The colonization of land by plants relied on fundamental biological innovations, among which was symbiosis with fungi to enhance nutrient uptake. Here we present evidence that several species representing the earliest groups of land plants are symbiotic with fungi of the Mucoromycotina. This finding brings up the possibility that terrestrialization was facilitated by these fungi rather than, as conventionally proposed, by members of the Glomeromycota. Since the 1970s it has been assumed, largely from the observation that vascular plant fossils of the early Devonian (400 Ma) show arbuscule-like structures, that fungi of the Glomeromycota were the earliest to form mycorrhizas, and evolutionary trees have, until now, placed Glomeromycota as the oldest known lineage of endomycorrhizal fungi. Our observation that Endogone -like fungi are widely associated with the earliest branching land plants, and give way to glomeromycotan fungi in later lineages, raises the new hypothesis that members of the Mucoromycotina rather than the Glomeromycota enabled the establishment and growth of early land colonists.

The discovery that Mucoromycotina, an ancient and partially saprotrophic fungal lineage, associates with the basal liverwort lineage Haplomitriopsida casts doubt on the widely held view that Glomeromycota formed the sole ancestral plant–fungus symbiosis. Whether this association is mutualistic, and how its functioning was affected by the fall in atmospheric CO2 concentration that followed plant terrestrialization in the Palaeozoic, remains unknown.We measured carbon-for-nutrient exchanges between Haplomitriopsida liverworts and Mucoromycotina fungi under simulated mid-Palaeozoic (1500 ppm) and near-contemporary (440 ppm) CO2 concentrations using isotope tracers, and analysed cytological differences in plant–fungal interactions. Concomitantly, we cultured both partners axenically, resynthesized the associations in vitro, and characterized their cytology.We demonstrate that liverwort–Mucoromycotina symbiosis is mutualistic and mycorrhiza-like, but differs from liverwort–Glomeromycota symbiosis in maintaining functional efficiency of carbon-for-nutrient exchange between partners across CO2 concentrations. Inoculation of axenic plants with Mucoromycotina caused major cytological changes affecting the anatomy of plant tissues, similar to that observed in wild-collected plants colonized by Mucoromycotina fungi.By demonstrating reciprocal exchange of carbon for nutrients between partners, our results provide support for Mucoromycotina establishing the earliest mutualistic symbiosis with land plants. As symbiotic functional efficiency was not compromised by reduced CO2, we suggest that other factors led to the modern predominance of the Glomeromycota symbiosis.

Background Molecular phylogeny has resolved the liverworts as the earliest-divergent clade of land plants and mosses as the sister group to hornworts plus tracheophytes, with alternative topologies resolving the hornworts as sister to mosses plus tracheophytes less well supported. The tracheophytes plus fossil plants putatively lacking lignified vascular tissue form the polysporangiophyte clade. Scope This paper reviews phylogenetic, developmental, anatomical, genetic and paleontological data with the aim of reconstructing the succession of events that shaped major land plant lineages. Conclusions Fundamental land plant characters primarily evolved in the bryophyte grade, and hence the key to a better understanding of the early evolution of land plants is in bryophytes. The last common ancestor of land plants was probably a leafless axial gametophyte bearing simple unisporangiate sporophytes. Water-conducting tissue, if present, was restricted to the gametophyte and presumably consisted of perforate cells similar to those in the early-divergent bryophytes Haplomitrium and Takakia. Stomata were a sporophyte innovation with the possible ancestral functions of producing a transpiration-driven flow of water and solutes from the parental gametophyte and facilitating spore separation before release. Stomata in mosses, hornworts and polysporangiophytes are viewed as homologous, and hence these three lineages are collectively referred to as the 'stomatophytes'. An indeterminate sporophyte body (the sporophyte shoot) developing from an apical meristem was the key innovation in polysporangiophytes. Poikilohydry is the ancestral condition in land plants; homoiohydry evolved in the sporophyte of polysporangiophytes. Fungal symbiotic associations ancestral to modern arbuscular mycorrhizas evolved in the gametophytic generation before the separation of major present-living lineages. Hydroids are imperforate water-conducting cells specific to advanced mosses. Xylem vascular cells in polysporangiophytes arose either from perforate cells or de novo. Food-conducting cells were a very early innovation in land plant evolution. The inferences presented here await testing by molecular genetics.

19We reconsider the existing paradigm and show that the symbiotic options available to 20 the first plants emerging onto the land were more varied than previously thought. 22The current paradigm 23 The colonisation of the terrestrial environment by plants was a major turning point in Earth's carbohydrates fixed from atmospheric carbon dioxide through photosynthesis [5]. 35Plant life diversified on land some 70 MY after the diversification of most major 36 animal lineages in the seas during the Cambrian explosion ( Fig. 1) [6]. Classic palaeoclimate 37 3 modelling shows that land plants diversified against a backdrop of falling atmospheric CO 2 38[7] (Figure 2), likely driven by growing demand and evolving capacity for carbon 39 assimilation of the burgeoning Earth flora [8,9]. Long before land plants emerged, however, 40 the terrestrial environment had been colonised by fungi [10,11]. Among the early-branching 41 fungal lineages were those that today form mutualistic associations with most plants (Figure 42 1). The macrofossil record for non-vascular plants is even more fragmentary than that for 43 vascular plants, though the study of microfossils is now providing key new data [12,13] lineages of land plants [24,25]. 60The resulting paradigm is that the earliest, rootless, terrestrial plants co-evolved with 61Glomeromycota fungi [4,[26][27][28][29][30] 153We now know that liverwort-Glomeromycota symbioses can be mutualistic, involving fungi within plant tissues. An alternative but untested hypothesis is that their evolution was 287 linked to harbouring an overlooked or extinct microbial symbiont of green algae [22]. 288The assumption that function of mycorrhizal and mycorrhiza-like fungal associations 289 is also conserved across land plant evolution has, until recently, been based on striking However, while we know for instance that functioning in modern vascular plant-

SummaryFungi (Eumycota) form close associations with plants, with which they have co-existed since the dawn of life on land, but their diversity in early terrestrial ecosystems is still poorly understood.We studied petrographic sections of exceptionally well-preserved petrified plants from the 407 million yr-old Rhynie Chert (Scotland, UK). For comparative purposes, we illustrate fungal associations in four extant lower land plants.We document two new endophytes in the plant Horneophyton lignieri: Palaeoglomus boullardii (sp. nov. Glomeromycota) colonizes parenchyma in a discontinuous zone of the outer cortex of the aerial axes, forming arbuscule-like structures, vesicles and spores; Palaeoendogone gwynne-vaughaniae (gen. nov., sp. nov. Mucoromycotina) colonizes parenchyma in the basal part of the plant, where it is present in intercellular spaces and as intracellular coils but absent from rhizoids.Critical comparisons between the newly discovered Horneophyton endophytes, fungi previously described from the Rhynie Chert and fungal colonization in extant lower land plants reveal several features characteristic of both Mucoromycotina and Glomeromycota. A reappraisal of fungal associations in early land plants indicates that they are more diverse than assumed hitherto, overturning the long-held paradigm that the early endophytes were exclusively Glomeromycota.

Most land plants form mutualistic associations with arbuscular mycorrhizal fungi of the Glomeromycota, but recent studies have found that ancient plant lineages form mutualisms with Mucoromycotina fungi. Simultaneous associations with both fungal lineages have now been found in some plants, necessitating studies to understand the functional and evolutionary significance of these tripartite associations for the first time. We investigate the physiology and cytology of dual fungal symbioses in the early-diverging liverworts Allisonia and Neohodgsonia at modern and Palaeozoic-like elevated atmospheric CO2 concentrations under which they are thought to have evolved. We found enhanced carbon cost to liverworts with simultaneous Mucoromycotina and Glomeromycota associations, greater nutrient gain compared with those symbiotic with only one fungal group in previous experiments and contrasting responses to atmospheric CO2 among liverwort–fungal symbioses. In liverwort–Mucoromycotina symbioses, there is increased P-for-C and N-for-C exchange efficiency at 440 p.p.m. compared with 1500 p.p.m. CO2. In liverwort–Glomeromycota symbioses, P-for-C exchange is lower at ambient CO2 compared with elevated CO2. No characteristic cytologies of dual symbiosis were identified. We provide evidence of a distinct physiological niche for plant symbioses with Mucoromycotina fungi, giving novel insight into why dual symbioses with Mucoromycotina and Glomeromycota fungi persist to the present day.

Hornworts are considered the sister group to vascular plants, but their fungal associations remain largely unexplored. The ancestral symbiotic condition for all plants is, nonetheless, widely assumed to be arbuscular mycorrhizal with Glomeromycota fungi. Owing to a recent report of other fungi in some non-vascular plants, here we investigate the fungi associated with diverse hornworts worldwide, using electron microscopy and molecular phylogenetics. We found that both Glomeromycota and Mucoromycotina fungi can form symbioses with most hornworts, often simultaneously. This discovery indicates that ancient terrestrial plants relied on a wider and more versatile symbiotic repertoire than previously thought, and it highlights the so far unappreciated ecological and evolutionary role of Mucoromycotina fungi.

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.