Aneuraceae (Metzgeriales) and tulasnelloid fungi (Basidiomycota): a model for early steps in fungal symbiosis

A total of 35 population samples of the liverwort genera Aneura (A. pinguis) and Riccardia (R.?latifrons, R. multifida, and R. palmata) were sampled from diverse habitats and geographical provenances in Germany, Austria, and Switzerland. Light and transmission electron microscopy were used to characterise the morphological features of the associations, and phylogenetic analyses based on internal transcribed spacers (ITS) and the D1/D2 regions of the fungal 28S rDNA were used to address diversity and phylogenetic relationships. By comparing the cellular structures of the plant-fungus interactions, we recognised the following states of fungal colonisation within the thalli: fungus-free, epiphytic, intercellular, and intracellular. Colonising hyphae showed dolipores with imperforate parenthesomes, slime bodies, and multilayered walls. Colonised liverwort cells had pleomorphic nuclei and elongated starch-free chloroplasts with distinctive grana. Our analyses revealed six phylogenetic groups of tulasnelloid fungi associated with liverworts, where major lineages mostly share similar host and/or ecological specialisations. The mode of colonisation of the tulasnelloid mycobionts in Aneura and Riccardia sharing identical fungal sequences is different. Consequently, the mode of colonisation may be host-dependent. Finally, our findings demonstrate that Aneuraceae are model organisms for evolutionary studies of symbiotic associations between liverworts and fungi.