Novel aspects in the life cycle and biotrophic interactions in Pezizomycetes (Ascomycota, Fungi)



The ascomycete class Pezizomycetes (single order Pezizales) is known for its cup-shaped fruit bodies and the evolution of edible truffles and morels, but little is known about the ontogeny and ecology of this large and ecologically diverse fungal group. In this issue of Molecular Ecology, Healy et al. (2013) make a great leap forward by describing and identifying asexual, anamorphic structures that produce mitotic spores in many ectomycorrhiza-forming truffle and nontruffle species on soil surfaces worldwide (Fig. 1). Although such anamorphic forms have been reported sporadically from certain ectomycorrhizal and saprotrophic Pezizomycetes (e.g. Warcup 1990), Healy et al. (2013) demonstrate that these terricolous asexual forms are both taxonomically and geographically more widespread and, in fact, much more common than previously understood. We anticipate that deeper insight into other substrates, provided by molecular analyses of materials such as dead wood and seeds, is likely to reveal numerous anamorphs of saprotrophic and pathogenic Pezizomycetes as well (see Marek et al. 2009).

The mitospores of these pezizomycetes anamorphs do not germinate on standard media, and therefore, their importance in the life cycle of Pezizomycetes cannot be directly evaluated. The authors argue that these small, thin-walled mitospores may function as spermatia to ensure outcrossing (Healy et al. 2013). Alternatively, mitospores may func-tion as dispersal propagules immediately after disturbance events: mitospores can be produced rapidly, whereas development of fruit bodies and the meiospores therein requires mating of compatible strains (at least in Tuber; Rubini et al. 2011) and development of the more complex fruit-body structure. The resulting meiospores, which are relatively thick-walled and large, resist digestion in the gut of small mammals and heat from moderate burning intensity, and potentially contribute to the formation of a persistent spore bank in soil (Bruns et al. 2005; Bonito et al. 2012). Despite the apparent benefits of this biphasic reproductive strategy, it appears that the widespread production of asexual structures in these Pezizomycetes is unique among ectomycorrhizal fungi (although known from other mycorrhizal ascomycetes Meliniomyces bicolor and Cadophora finlandica of the Helotiales). Ectomycorrhizal basidiomycetes lack such an asexual propagation stage in their life cycle (Hutchison 1989).

Despite recent discoveries regarding the genetics and genomics of truffles, especially the model species black Périgord truffle (Tuber melanosporum) (Martin et al. 2010; Rubini et al. 2011), much of the life history and ecology of truffles and other Pezizomycetes remain shrouded in mystery. Until recently, most Pezizomycetes that fruit epigeously have been considered primarily saprobic and rarely plant pathogenic (reviewed in Hansen & Pfister 2006). However, new studies using molecular techniques have unveiled the roles of many taxa as ectomycorrhizal symbionts (Tedersoo et al. 2006, 2010; Smith et al. 2007), orchid root symbionts (Waterman et al. 2011; Tešitelova et al. 2012), and endophytes and endolichenic fungi in living photosynthetic tissues of plants and lichens, respectively (U'Ren et al. 2012). Here we highlight several new and previously unpublished discoveries on the ecology of Pezizomycetes, particularly in the ectomycorrhizal, orchid mycorrhizal, endophytic and endolichenic Pyronemataceae, which is the largest and most heterogeneous family within cup fungi (Perry et al. 2007).

New belowground discoveries

At present, Pezizomycetes appear to include 16 phylogenetically distinct lineages of ectomycorrhizal fungi that mostly inhabit temperate ecosystems of the Northern Hemisphere, but are scarce in tropical habitats (Tedersoo et al. 2010, 2012). Several recent studies have reported multiple ectomycorrhizal groups within Pezizomycetes that do not conform to these hitherto known lineages (e.g. Bonito et al. 2012; Healy et al. 2013). Re-analysis of large subunit rDNA sequence data from previous studies and unpublished work revealed four additional and previously unreported ectomycorrhizal lineages within the Pyronemataceae (Fig. 2). One of these ectomycorrhizal lineages comprises the circumboreal species Pustularia patavina, which forms a strongly supported monophyletic group with an undescribed species of Pyronemataceae (PP = 1.00). Rhodoscypha ovilla, which also has a circumboreal distribution, forms the second lineage. The sister taxa of Rhodoscypha remain unresolved, but constitute Leucoscypha, Neottiella, Rhodotarzetta or Octospora. The third group comprises a well-supported clade (PP = 1.00) of the hypogeous truffle-like Unicava sp. (gen. ined.) and Aleurina imaii. This clade exhibits a southern temperate distribution with Nothofagus and potentially Eucalyptus. The fourth group of ectomycorrhizal symbionts, from West Africa, USA and Argentina (PP = 0.98), is nested within a well-supported clade of Pulvinula spp. and Lazuardia lobata. This small ectomycorrhizal group lacks sequenced specimens and its identity and distribution are not yet known. It is distinct from a group of ectomycorrhizal Pulvinula species (corresponding to the/pulvinula lineage sensu Tedersoo et al. 2010) that are restricted to the Northern Hemisphere, and from Lazuardia and Pulvinula globifera, which presumably are saprotrophic. Profound taxonomic investigations into this relatively large and ecologically heterogeneous clade of Pyronemataceae could shed light into the evolution of symbiosis in this group.

Sequences belonging to these hitherto unrecognized ectomycorrhizal lineages have never been recovered from other plant tissues, roots of nonectomycorrhizal plants or agricultural soils. Moreover, root tip collections available to us revealed strong morphological similarity within these groups, as is commonly seen in other ectomycorrhizal lineages of Pezizomycetes and Leotiomycetes (Tedersoo et al. 2006, 2009). Although proof of synthesis is lacking, consistent association with root tips and similarities in morphology suggest that these root-associated groups are truly ectomycorrhizal. These findings also indicate that further ectomycorrhizal lineages of Pezizomycetes almost certainly await discovery, particularly in temperate and subtropical forests of the Southern Hemisphere that are relatively poorly covered in taxonomic and mycorrhizal surveys coupled with molecular identification (Nouhra et al. 2012).

Whereas many orchid species acquire their mineral nutrients via saprotrophic fungi of the Ceratobasidiaceae, Tulasnellaceae and Serendipitaceae families, certain orchid groups have evolved associations with ectomycorrhizal fungi, particularly basidiomycetes. Many of these ectomycorrhizal basidiomycete-associated orchids have partly or fully lost their photosynthetic capacity, relying on their fungi both for carbon and mineral nutrition (Dearnaley et al. 2012). However, specific clades of partly or fully autotrophic groups have switched their basidiomycete root symbionts to specific groups within Pezizomycetes. For example, many partly autotrophic species of Epipactis associate nearly exclusively with ectomycorrhizal Tuberaceae and several groups of Pyronemataceae, including Wilcoxina, Genea, Geopora and Trichophaea woolhopeia (Tešitelova et al. 2012; Fig. 2). The fully autotrophic orchid Gymnadenia conopsea predominately associates with saprotrophic Pezizaceae, in addition to Tulasnellaceae and Ceratobasidiaceae in Germany (Stark et al. 2009). South African photosynthetic orchids from the genera Corycium and Pterygodium (sect. Ommatidium) almost exclusively associate with root symbionts that are closely related to mycobionts of Gymnadenia, and additionally with saprotrophic Tricharina spp. (Waterman et al. 2011).

Instead of mycorrhizal associations, truffle-like forms of the Pezizaceae genera Mattirolomyces and Kalaharituber inhabit roots of nonectomycorrhizal plants, forming differentiated, arbuscular mycorrhiza-like interactions within root cells (Bratek et al. 1996; Kagan-Zur et al. 1999). These specialized structures and the formation of relatively large fruit bodies render these associations distinct from typical root endophytic interactions that lack differentiated structures for nutrient exchange. These root endophytes are not expected to access the large carbon and nitrogen sources required for fruit-body production. Such truffle-like fungi and morels (Morchella spp.) seem to inhabit root tissues in a facultative manner, relying instead on carbon and mineral nutrients in soil organic matter.

Endophytic and endolichenic associations

The vast majority of endophytic fungi in roots and aboveground tissues of plants belong to the subphylum Pezizomycotina within the Ascomycota (Arnold et al. 2009). Traditional culture-based techniques have revealed that relatively fast-growing groups such as Helotiales, Sordariales, Xylariales, and Dothideales often dominate surveys, but communities differ markedly as a function of plant tissue, host taxon and biome (Arnold 2007; U'Ren et al. 2012). The importance of relatively slow-growing Pezizomycetes as foliar endophytes of trees and as endolichenic fungi has been only recently uncovered by combining culturing and sequence analysis (Arnold et al. 2009; U'Ren et al. 2012). One recent study showed that phylogenetically diverse Pyronemataceae (Fig. 2) and related Pezizomycetes comprised >50% of isolates from lichens and photosynthetic tissues of plants in the seasonally dry, fire-dominated montane forests of Arizona (U'Ren et al. 2012). Overall, endophytic and endolichenic Pezizomycetes have proved less common in humid temperate and subtropical sites, and in those cases, families other than Pyronemataceae typically dominate the pezizalean strains (particularly Pezizaceae, Sarcosomataceae and Sarcoscyphaceae). The cause of pezizalean dominance in Arizona forests, a phenomenon observed across a wide geographic range and in mountains with different floristic structures (i.e., Madrean Archipelago and Petran floras), remains unknown.

Most of the Pyronemataceae that occur as endophytes and endolichenic fungi in these mountains are affiliated with Tricharina, Trichophaea and Geopyxis (Fig. 2) (U'Ren et al. 2012), which are phylogenetically distinct from ectomycorrhizal fungi (except a single isolation of Trichophaea brunnea). Although found in vascular plants (e.g. Juniperus, Pinus and Pseudotsuga; Fig. 2) these and related Pezizomycetes are especially common in mosses and lichens, including species that grow on the ground (U'Ren et al. 2010). As such, their distinctiveness from ectomycorrhizal fungi that are present in soil is striking (Fig. 2).

Importantly, symbiotic Pezizomycetes are not common only at the soil level: they have been isolated metres above the forest floor in healthy foliage or in lichens growing on tree trunks or hanging from elevated branches. Cup-shaped or sometimes truffle-like fruit bodies of many Pezizomycetes commonly fruit on bare soil, among litter or strongly decayed wood—but what takes these typically soil-inhabiting groups of fungi into the heights? Pezizalean fruit bodies and asexual mitosporic structures have never been observed on healthy leaves, such that plant tissues must be colonized by propagules originating from other substrates. Indeed, many other ascomycete groups start their life cycle as foliar endophytes in the canopy, but then account for degradation of litter for several months in the forest floor (Osono 2006; U'Ren 2011). Colonization of healthy leaves likely enables them to establish in the substrate and perhaps compete effectively against potentially more efficient saprobes in the litter and humus layers (Vořiškova & Baldrian 2012). These fungi typically fruit or produce mitospores in the litter that infect the next generation of young leaves, and in many cases, the same genotypes can be recovered from dead leaves of the same hosts in which they are found as endophytes (U'Ren 2011). However, it is not yet clear whether the pezizalean endophytes and endolichenic fungi generally follow this dual ecological strategy, nor what is the relative contribution of sexual and asexual propagules in the infection process.

Interestingly, several genera of Pyronemataceae (such as Lamprospora, Octosporella and Octospora) are obligately symbiotic with bryophytes and hepatics. Those species studied are characterized by hyphae that attach to subterranean, living rhizoids by means of appressoria connected to intracellular haustoria, suggesting a rhizoid parasitism in nature (Benkert 1993; Döbbeler 2002). Many species exhibit restricted host ranges and associate with a single bryophyte species or genus, whereas others have a wide spectrum of hosts (Benkert 1993). A recent molecular identification study of moss-associated fungi from a boreal biome revealed Pezizomycetes to account for <1% of operational taxonomic units and sequences (Davey et al. 2012). By contrast in another study, >70% of the fungal isolates from photosynthetic tissues of mosses belonged to the Pezizomycetes (see U'Ren et al. 2010). These were isolated from two of the same boreal region mosses as in the previous study, Polytrichum commune and Pleurozium schreberi, and additionally from Ceratodon purpureus and Leucobryum sp. in Arizona (U'Ren et al. 2012). None of the recovered pezizomycetes were members of the obligately bryophilous symbionts, underscoring the wealth of diversity in Pezizomycetes yet to be uncovered from plants and lichens (U'Ren et al. 2012).

Towards functional studies

These recently revealed biotrophic associations of Pezizomycetes suggest that this group has a hitherto underestimated role in nature. Much remains to be documented in fire-dominated habitats (such as ecosystems of Arizona, per above), where many pyrophilous groups of Pezizomycetes proliferate (Petersen 1970) and form a continuum of biotrophic associations with emerging tree seedlings (Egger & Paden 1986; Warcup 1990). These studies also revealed that both saprotrophic and ectomycorrhizal groups exhibit weak to moderate abilities to degrade components of plant cell walls. Cellulolytic activity of diverse endophytic and endolichenic Pyronemataceae were recently detected as well (A.E. Arnold, unpublished). The nutritional relevance of these capacities to the biotrophic interactions with plants and lichens, and the insights they provide for the ecology and evolution of Pezizomycetes more broadly, remains to be evaluated by use of genomic tools.

Although the endophytic and ectomycorrhizal interactions involving particular groups of Pezizomycetes are probably facultative to most plants, the orchid root-symbiotic fungi in this group are exclusive mutualists of certain orchids, indicating the unique capacity for pezizalean fungi to support plant diversity. We believe that further investigations particularly in poorly studied habitats and using culture-independent, high-throughput identification methods will reveal many interesting associations involving Pezizomycetes in parts or all of their life cycles. This returns us to the black box of the ecology of Pezizomycetes, in particular their abilities to propagate, to be dispersed by wind and small mammals, to persist in soil and to associate with various lichens, plants and other organisms. Discoveries below- and above-ground await.

L.T. and A.E.A. compiled data on ectomycorrhizal and endophytic/endolichenic fungi, respectively; K.H. analysed the data and introduced up-to-date taxonomy. All authors contributed to writing the manuscript.

Data accessibility

The DNA sequences are publicly available in INSDc and UNITE sequence databases. Phylogenetic data are stored in TreeBASE (accession no. S13709). Methods are profoundly described in Supporting information.

Figure 1.

Photographs of vegetative and sexual structures of Pachyphloeus OTU13 (sensu Healy et al. 2013): (a) mitosporic spore mat (RHAM282, GenBank JN102395); (b) ectomycorrhiza with Carya illinoensis (GenBank HQ541834); and (c) mature fruit body (RH1240, GenBank JN409345). Scale bar refers to 10 mm (a), 0.5 mm (b), and 5 mm (c). Photo credit: (a,c) R.A. Healy; (b) G.M. Bonito.

Figure 2.

Phylogenetic placement of ectomycorrhizal (highlighted in bold; newly described groups, red), endophytic (green) and endolichenic (blue) isolates among Pyronemataceae species as inferred from a Bayesian analysis of 126 sequences of nuclear ribosomal large subunit (LSUrDNA). The phylogram represents a 50% majority rule consensus tree of 36 000 trees from the last 75% of the posterior obtained from a 12 million generation analysis with four parallel runs using Bayesian Metropolis-coupled Markov chain Monte Carlo (MCMCMC) with model jumping (see Supplementary information for methods). Green and black asterisks denote the approximate phylogenetic position of root symbionts of autotrophic orchids in South Africa and partly photosynthetic Epipactis spp., respectively, as based on publicly available ITS sequences. Bar indicates 0.2 changes. Posterior probabilities >0.94 are shown above branches.