Dual colonization of Mucoromycotina and Glomeromycotina fungi in the basal liverwort, Haplomitrium mnioides (Haplomitriopsida).

In general, Glomeromycotina was thought to be the earliest fungi forming mycorrhiza-like structure (MLS) in land plant evolution. In contrast, because the earliest divergent lineage of extant land plants, i.e. Haplomitriopsida liverworts, associates only with Mucoromycotina mycobionts, recent studies suggested that those fungi are novel candidates for the earliest mycobionts. Therefore, Mucoromycotina-Haplomitriopsida association currently attracts attention as an ancient mycorrhiza-like association. However, mycobionts were identified in only 7 of 16 Haplomitriopsida species and the mycobionts diversity of this lineage is largely unclarified. To clarify the taxonomic composition of mycobionts in Haplomitriopsida, we observed MLSs in the rhizome of Haplomitrium mnioides (Haplomitriopsida), the Asian representative Haplomitriopsida species, and conducted molecular identification of mycobionts. It was recorded for the first time that Glomeromycotina and Mucoromycotina co-occur in Haplomitriopsida as mycobionts. Significantly, the arbuscule-like branching (ALB) of Glomeromycotina was newly described. As the Mucoromycotina fungi forming MLSs in H. mnioides, Endogonaceae and Densosporaceae were detected, in which size differences of hyphal swelling (HS) were found between the fungal families. This study provides a novel evidence in the MLS of Haplomitriopsida, i.e. the existence of Glomeromycotina association as well as the dominant Mucoromycotina association. In addition, since hyphal characteristics of the HS-type MLS were quite similar to those of fine endophytes (FE) of Endogonales in other bryophytes and vascular plants previously described, this MLS is suggested to be included in FE. These results suggest that Glomeromycotina and Mucoromycotina were acquired concurrently as the mycobionts by the earliest land plants evolved into arbuscular mycorrhizae and FE. Therefore, dual association of Haplomitriopsida, with Endogonales and Glomeromycotina will provide us novel insight on how the earliest land plants adapted to terrestrial habitats with fungi.