Reflexicalyptrales ordo nov., a distinct Agaricomycetes lineage discovered from orchid mycorrhizae

Uncovering the identity of "dark fungi" is crucial for understanding fungal taxonomy, diversity, and phylogenetic relationships. Previous exploratory analysis of the mycorrhizal fungi of the partially mycoheterotrophic orchid Nervilia nipponica revealed an association with a particular group of Agaricomycetes, tentatively designated "unidentified Agaricomycetes X" (uAX). Two culture strains were successfully isolated from the mycorrhiza, which enabled the phylogenetic position of this fungus to be determined using ultrastructural and multigene phylogenetic analyses. Transmission electron microscopy observations of the two cultures of uAX revealed that the dolipore septum is associated with perforate septal pore caps (SPCs), the margins of which are reflexed outwards. This combination of characters has not previously been reported in any order of Basidiomycota. Combined phylogenetic analyses based on sequence data from four loci [5.8S and 28S ribosomal DNA (rDNA), translation elongation factor 1-α, and RNA polymerase II second large subunit] indicate that uAX is an early-diverging clade of Agaricomycetes that cannot be accommodated in any known order of Basidiomycota. The name Reflexicalyptrales is proposed here for this new order of Basidiomycota. Furthermore, BLAST searches of the internal transcribed spacers of nuclear ribosomal DNA (nrDNA) and phylogenetic analyses reveal that closely related taxa have previously been detected in soil, leaf litter, and living root samples collected at various locations worldwide. This suggests that the group is ubiquitously distributed and probably exhibits a saprotrophic lifestyle.