The distribution and evolution of fungal symbioses in ancient lineages of land plants.

An accurate understanding of the diversity and distribution of fungal symbioses in land plants is essential for mycorrhizal research. Here we update the seminal work of Wang and Qiu (Mycorrhiza 16:299-363, 2006) with a long-overdue focus on early-diverging land plant lineages, which were considerably under-represented in their survey, by examining the published literature to compile data on the status of fungal symbioses in liverworts, hornworts and lycophytes. Our survey combines data from 84 publications, including recent, post-2006, reports of Mucoromycotina associations in these lineages, to produce a list of at least 591 species with known fungal symbiosis status, 180 of which were included in Wang and Qiu (Mycorrhiza 16:299-363, 2006). Using this up-to-date compilation, we estimate that fewer than 30% of liverwort species engage in symbiosis with fungi belonging to all three mycorrhizal phyla, Mucoromycota, Basidiomycota and Ascomycota, with the last being the most widespread (17%). Fungal symbioses in hornworts (78%) and lycophytes (up to 100%) appear to be more common but involve only members of the two Mucoromycota subphyla Mucoromycotina and Glomeromycotina, with Glomeromycotina prevailing in both plant groups. Our fungal symbiosis occurrence estimates are considerably more conservative than those published previously, but they too may represent overestimates due to currently unavoidable assumptions.

Evolution and networks in ancient and widespread symbioses between Mucoromycotina and liverworts.

Like the majority of land plants, liverworts regularly form intimate symbioses with arbuscular mycorrhizal fungi (Glomeromycotina). Recent phylogenetic and physiological studies report that they also form intimate symbioses with Mucoromycotina fungi and that some of these, like those involving Glomeromycotina, represent nutritional mutualisms. To compare these symbioses, we carried out a global analysis of Mucoromycotina fungi in liverworts and other plants using species delimitation, ancestral reconstruction, and network analyses. We found that Mucoromycotina are more common and diverse symbionts of liverworts than previously thought, globally distributed, ancestral, and often co-occur with Glomeromycotina within plants. However, our results also suggest that the associations formed by Mucoromycotina fungi are fundamentally different because, unlike Glomeromycotina, they may have evolved multiple times and their symbiotic networks are un-nested (i.e., not forming nested subsets of species). We infer that the global Mucoromycotina symbiosis is evolutionarily and ecologically distinctive.

Mucoromycotina Fine Root Endophyte Fungi Form Nutritional Mutualisms with Vascular Plants.

Fungi and plants have engaged in intimate symbioses that are globally widespread and have driven terrestrial biogeochemical processes since plant terrestrialization >500 million years ago. Recently, hitherto unknown nutritional mutualisms involving ancient lineages of fungi and nonvascular plants have been discovered, although their extent and functional significance in vascular plants remain uncertain. Here, we provide evidence of carbon-for-nitrogen exchange between an early-diverging vascular plant (Lycopodiella inundata) and Mucoromycotina (Endogonales) fine root endophyte fungi. Furthermore, we demonstrate that the same fungal symbionts colonize neighboring nonvascular and flowering plants. These findings fundamentally change our understanding of the physiology, interrelationships, and ecology of underground plant-fungal symbioses in modern terrestrial ecosystems by revealing the nutritional role of Mucoromycotina fungal symbionts in vascular plants.

Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts.

Liverworts, which are amongst the earliest divergent plant lineages and important ecosystem pioneers, often form nutritional mutualisms with arbuscular mycorrhiza-forming Glomeromycotina and fine-root endophytic Mucoromycotina fungi, both of which coevolved with early land plants. Some liverworts, in common with many later divergent plants, harbour both fungal groups, suggesting these fungi may complementarily improve plant access to different soil nutrients. We tested this hypothesis by growing liverworts in single and dual fungal partnerships under a modern atmosphere and under 1500 ppm [CO2 ], as experienced by early land plants. Access to soil nutrients via fungal partners was investigated with 15 N-labelled algal necromass and 33 P orthophosphate. Photosynthate allocation to fungi was traced using 14 CO2 . Only Mucoromycotina fungal partners provided liverworts with substantial access to algal 15 N, irrespective of atmospheric CO2 concentration. Both symbionts increased 33 P uptake, but Glomeromycotina were often more effective. Dual partnerships showed complementarity of nutrient pool use and greatest photosynthate allocation to symbiotic fungi. We show there are important functional differences between the plant-fungal symbioses tested, providing new insights into the functional biology of Glomeromycotina and Mucoromycotina fungal groups that form symbioses with plants. This may explain the persistence of the two fungal lineages in symbioses across the evolution of land plants.

Ancient plants with ancient fungi: liverworts associate with early-diverging arbuscular mycorrhizal fungi.

Arbuscular mycorrhizas are widespread in land plants including liverworts, some of the closest living relatives of the first plants to colonize land 500 million years ago (MYA). Previous investigations reported near-exclusive colonization of liverworts by the most recently evolved arbuscular mycorrhizal fungi, the Glomeraceae, indicating a recent acquisition from flowering plants at odds with the widely held notion that arbuscular mycorrhizal-like associations in liverworts represent the ancestral symbiotic condition in land plants. We performed an analysis of symbiotic fungi in 674 globally collected liverworts using molecular phylogenetics and electron microscopy. Here, we show every order of arbuscular mycorrhizal fungi colonizes early-diverging liverworts, with non-Glomeraceae being at least 10 times more common than in flowering plants. Arbuscular mycorrhizal fungi in liverworts and other ancient plant lineages (hornworts, lycopods, and ferns) were delimited into 58 taxa and 36 singletons, of which at least 43 are novel and specific to liverworts. The discovery that early plant lineages are colonized by early-diverging fungi supports the hypothesis that arbuscular mycorrhizas are an ancestral symbiosis for all land plants.

A mycorrhizal revolution.

It has long been postulated that symbiotic fungi facilitated plant migrations onto land through enhancing the scavenging of mineral nutrients and exchanging these for photosynthetically fixed organic carbon. Today, land plant-fungal symbioses are both widespread and diverse. Recent discoveries show that a variety of potential fungal associates were likely available to the earliest land plants, and that these early partnerships were probably affected by changing atmospheric CO2 concentrations. Here, we evaluate current hypotheses and knowledge gaps regarding early plant-fungal partnerships in the context of newly discovered fungal mutualists of early and more recently evolved land plants and the rapidly changing views on the roles of plant-fungal symbioses in the evolution and ecology of the terrestrial biosphere.

Multigene phylogeny of Endogonales, an early diverging lineage of fungi associated with plants.

Endogonales is a lineage of early diverging fungi within Mucoromycota. Many species in this order produce small sporophores ("sporocarps") containing a large number of zygospores, and many species form symbioses with plants. However, due to limited collections, subtle morphological differentiation, difficulties in growing these organisms in vitro, and idiosyncrasies in their rDNA that make PCR amplification difficult, the systematics and character evolution of these fungi have been challenging to resolve. To overcome these challenges we generated a multigene phylogeny of Endogonales using sporophores collected over the past three decades from four continents. Our results show that Endogonales harbour significant undescribed diversity and form two deeply divergent and well-supported phylogenetic clades, which we delimit as the families Endogonaceae and Densosporaceae fam. nov. The family Densosporaceae consists of the genus Densospora,Sphaerocreas pubescens, and many diverse lineages known only from environmental DNA sequences of plant-endosymbiotic fungi. Within Endogonaceae there are two clades. One corresponds to Endogone and includes the type species, E. pisiformis. Species of Endogone are characterized by above- and below-ground sporophores, a hollow and infolded sporophore form, a loose zygosporangial hyphal mantle, homogeneous gametangia, and an enigmatic trophic mode with no evidence of ectomycorrhizal association for most species. For the other clade we introduce a new generic name, Jimgerdemannia gen. nov. Members of that genus (J. flammicorona and J. lactiflua species complexes, and an undescribed species) are characterized by hypogeous sporophores with a solid gleba, a well-developed zygosporangial hyphal mantle, heterogeneous gametangia, and an ectomycorrhizal trophic mode. Future studies on Densosporaceae and Endogonaceae will be important for understanding fungal innovations including evolution of macroscopic sporophores and symbioses with plants.

Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline.

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.

Symbiotic options for the conquest of land.

The domination of the landmasses of Earth by plants starting during the Ordovician Period drastically altered the development of the biosphere and the composition of the atmosphere, with far-reaching consequences for all life ever since. It is widely thought that symbiotic soil fungi facilitated the colonization of the terrestrial environment by plants. However, recent discoveries in molecular ecology, physiology, cytology, and paleontology have brought into question the hitherto-assumed identity and biology of the fungi engaged in symbiosis with the earliest-diverging lineages of extant land plants. Here, we reconsider the existing paradigm and show that the symbiotic options available to the first plants emerging onto the land were more varied than previously thought.

From mycoheterotrophy to mutualism: mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes.

Mycorrhizal functioning in the fern Ophioglossum is complex and poorly understood. It is unknown whether mature O. vulgatum sporophytes form mutualistic associations with fungi of the Glomeromycota and with what specificity. Are green sporophytes able to 'repay' fungal carbon (C) invested in them by mycorrhizal partners during the initially heterotrophic gametophyte and early sporophyte stages of the lifecycle? We identified fungal partners of O. vulgatum sporophytes using molecular techniques and supplied them with (33) P-orthophosphate and O. vulgatum sporophytes with (14) CO2 . We traced the movement of fungal-acquired nutrients and plant-fixed C between symbionts and analysed natural abundance (13) C and (15) N isotope signatures to assess nutritional interactions. We found fungal specificity of O. vulgatum sporophytes towards a mycorrhizal fungus closely related to Glomus macrocarpum. Our radioisotope tracers revealed reciprocal C-for-phosphorus exchange between fern sporophytes and fungal partners, despite competition from surrounding vegetation. Monocultures of O. vulgatum were enriched in (13) C and (15) N, providing inconclusive evidence of mycoheterotrophy when experiencing competition from the surrounding plant community. We show mutualistic and specific symbiosis between a eusporangiate fern and fungi of the Glomeromycota. Our findings suggest a 'take now, pay later' strategy of mycorrhizal functioning through the lifecycle O. vulgatum, from mycoheterotrophic gametophyte to mutualistic aboveground sporophyte.

First evidence of mutualism between ancient plant lineages (Haplomitriopsida liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2.

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.