Fungal symbioses in hornworts: a chequered history

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.     

The dawn of symbiosis between plants and fungi

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.     

Molecular evolution patterns reveal life history features of mycoplasma‐related endobacteria associated with arbuscular mycorrhizal fungi

The mycoplasma‐related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant‐assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota–MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.     

Programming good relations--development of the arbuscular mycorrhizal symbiosis

The majority of plants live in symbiotic associations with fungi or bacteria that improve their nutrition. Critical steps in a symbiosis are mutual recognition and subsequently the establishment of an intimate association, which involves the penetration of plant tissues and, in many cases, the invasion of individual host cells by the microbial symbiont. Recent advances revealed that in the arbuscular mycorrhizal symbiosis with soil fungi of the order Glomeromycota, plant-derived signals attract fungal hyphae and stimulate their growth. Upon physical attachment of the fungal symbiont to the root surface, an active plant developmental program prepares the epidermal cells for penetration by the fungus. Thus, plants actively help symbiotic fungi to colonize their roots rather than just tolerating them.     

Sharing of Diverse Mycorrhizal and Root-Endophytic Fungi among Plant Species in an Oak-Dominated Cool–Temperate Forest

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant–fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant–mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant–fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant–fungal associations. Such insights into the overall structure of belowground plant–fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities.     

Trait‐based partner selection drives mycorrhizal network assembly

Plants and their microbial symbionts are often found to interact non‐randomly in nature, but we have yet to understand the mechanisms responsible for such preferential species associations. Theory predicts that host plants should select symbiotic partners bearing traits complementary to their own, as this should favor cooperation and evolutionary stability of mutualisms. Here, we present the first field‐based empirical test for this hypothesis using arbuscular mycorrhizas (AM), the oldest and most widespread plant symbiosis. Preferential associations occurring within a local plant–AM fungal community could not be predicted by the spatial distributions of interacting partners, nor by gradients in soil properties. Rather, plants with similar traits preferentially hosted similar AM fungi and, likewise, phylogenetically related AM fungi (assumed to have similar functional traits) interacted with similar plants. Our results suggest that trait‐based partner selection may have been a strong force in maintaining plant–AM fungal symbioses since the evolution of land plants.     

The interactive effects of plant microbial symbionts: a review and meta-analysis

In nature, plants often associate with multiple symbionts concurrently, yet the effects of tripartite symbioses are not well understood. We expected synergistic growth responses from plants associating with functionally distinct symbionts. In contrast, symbionts providing similar benefits to a host may reduce host plant growth. We reviewed studies investigating the effect of multiple interactions on host plant performance. Additionally, we conducted a meta-analysis on the studies that performed controlled manipulations of the presence of two microbial symbionts. Using response ratios, we investigated the effects on plants of pairs of symbionts (mycorrhizal fungi, fungal endophytes, and nitrogen-fixers). The results did not support the view that arbuscular mycorrhizal (AM) fungi and rhizobia should interact synergistically. In contrast, we found the joint effects of fungal endophytes and arbuscular mycorrhizal fungi to be greater than expected given their independent effects. This increase in plant performance only held for antagonistic endophytes, whose negative effects were alleviated when in association with AM fungi, while the impact of beneficial endophytes was not altered by infection with AM fungi. Generalizations from the meta-analysis were limited by the substantial variation within types of interactions and the data available, highlighting the need for more research on a range of plant systems.     

Successful joint ventures of plants: arbuscular mycorrhiza and beyond

Among the oldest symbiotic associations of plants are arbuscular mycorrhiza (AM) with fungi of the phylum Glomeromycota. Although many of the symbiotic signaling components have been identified on the side of the plant, AM fungi have long evaded genetic analysis owing to their strict biotrophy and their exceptional genetics. Recently, the identification of the fungal symbiosis signal (Myc factor) and of a corresponding Myc factor receptor, and new insights into AM fungal genetics, have opened new avenues to address early communication and functional aspects of AM symbiosis. These advances will pave the way for breeding programs towards adapted AM fungi for crop production, and will shed light on the ecology and evolution of this remarkably successful symbiosis.     

How are plant and fungal communities linked to each other in belowground ecosystems? A massively parallel pyrosequencing analysis of the association specificity of root-associated fungi and their host plants

In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root‐associated fungi and their host plants influence each other. In an oak‐dominated forest in Japan, we investigated the root‐associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root‐endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on “non‐ectomycorrhizal” plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant–fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root‐endophytic fungi may be important features of belowground linkage between plant and fungal communities.     

Phylogeny of the glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers

The fungal symbionts of arbuscular mycorrhiza form a monophyletic group in the true Fungi, the phylum Glomeromycota. Fewer than 200 described species currently are included in this group. The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene phylogenies has been slow and the nuclear-encoded ribosomal RNA genes have remained the only widely accessible molecular markers. rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that has been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recovered an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA-supported sister group relationship of the Glomeromycota with Asco/Basidiomycota. However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships. We use nuclear-encoded rDNA and rpb1 protein gene sequences to reassess the phylogeny of the Glomeromycota and discuss possible implications.     

Diversity and Spatial Structure of Belowground Plant–Fungal Symbiosis in a Mixed Subtropical Forest of Ectomycorrhizal and Arbuscular Mycorrhizal Plants

Plant–mycorrhizal fungal interactions are ubiquitous in forest ecosystems. While ectomycorrhizal plants and their fungi generally dominate temperate forests, arbuscular mycorrhizal symbiosis is common in the tropics. In subtropical regions, however, ectomycorrhizal and arbuscular mycorrhizal plants co-occur at comparable abundances in single forests, presumably generating complex community structures of root-associated fungi. To reveal root-associated fungal community structure in a mixed forest of ectomycorrhizal and arbuscular mycorrhizal plants, we conducted a massively-parallel pyrosequencing analysis, targeting fungi in the roots of 36 plant species that co-occur in a subtropical forest. In total, 580 fungal operational taxonomic units were detected, of which 132 and 58 were probably ectomycorrhizal and arbuscular mycorrhizal, respectively. As expected, the composition of fungal symbionts differed between fagaceous (ectomycorrhizal) and non-fagaceous (possibly arbuscular mycorrhizal) plants. However, non-fagaceous plants were associated with not only arbuscular mycorrhizal fungi but also several clades of ectomycorrhizal (e.g., Russula) and root-endophytic ascomycete fungi. Many of the ectomycorrhizal and root-endophytic fungi were detected from both fagaceous and non-fagaceous plants in the community. Interestingly, ectomycorrhizal and arbuscular mycorrhizal fungi were concurrently detected from tiny root fragments of non-fagaceous plants. The plant–fungal associations in the forest were spatially structured, and non-fagaceous plant roots hosted ectomycorrhizal fungi more often in the proximity of ectomycorrhizal plant roots. Overall, this study suggests that belowground plant–fungal symbiosis in subtropical forests is complex in that it includes “non-typical” plant–fungal combinations (e.g., ectomycorrhizal fungi on possibly arbuscular mycorrhizal plants) that do not fall within the conventional classification of mycorrhizal symbioses, and in that associations with multiple functional (or phylogenetic) groups of fungi are ubiquitous among plants. Moreover, ectomycorrhizal fungal symbionts of fagaceous plants may “invade” the roots of neighboring non-fagaceous plants, potentially influencing the interactions between non-fagaceous plants and their arbuscular-mycorrhizal fungal symbionts at a fine spatial scale.     

Evolutionary diversification of the gall midge genus Asteromyia (Cecidomyiidae) in a multitrophic ecological context

Gall-forming insects provide ideal systems to analyze the evolution of host–parasite interactions and understand the ecological interactions that contribute to evolutionary diversification. Flies in the family Cecidomyiidae represent the largest radiation of gall-forming insects and are characterized by complex trophic interactions with plants, fungal symbionts, and predators. We analyzed the phylogenetic history and evolutionary associations of the North American cecidomyiid genus Asteromyia, which is engaged in a complex and perhaps co-evolving community of interactions with host-plants, fungi, and parasitoids. Mitochondrial gene trees generally support current classifications, but reveal extensive cryptic diversity within the eight named species. Asteromyia likely radiated after their associated host-plants in the Astereae, but species groups exhibit strong associations with specific lineages of Astereae. Evolutionary associations with fungal mutualists are dynamic, however, and suggest rapid and perhaps coordinated changes across trophic levels.     

Guilds of mycorrhizal fungi and their relation to trees,ericads, orchids and liverworts in a neotropical mountain rain forest

Mycorrhizas of vascular plants and mycorrhiza-like associations of liverworts and hornworts are integral parts of terrestrial ecosystems, but have rarely been studied in tropical mountain rain forests. The tropical mountain rain forest area of the Reserva Biológica San Francisco in South Ecuador situated on the eastern slope of the Cordillera El Consuelo is exceptionally rich in tree species, ericads and orchids, but also in liverworts. Previous light and electron microscopical studies revealed that tree roots are well colonized by structurally diverse Glomeromycota, and that epiphytic, pleurothallid orchids form mycorrhizas with members of the Tulasnellales and the Sebacinales (Basidiomycota). Sebacinales also occurred in mycorrhizas of hemiepiphytic ericads and Tulasnellales were found in liverworts belonging to the Aneuraceae. On the basis of these findings, we hypothesized that symbiotic fungi with a broad host range created shared guilds or even fungal networks between different plant species and plant families. To test this hypothesis, molecular phylogenetic studies of the fungi associated with roots and thalli were carried out using sequences of the nuclear rDNA coding for the small subunit rRNA (nucSSU) of Glomeromycota and the large subunit rRNA (nucLSU) of Basidiomycota. Sequence analyses showed that Sebacinales and Tulasnellales were only shared within but not between ericads and orchids or between liverworts and orchids, respectively. Regarding arbuscular-mycorrhiza-forming trees, however, 18 out of 33 Glomus sequence types were shared by two to four tree species belonging to distinct families. Nearly all investigated trees shared one sequence type with another tree individual. Host range and potential shared guilds appeared to be restricted to the plant family level for Basidiomycota, but were covering diverse plant families in case of Glomeromycota. Given that the sequence types as defined here correspond to fungal species, our findings indicate potential fungal networks between trees.     

Genetic processes in arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi (Glomeromycota) colonize roots of the majority of land plants and facilitate their mineral nutrient uptake. Consequently, AM fungi play an important role in terrestrial ecosystems and are becoming a component of sustainable land management practices. The absence of sexual reproductive structures in modern Glomeromycota combined with their long evolutionary history suggest that these fungi may represent an ancient asexual lineage of great potential interest to evolutionary biology. However, many aspects of basic AM fungal biology, including genome structure, within-individual genetic variation, and reproductive mode are poorly understood. These knowledge gaps hinder research on the mechanisms of AM fungal interactions with individual plants and plant communities, and utilization of AM fungi in agricultural practices. I present here the current state of research on the reproduction in AM fungi and indicate what new findings can be expected in the future.     

Plant signals and fungal perception during arbuscular mycorrhiza establishment

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that need their plant hosts to complete their life cycle. In the absence of the plant, germlings arrest growth after a few days and retract most of their cytoplasm back into the multinuclear spores. The spores can germinate again during more favorable conditions. How AM fungi recognize compatible host roots and activate their symbiotic program is not yet understood. However, research in this field in the last years has shed light into this topic. We, and others, have approached some of these aspects by studying changes in fungal gene expression observed at early stages of development, before and at the plant recognition stage in an attempt to identify genes and proteins featuring as key regulators in the switch between the asymbiotic and symbiotic style of life. The molecular bases of this recognition process are now starting to be understood and point to common signaling pathways shared with other microbe-plant associations and to arbuscular mycorrhiza specific signaling pathways.     

Mycorrhizas and global environmental change: research at different scales

Global environmental change (GEC), in particular rising atmospheric CO₂ concentration and temperature, will affect most ecosystems. The varied responses of plants to these aspects of GEC are well documented. As with other key below-ground components of terrestrial ecosystems, the response of the ubiquitous mycorrhizal fungal root symbionts has received limited attention. Most of the research on the effects of GEC on mycorrhizal fungi has been pot-based with a few field (especially monoculture) studies. A major question that arises in all these studies is whether the GEC effects on the mycorrhizal fungi are independent of the effects on their plant hosts. We evaluate the current knowledge on the effects of elevated CO₂ and increased temperature on mycorrhizal fungi and focus on the few available field examples. The value of using long-term and large-scale field experiments is emphasised. We conclude that the laboratory evidence to date shows that the effect of elevated CO₂ on mycorrhizal fungi is dependent on plant growth and that temperature effects seen in the past might have reflected a similar dependence. Therefore, how temperature directly affects mycorrhizal fungi remains unknown. In natural ecosystems, we predict that GEC effects on mycorrhizal fungal communities will be strongly mediated by the effects on plant communities to the extent that community level interactions will prove to be the key mechanism for determining GEC-induced changes in mycorrhizal fungal communities.     

Parasites and phytoplankton, with special emphasis on dinoflagellate infections

Planktonic members of most algal groups are known to harbor intracellular symbionts, including viruses, bacteria, fungi, and protozoa. Among the dinoflagellates, viral and bacterial associations were recognized a quarter century ago, yet their impact on host populations remains largely unresolved. By contrast, fungal and protozoan infections of dinoflagellates are well documented and generally viewed as playing major roles in host population dynamics. Our understanding of fungal parasites is largely based on studies for freshwater diatoms and dinoflagellates, although fungal infections are known for some marine phytoplankton. In freshwater systems, fungal chytrids have been linked to mass mortalities of host organisms, suppression or retardation of phytoplankton blooms, and selective effects on species composition leading to successional changes in plankton communities. Parasitic dinoflagellates of the genus Amoebophrya and the newly described Perkinsozoa, Parvilucifera infectans, are widely distributed in coastal waters of the world where they commonly infect photosynthetic and heterotrophic dinoflagellates. Recent work indicates that these parasites can have significant impacts on host physiology, behavior, and bloom dynamics. Thus, parasitism needs to be carefully considered in developing concepts about plankton dynamics and the flow of material in marine food webs.     

Forms of nitrogen uptake, translocation, and transfer via arbuscular mycorrhizal fungi: A review

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the roots of more than 80% of land plants. Experiments on the relationship between the host plant and AM in soil or in sterile root-organ culture have provided clear evidence that the extraradical mycelia of AM fungi uptake various forms of nitrogen (N) and transport the assimilated N to the roots of the host plant. However, the uptake mechanisms of various forms of N and its translocation and transfer from the fungus to the host are virtually unknown. Therefore, there is a dearth of integrated models describing the movement of N through the AM fungal hyphae. Recent studies examined Ri T-DNA-transformed carrot roots colonized with AM fungi in ¹⁵N tracer experiments. In these experiments, the activities of key enzymes were determined, and expressions of genes related to N assimilation and translocation pathways were quantified. This review summarizes and discusses the results of recent research on the forms of N uptake, transport, degradation, and transfer to the roots of the host plant and the underlying mechanisms, as well as research on the forms of N and carbon used by germinating spores and their effects on amino acid metabolism. Finally, a pathway model summarizing the entire mechanism of N metabolism in AM fungi is outlined.     

Peeking through a frosty window: molecular insights into the ecology of Arctic soil fungi

Fungi are ubiquitous in Arctic soils, where they function as symbionts and decomposers and may affect the carbon balance of terrestrial ecosystems subjected to climate change, and yet little is known about soil fungi at high latitudes. Here we review data from recent molecular studies to determine broad patterns in Arctic soil fungal ecology. The data indicate comparatively high fungal diversity in Arctic soils, with currently no evidence for lower species richness at higher latitudes. The dominant fungi, and particularly ectomycorrhizal-forming fungi, appear to be cosmopolitan species. Arctic soil fungi are capable of growth at sub-zero temperatures, melanized forms are frequent, host specificity is low and there is evidence that community composition alters under experimental warming. Future challenges are to determine the drivers of fungal diversity, whether or not diversity alters at higher latitudes and how apparently cosmopolitan fungi are able to survive the extreme environments encountered in Arctic habitats.