaeriaceae, while the position of Graphiaceae remained unclear. Ceratocystidaceae, Gondwanamycetaceae, Graphiaceae and Halosphaeriaceae were recovered as monophyletic. Microascaceae was also monophyletic but lacked strong support, although many of its genera formed well-supported groups.

The Ophiostomatales tree confirmed most genera and species complexes from a recent genus-level revision. It recovered the Ophiostoma–Sporothrix and Leptographium–Grosmannia clades, although neither received ultrafast bootstrap support. Graphilbum was placed as sister to the Ophiostoma–Sporothrix clade, a placement that conflicts with results from other studies and leaves that relationship as an area of disagreement rather than a settled conclusion.

Ecological annotations showed contrasting patterns between the orders. Ophiostomatales taxa were generally classified as plant pathogens, wood saprotrophs or both, and beetle associations were reported for almost all genera. Microascales displayed a broader range of lifestyles and a more uneven distribution of beetle associations. Ambrosia lifestyles appeared in three distantly related Ophiostomatales clades and in two Microascales clades containing Ambrosiella, Toshionella, Phialophoropsis and Wolfgangiella.

These ecological comparisons have important limitations. Lifestyle records came largely from FUNGuild and were assigned at species, genus or family level when more specific data were unavailable. Such hierarchical assignments can suggest ecological transitions that do not necessarily reflect biological changes, so the study used them to contextualize available ecological knowledge rather than directly infer trait evolution. Sampling also remained incomplete for two small Microascales families, and some deeper placements and species-complex relationships received weak support.

The study’s open-access alignments, trees, supplementary taxon information and analysis code establish a standardized community resource. The authors position the framework as a foundation for placing environmental sequences, identifying stable clades, tracing lineage-specific lifestyles and examining the phylogenetic context of emerging plant pathogens, while noting that genome-scale data and more complete species-level ecological records are needed to resolve some uncertainties.

Subject of Research: Computational phylogenomics and ecological mapping of the fungal orders Ophiostomatales and Microascales

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

Article References: Llewellyn, T., & Vogler, A. P. (2026). A robust phylogenetic framework for ophiostomatoid fungi: orders Ophiostomatales and Microascales (Sordariomycetes, Ascomycota). MycoKeys, 140, 103-129. https://doi.org/10.3897/mycokeys.140.201772

Image Credits: AI Generated

DOI: 10.3897/mycokeys.140.201772

Keywords: ophiostomatoid fungi, Ophiostomatales, Microascales, phylogenetic framework, multi-marker analysis, fungal systematics, ecological guilds, beetle associations, maximum-likelihood phylogeny, sequence curation, robust, phylogenetic