Variation in arbuscular mycorrhizal status among cordate fern gametophytes in relation to phylogeny, habitat, and gametophyte structure

Ferns have two free-living generations in their life cycle, the sporophyte and the gametophyte, yet mycorrhizal associations in the gametophyte generation remain poorly understood. Arbuscular mycorrhizal (AM) symbiosis in cordate gametophytes—the most widespread gametophyte morphology in ferns—has not been comprehensively evaluated at the species level. In this study, we investigated 40 species of cordate gametophytes representing five orders and 16 families collected from various regions of Japan. Species were identified using DNA barcoding, and mycorrhizal status was assessed based on AM fungal colonization rates in wild individuals. Species were classified as obligately mycorrhizal (OM), facultatively mycorrhizal (FM), or non-mycorrhizal (NM). All three mycorrhizal statuses were detected, and their distribution was associated with phylogeny and habitat. OM and NM tended to correspond to specific lineages, whereas FM species were distributed across multiple clades. Notably, all NM species were non-terrestrial (epiphytic or epilithic). Controlled soil co-culture experiments conducted on 16 species revealed marked interspecific variation in mycorrhizal growth response (MGR), indicating substantial differences in physiological dependence on AM symbiosis. Overall, these findings demonstrate that AM associations in cordate gametophytes are phylogenetically structured and ecologically diversified, providing new insights into plant–fungal symbiosis across the fern life cycle.