Mollicutes-related endobacteria thrive inside liverwort-associated arbuscular mycorrhizal fungi

Arbuscular mycorrhizal fungi (AMF) can host Gram-positive endobacteria (BLOs) in their cytoplasm. These have been identified as Mollicutes-related microbes based on an inventory of AMF spores from fungal collections. Bacteria-like organisms (BLOs) of unknown identity have also been reported in the cytoplasm of AMF associated with liverworts, the earliest-diverged extant lineage of land plants. A combination of morphological, molecular and phylogenetic analyses revealed that three samples of two liverwort species (Conocephalum conicum and Lunularia cruciata) growing spontaneously in a botanical garden harboured AMF belonging to Glomerales, and these, in turn, hosted coccoid BLOs. 16S rDNA sequences from these BLOs clustered with the Mollicutes sequences identified from the spore collections but revealed the presence of novel phylotypes. Electron microscopy and fluorescence in situ hybridization (FISH) confirmed the presence of BLOs inside the cytoplasm of AMF hyphae colonizing the liverwort thalli. The high genetic variability of BLOs in liverwort-AMF associations thriving in the same ecological niche raises questions about the mechanisms underlying such diversity.