Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes of Diphasiastrum alpinum (Lycopodiaceae).

Premise Of The Study: To understand the early evolution of mycorrhizal symbioses, it is important to know the fungal partners of gametophytes and sporophytes for basal lineages of vascular plants. Subterranean mycotrophic gametophytes of the clubmoss Diphasiastrum alpinum found at three localities gave an opportunity to study their morphology and anatomy and to identify and describe their hitherto unknown fungal endophytes. In addition, sporophytes were screened for fungal partners.

Methods: Gametophytes with attached young sporophytes were excavated, and their anatomy and their associated fungi were studied by light microscopy. DNA was isolated and amplified with both universal and group-specific fungal primers for the ITS region, the large subunit and small subunit of the nuclear rDNA, respectively, to identify the fungal partner.

Key Results: Gametophytes were uniformly colonized by a fungus with septate hyphae forming coils and vesicles. Its morphology resembles that of the sebacinoid genus Piriformospora. Both ITS and LSU sequences were identified as Sebacinales group B, a basal clade of the Agaricomycetes (Basidiomycota). This fungus was detected in 11 gametophytes from two localities and in rootlets of adjacent Calluna vulgaris (Ericaceae) plants, but was absent in roots of sporophytes. In addition, several ascomycetes and glomeromycetes were found by DNA sequencing.

Conclusions: Our study suggests a fungus belonging to Sebacinales group B as the main fungal host of the D. alpinum gametophytes. However, Sebacinales group B fungi occur as well in adjacent Ericaceae plants; therefore, we assume the mycoheterotrophic gametophyte to be epiparasitic on Ericaceae, which would explain the steady association of these plants.