Evolutionary histories and mycorrhizal associations of mycoheterotrophic plants dependent on saprotrophic fungi

Mycoheterotrophic plants (MHPs) are leafless, achlorophyllous, and completely dependent on mycorrhizal fungi for their carbon supply. Mycorrhizal symbiosis is a mutualistic association with fungi that is undertaken by the majority of land plants, but mycoheterotrophy represents a breakdown of this mutualism in that plants parasitize fungi. Most MHPs are associated with fungi that are mycorrhizal with autotrophic plants, such as arbuscular mycorrhizal (AM) or ectomycorrhizal (ECM) fungi. Although these MHPs gain carbon via the common mycorrhizal network that links the surrounding autotrophic plants, some mycoheterotrophic lineages are associated with saprotrophic (SAP) fungi, which are free-living and decompose leaf litter and wood materials. Such MHPs are dependent on the forest carbon cycle, which involves the decomposition of wood debris and leaf litter, and have a unique biology and evolutionary history. MHPs associated with SAP fungi (SAP-MHPs) have to date been found only in the Orchidaceae and likely evolved independently at least nine times within that family. Phylogenetically divergent SAP Basidiomycota, mostly Agaricales but also Hymenochaetales, Polyporales, and others, are involved in mycoheterotrophy. The fungal specificity of SAP-MHPs varies from a highly specific association with a single fungal species to a broad range of interactions with multiple fungal orders. Establishment of symbiotic culture systems is indispensable for understanding the mechanisms underlying plant–fungus interactions and the conservation of MHPs. Symbiotic culture systems have been established for many SAP-MHP species as a pure culture of free-living SAP fungi is easier than that of biotrophic AM or ECM fungi. Culturable SAP-MHPs are useful research materials and will contribute to the advancement of plant science.

     

Asynchronous origins of ectomycorrhizal clades of Agaricales

The ectomycorrhizal (ECM) symbiosis is the most widespread biotrophic nutritional mode in mushroom-forming fungi. ECM fungi include, though are not limited to, about 5000 described species of Agaricales from numerous, independently evolved lineages. Two central hypotheses suggest different explanations for the origin of ECM fungal diversity: (i) dual origins, initially with the Pinaceae in the Jurassic and later with angiosperms during the Late Cretaceous, and (ii) a simultaneous and convergent radiation of ECM lineages in response to cooling climate during the Palaeogene and advancing temperate ECM plant communities. Neither of these hypotheses is supported here. While we demonstrate support for asynchronous origins of ECM Agaricales, the timing of such events appears to have occurred more recently than suggested by the first hypothesis, first during the Cretaceous and later during the Palaeogene. We are also unable to reject models of rate constancy, which suggests that the diversity of ECM Agaricales is not a consequence of convergent rapid radiations following evolutionary transitions from saprotrophic to ECM habits. ECM lineages of Agaricales differ not only in age, but also in rates of diversification and rate of substitution at nuclear ribosomal RNA loci. These results question the biological uniformity of the ECM guild.     

Blurred boundaries: lifestyle lessons from ectomycorrhizal fungal genomes

Soils contain a multitude of fungi with vastly divergent lifestyles ranging from saprotrophic to mutualistic and pathogenic. The recent release of many fungal genomes has led to comparative studies that consider the extent to which these lifestyles are encoded in the genome. The genomes of the symbiotic fungi Laccaria bicolor and Tuber melanosporum are proving especially useful in characterizing the genetic foundation of mutualistic symbiosis. New insights gleaned from these genomes, as compared to their saprotrophic and pathogenic cousins, have helped to redefine and shape our understanding of the nature of the symbiotic lifestyle. Here we detail the current state of research into this complex relationship and discuss avenues for future exploration.     

Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens

Glycosyl hydrolase family 28 (GH28) is a set of structurally related enzymes that hydrolyze glycosidic bonds in pectin, and are important extracellular enzymes for both pathogenic and saprotrophic fungi. Yet, very little is understood about the evolutionary forces driving the diversification of GH28s in fungal genomes. We reconstructed the evolutionary history of family GH28 in fungi by examining the distribution of GH28 copy number across the phylogeny of fungi, and by reconstructing the phylogeny of GH28 genes. We also examined the relationship between lineage-specific GH28 expansions and fungal ecological strategy, testing the hypothesis that GH28 evolution in fungi is driven by ecological strategy (pathogenic vs. non-pathogenic) and pathogenic niche (necrotrophic vs. biotrophic). Our results showed that GH28 phylogeny of Ascomycota and Basidiomycota sequences was structured by specific biochemical function, with endo-polygalacturonases and endo-rhamnogalacturonases forming distinct, apparently ancient clades, while exo-polygalacturonases are more widely distributed. In contrast, Mucoromycotina and Stramenopile sequences formed taxonomically-distinct clades. Large, lineage-specific variation in GH28 copy number indicates that the evolution of this gene family is consistent with the birth-and-death model of gene family evolution, where diversity of GH28 loci within genomes was generated through multiple rounds of gene duplication followed by functional diversification and loss of some gene family members. Although GH28 copy number was correlated with genome size, our findings suggest that ecological strategy also plays an important role in determining the GH28 repertoire of fungi. Both necrotrophic and biotrophic fungi have larger genomes than non-pathogens, yet only necrotrophs possess more GH28 enzymes than non-pathogens. Hence, lineage-specific GH28 expansion is the result of both variation in genome size across fungal species and diversifying selection within the necrotrophic plant pathogen ecological niche. GH28 evolution among necrotrophs has likely been driven by a co-evolutionary arms race with plants, whereas the need to avoid plant immune responses has resulted in purifying selection within biotrophic fungi.     

Ectomycorrhizal fungi and their enzymes in soils: is there enough evidence for their role as facultative soil saprotrophs?

Although ectomycorrhizal (ECM) fungi are generally regarded as dependent upon the supply of carbon from their plant hosts, some recent papers have postulated a role for these fungi in the saprotrophic acquisition of carbon from soil. This theory was mainly based on the increase in enzymatic activity during periods of low photosynthate supply from tree hosts and emergence of the theory has led to a question about the overall influence of saprotrophy by ECM fungi on soil carbon turnover. However, I argue here that there is still not enough evidence to confirm this proposed function. My argument is based on inference from several lines of observation and concern over several aspects of the past studies. First, ECM fungi mainly inhabit deeper soil horizons, in which the availability of carbon compounds with positive energetic value is low. Second, the ability of ECM fungi to produce ligninolytic enzymes and cellulases is much weaker than that of saprotrophic basidiomycetes. This is most apparent in the low copy abundance of corresponding genes in the sequenced genomes of ECM species Laccaria bicolor and Amanita bisporigenes compared to the saprotrophic species Galerina marginata. I offer alternative hypotheses to explain the past observations of increased enzyme activity during starvation periods. These include, the induction of autolytic processes in ECM fungal mycelia or an attack on the host tissues to support escape from a dying root and to allow for a search for new hosts.     

New evidence for broad trophic status of leaf endophytic fungi of Quercus gambelii

Endophytic fungi inhabit the living tissues of every terrestrial plant species thus far examined. In at least some cases they significantly improve stress tolerance of their hosts. We asked whether endophytic fungi play other ecological roles, specifically whether the leaf endophytes of Quercus gambelii persist during the course of leaf decomposition, requiring a transition from a biotrophic to a saprotrophic mode of nutrition. Using automated ribosomal intergenic spacer analysis (ARISA), we found that endophyte fungal OTU diversity declined as decomposition commenced, but some endophytes persisted for months during which leaves were decomposing. In contrast, saprotroph fungi OTU diversity increased as decomposition progressed. These results are consistent with the hypothesis that some biotrophic endophytes persist in leaves during decomposition by becoming saprotrophic, and that the niche occupied by them is broader than expected.     

The irreversible loss of a decomposition pathway marks the single origin of an ectomycorrhizal symbiosis

Microbial symbioses have evolved repeatedly across the tree of life, but the genetic changes underlying transitions to symbiosis are largely unknown, especially for eukaryotic microbial symbionts. We used the genus Amanita, an iconic group of mushroom-forming fungi engaged in ectomycorrhizal symbioses with plants, to identify both the origins and potential genetic changes maintaining the stability of this mutualism. A multi-gene phylogeny reveals one origin of the symbiosis within Amanita, with a single transition from saprotrophic decomposition of dead organic matter to biotrophic dependence on host plants for carbon. Associated with this transition are the losses of two cellulase genes, each of which plays a critical role in extracellular decomposition of organic matter. However a third gene, which acts at later stages in cellulose decomposition, is retained by many, but not all, ectomycorrhizal species. Experiments confirm that symbiotic Amanita species have lost the ability to grow on complex organic matter and have therefore lost the capacity to live in forest soils without carbon supplied by a host plant. Irreversible losses of decomposition pathways are likely to play key roles in the evolutionary stability of these ubiquitous mutualisms.     

Trait‐dependent distributional shifts in fruiting of common British fungi

Despite the dramatic phenological responses of fungal fruiting to recent climate warming, it is unknown whether spatial distributions of fungi have changed and to what extent such changes are influenced by fungal traits, such as ectomycorrhizal (ECM) or saprotrophic lifestyles, spore characteristics, or fruit body size. Our overall aim was to understand how climate and fungal traits determine whether and how species‐specific fungal fruit body abundances have shifted across latitudes over time, using the UK national database of fruiting records. The data employed were recorded over 45 yr (1970–2014), and include 853 278 records of Agaricales, Boletales and Russulales, though we focus only on the most common species (with more than 3000 records each). The georeferenced observations were analysed by a Bayesian inference as a Gaussian additive model with a specification following a joint species distribution model. We used an offset, random contributions and fixed effects to isolate different potential biases from the trait‐specific interactions with latitude/climate and time. Our main aim was assessed by examination of the three‐way‐interaction of trait, predictor (latitude or climate) and time. The results show a strong trait‐specific shift in latitudinal abundance through time, as ECM species have become more abundant relative to saprotrophic species in the north. Along precipitation gradients, phenology was important, in that species with shorter fruiting seasons have declined markedly in abundance in oceanic regions, whereas species with longer seasons have become relatively more common overall. These changes in fruit body distributions are correlated with temperature and rainfall, which act directly on both saprotrophic and ECM fungi, and also indirectly on ECM fungi, through altered photosynthate allocation from their hosts. If these distributional changes reflect fungal activity, there will be important consequences for the responses of forest ecosystems to changing climate, through effects on primary production and nutrient cycling.     

The potential saprotrophic capacity of foliar endophytic fungi from Quercus gambelii

Endophytic fungi occur in living tissues of terrestrial plants. Many of these fungi are primarily biotrophic, but the trophic range of endophytic fungi as a group may not be fully appreciated. In this study, our goals were (1) for the Class 3 foliar endophytic fungi isolated from Quercus gambelii, determine their potential saprotrophic capacity, which we define as the difference in growth rate in culture on Quercus gambelii leaf litter medium and control medium lacking leaf litter and (2) quantify sources of variation among isolates of these endophytic fungi in potential saprotrophic capacity, including variation due to microsite within host trees (leaves receiving full sun vs. shade) and variation within and among fungal genera. We found that 48 of the 49 tested endophytic fungal isolates have significant potential saprotrophic capacity. Contrary to expectation, the amount of solar radiation available to the leaf from which the fungi were isolated had no significant impact on potential saprotrophic capacity and there was more variability in potential saprotrophic capacity among isolates within a genus than among genera. Our results suggest that some Class 3 endophytic fungi may have the potential to function as saprotrophic fungi within plant litter, but this remains to be seen for these Quercus gambelii isolates under more natural circumstances.     

Ectomycorrhizal fungal diversity and community structure on three co-occurring leguminous canopy tree species in a Neotropical rainforest

? The ectomycorrhizal (ECM) symbiosis was historically considered restricted to the temperate zones, but recent studies have shown the importance of this symbiosis across the tropics. We examined ECM fungal diversity, host plant phylogeny and ECM host preferences in a rainforest dominated by the leguminous host plants Dicymbe corymbosa, Dicymbe altsonii and Aldina insignis. ? Ectomycorrhizal fungi were identified by internal transcribed spacer rDNA sequencing and host species were verified with chloroplast trnL sequencing. To test whether Dicymbe and Aldina represent independent gains of the ECM symbiosis, we constructed a Fabaceae phylogeny using MatK and trnL. We identified four independent ECM lineages within the Fabaceae. ? We detected a diverse community of 118 ECM species dominated by the /clavulina, /russula-lactarius, /boletus, and /tomentella-thelephora lineages. Ectomycorrhizal species in Agaricales, Atheliales and Polyporales may represent previously unrecognized tropical-endemic ECM lineages. Previous studies suggested that ECM fungi did not diversify in the tropics, but the /clavulina lineage appears to have a center of diversity in tropical South America. ? Dicymbe and Aldina represent independent gains of the ECM symbiosis in Fabaceae but their fungal symbionts showed no host preferences. Spatial factors are more important than hosts in structuring the ECM fungal community in this ecosystem.     

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Background  

Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi.     

Mean reproductive traits of fungal assemblages are correlated with resource availability

Organisms have evolved a fascinating variety of strategies and organs for successful reproduction. Fruit bodies are the reproductive organ of fungi and vary considerably in size and shape among species. Our understanding of the mechanisms underlying the differences in fruit body size among species is still limited. Fruit bodies of saprotrophic fungi are smaller than those of mutualistic ectomycorrhizal fungi. If differences in fruit body size are determined by carbon acquisition, then mean reproductive traits of saprotrophic and ectomycorrhizal fungi assemblages should vary differently along gradients of resource availability as carbon acquisition seems more unpredictable and costly for saprotrophs than for ectomycorrhizal fungi. Here, we used 48 local inventories of fungal fruit bodies (plot size: 0.02 ha each) sampled along a gradient of resource availability (growing stock) across 3 years in the Bavarian Forest National Park in Germany to investigate regional and local factors that might influence the distribution of species with different reproductive traits, particularly fruit body size. As predicted, mean fruit body size of local assemblages of saprotrophic fungi was smaller than expected from the distribution of traits of the regional species pool across central and northern Europe, whereas that of ectomycorrhizal fungi did not differ from random expectation. Furthermore and also as expected, mean fruit body size of assemblages of saprotrophic fungi was significantly smaller than for assemblages of ectomycorrhizal species. However, mean fruit body sizes of not only saprotrophic species but also ectomycorrhizal species increased with resource availability, and the mean number of fruit bodies of both assemblages decreased. Our results indicate that the differences in carbon acquisition between saprotrophs and ectomycorrhizal species lead to differences in basic reproductive strategies, with implications for the breadth of their distribution. However, the differences in resource acquisition cannot explain detailed species distribution patterns at a finer, local scale based on their reproductive traits.     

Intra-species genetic variability drives carbon metabolism and symbiotic host interactions in the ectomycorrhizal fungus Pisolithus microcarpus

Ectomycorrhizal (ECM) fungi are integral to boreal and temperate forest ecosystem functioning and nutrient cycling. ECM fungi, however, originate from diverse saprotrophic lineages and the impacts of genetic variation across species, and especially within a given ECM species, on function and interactions with the environment is not well understood. Here, we explore the extent of intra-species variation between four isolates of the ECM fungus Pisolithus microcarpus, in terms of gene regulation, carbon metabolism and growth, and interactions with a host, Eucalyptus grandis. We demonstrate that, while a core response to the host is maintained by all of the isolates tested, they have distinct patterns of gene expression and carbon metabolism, resulting in the differential expression of isolate-specific response pathways in the host plant. Together, these results highlight the importance of using a wider range of individuals within a species to understand the broader ecological roles of ECM fungi and their host interactions.     

Meeting a non-host: the behaviour of AM fungi

 Arbuscular mycorrhizal (AM) fungi are obligately biotrophic organisms that live symbiotically with the roots of most plants. The establishment of a functional symbiosis between AM fungi and host plants involves a sequence of recognition events leading to the morphological and physiological integration of the two symbionts. The developmental switches in the fungi are triggered by host signals which induce changes in gene expression and a process leading to unequivocal recognition between the two partners of the symbiosis. It has been calculated that about 80% of plant families from all phyla of land plants are hosts of AM fungi. The remaining plant species are either non-mycorrhizal or hosts of mycorrhizas other than the arbuscular type. Non-host plants have been used to obtain information on the factors regulating the development of a functional symbiosis. The aim of this present review is to highlight present-day knowledge of the fungal developmental switches involved in the process of host/non-host discrimination. The following stages of the life cycle of AM fungi are analysed in detail: spore germination, presymbiotic mycelial growth, differential branching pattern and chemotropism, appressorium formation, root colonization. Accepted: 17 June 1998     

Chromosome-level genomes provide insights into genome evolution,organization and size in Epichloe fungi

Epichloe fungi are endophytes of cool season grasses, both wild species and commercial cultivars, where they may exhibit mutualistic or pathogenic lifestyles. The Epichloe-grass symbiosis is of great interest to agricultural research for the fungal bioprotective properties conferred to host grasses but also serves as an ideal system to study the evolution of fungal plant-pathogens in natural environments. Here, we assembled and annotated gapless chromosome-level genomes of two pathogenic Epichloe sibling species. Both genomes have a bipartite genome organization, with blocks of highly syntenic gene-rich regions separated by blocks of AT-rich DNA. The AT-rich regions show an extensive signature of RIP (repeat-induced point mutation) and the expansion of this compartment accounts for the large difference in genome size between the two species. This study reveals how the rapid evolution of repeat structure can drive divergence between closely related taxa and highlights the evolutionary role of dynamic compartments in fungal genomes.     

The effects of grazing history,soil properties and stand structure on the communities of saprotrophic fungi in wood-pastures

Wood-pastures are threatened anthropogenic biotopes that provide habitat for an extensive group of species. Here we studied the effect of management, grazing intensity, time since abandonment, historical land-use intensity, soil properties and stand conditions on communities of saprotrophic fungi in wood-pastures in Central Finland. We found that the proportion of broadleaved trees and soil pH are the major drivers in the communities of saprotrophic fungi in these boreal wood-pastures. In addition, tree species richness, soil moisture, historical land-use intensity and time since abandonment affected the communities of saprotrophic fungi. Current management or grazing intensity did not have a clear effect on saprotrophic fungal species richness, although dung-inhabiting fungal species richness was highest at intermediate to high grazing intensity. Obviously, there were many more dung-inhabiting fungal species on grazed than on abandoned sites. Our study highlights the conservation value of wood-pastures as hotspots of saprotrophic fungi.     

Plant identity strongly structures the root-associated fungal community in a diverse subtropical forest

Revealing the relationship between plants and root-associated fungi is very important in understanding diversity maintenance and community assembly in ecosystems. However, the community assembly of root-associated fungi of focal plant species along a subtropical plant species diversity gradient is less documented. Here, we examined root-associated fungal communities associated with five ectomycorrhizal (EM) plant species (Betula luminifera, Castanea henryi, Castanopsis fargesii, C. sclerophylla, and Quercus serrate) in a Chinese subtropical woody plant species diversity (1, 2, 4, 8, 16 and 24 species) experiment, using paired-end Illumina MiSeq sequencing of the ITS2 region. In total, we detected 1933 root-associated fungal operational taxonomic units (OTUs) at a 97% sequence similarity level. Plant identity had a significant effect on total and saprotrophic fungal OTU richness, but plant species diversity level had a significant effect on saprotrophic and pathogenic fungal OTU richness. The community composition of total, saprotrophic and EM fungi was structured by plant identity and plant species diversity level. However, the community composition of pathogenic fungi was only shaped by plant identity. This study highlights that plant identity has a stronger effect on the root-associated fungal community than plant species diversity level in a diverse subtropical forest ecosystem.     

Studies on Ectomycorrhiza: An Appraisal

Ectomycorrhizal (ECM) fungi are obligate symbionts of dominant vascular plants, liverworts and hornworts. There are reports of about 20,000 to 25,000 ECM fungi that promote plant growth by facilitating enhanced water and nutrient absorption, and provide tolerance to environmental stresses. These below-ground fungi play a key role in terrestrial ecosystems as they regulate plant diversity, nutrient and carbon cycles, and influence soil structure and ecosystem multifunctionality. Because ECM fungi are obligate root symbionts, host plant can have a strong effect on ECM species richness and community composition. The biogeographic pattern and detailed functioning and regulation of these mycorrhizosphere processes are still poorly understood and require detailed study. More recent researches have placed emphasis on a wider, multifunctional perspective, including the effects of ectomycorrhizal symbiosis on plant and microbial communities, and on ecosystem processes. Over the years the main focus in ECM research has been on the study of diversity and specificity of ECM strains, the role of ECM in regeneration of degraded ecosystem, the growth and establishment of seedlings through nutrient acquisition and the mediation of plant responses to various types of stress. In this review, recent progresses in ectomycorrhizal biology are presented, especially the potential role of ECM symbioses in resistance or tolerance to various biotic and abiotic stresses, and in maintinance of plant diversity for proper ecosystem functioning.     

Ultrastructural and cytochemical aspects of the interaction between the ectomycorrhizal fungus Laccaria laccata and the saprotrophic fungi, Trichoderma harzianum and T. virens

Different interactions between soil fungi competing in the rhizosphere with each other are necessary to understand their influence on plant growth and health. The interactions between the ectomycorrhizal (ECM) fungus Laccaria laccata and soil saprotrophic fungi (T. harzianum, T. virens) were studied by transmission electron microscopy, and by gold cytochemistry to assess the potential role of cell wall lytic enzymes in mycoparasitism. Anti-β-1,3-glucan antibody, WGA/ovomucoid-gold complex and PATAg test were used to localize β-1,3-glucan, chitin and polysaccharides. Cytoplasm disorganisation of the saprotrophic fungi occurred concurrently with dissolution of β-1,3-glucan in walls of hyphae and conidia of the saprotrophic fungi. Then digestion of polysaccharides and chitin of colonised fungal structures occurred. The studies suggest sequential contribution of cell wall lytic enzymes and importance of disturbing the host's cell integrity during mycoparasitism. We conclude that the ECM fungus can parasitise on the saprotrophic fungi not only in dual culture on artificial medium but also in the rhizosphere of Scots pine.     

Mycorrhizas: Gene to Function

Substantial progress has been made toward development of molecular tools for identification and quantification of mycorrhizal fungi in roots and evaluation of the diversity of ectomycorrhizal (ECM) fungi and the phylogeny and genetic structure of arbuscular mycorrhizal (AM) fungi. rDNA analysis confirms high diversity of ECM fungi on their hosts, and for AM fungi has revealed considerable genetic variation within and among morphologically similar AM fungal species. The fungal and plant genes, regulation of their expression, and biochemical pathways for nutrient exchange between symbiotic partners are now coming under intense study and will eventually be used to define the ecological nutritional role of the fungi. While molecular biological approaches have increased understanding of the mycorrhizal symbiosis, such knowledge about these lower-scale processes has yet to influence our understanding of larger-scale responses to any great extent.