Liverworts form endophytic associations with fungi that mirror mycorrhizal associations in tracheophytes. Here we report a worldwide survey of liverwort associations with glomeromycotean fungi (GAs), together with a comparative molecular and cellular analysis in representative species. Liverwort GAs are circumscribed by a basal assemblage embracing the Haplomitriopsida, the Marchantiopsida (except a few mostly derived clades), and part of the Metzgeriidae. Fungal endophytes from Haplomitrium, Conocephalum, Fossombronia, and Pellia were related to Glomus Group A, while the endophyte from Monoclea was related to Acaulospora. An isolate of G. mosseae colonized axenic thalli of Conocephalum, producing an association similar to that in the wild. Fungal colonization in marchantialean liverworts suppressed cell wall autofluorescence and elicited the deposition of a new wall layer that specifically bound the monoclonal antibody CCRC-M1 against fucosylated side groups associated with xyloglucan and rhamnogalacturonan I. The interfacial material covering the intracellular fungus contained the same epitopes present in host cell walls. The taxonomic distribution and cytology of liverwort GAs suggest an ancient origin and multiple more recent losses, but the occurence in widely separated liverwort taxa of fungi related to glomeromycotean lineages that form arbuscular mycorrhizas in tracheophytes, notably the Glomus Group A, is better explained by host shifting from tracheophytes to liverworts.

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Thallus anatomy in three species of the primitive liverwort genus Treubia (Metzgeriidae, Treubiales) was studied by light and electron microscopy. The thallus exudes copious mucilage, a feature shared elsewhere in liverworts only with the mycotrophic subterranean axes of the allied genus Haplomitrium. The central strand in the thallus midrib has a unique histological organization and harbors an intra- and intercellular infection by a glomeromycotean fungus that is far more highly differentiated than most of the glomeromycotean associations described to date. The fungus enters the thallus via clefts in the ventral epidermis along the midrib and colonizes the parenchyma above, forming intracellular coils and prominent, relatively short-lived, hyphal swellings. Above the zone with intracellular colonization is a tissue area containing mucilage-filled intercellular spaces; here the fungus is entirely intercellular and forms abundant pseudoparenchymatous structures and, in more mature parts of the thalli, large hyphae with thick multistratose walls. Mucilage in Treubia differs in histochemistry and origin from that produced by apical papillae, via hypertrophied Golgi, in all other bryophytes. Remarkable parallels between fungal associations in Treubia, Haplomitrium, and Lycopodium, all members of very ancient lineages, suggest that these associations epitomize very early stages in the evolution of glomeromycotean symbioses.

Summary• Xylans are known to be major cellulose-linking polysaccharides in secondary cell walls in higher plants.• We used two monoclonal antibodies (LM10 and LM11) for a comparative immunocytochemical analysis of tissue and cell distribution of xylans in a number of taxa representative of all major tracheophyte and bryophyte lineages.• The results show that xylans containing the epitopes recognized by LM10 and LM11 are ubiquitous components of secondary cell walls in vascular and mechanical tissues in all present-living tracheophytes. In contrast, among the three bryophyte lineages, LM11 binding was detected in specific cell-wall layers in pseudoelaters and spores in the sporophyte of hornworts, while no binding was observed with either antibody in the gametophyte or sporophyte of liverworts and mosses.• The ubiquitous occurrence of xylans containing LM10 and LM11 epitopes in tracheophytes suggests that the appearance of these polysaccharides has been a pivotal event for the evolution of highly efficient vascular and mechanical tissues. LM11 binding in the sporophyte of hornworts, indicating the presence of relatively highly substituted xylans (possibly arabinoxylans), separates these from the other bryophytes and is consistent with recent molecular data indicating a sister relationship of the hornworts with tracheophytes.