Introduction

Fungal endophytes live asymptomatically within plants and are widespread inhabitants of leaves and other organs (Wilson, 1995). Likewise, endolichenic fungi live asymptomatically within lichens, occurring in healthy lichen thalli worldwide (Arnold et al., 2009). Endophytes and endolichenic fungi are ecologically similar, living in symbiosis with either a plant or the photobionts of lichens (Arnold et al., 2009), and both functional groups represent the same major lineages of fungi (U’Ren et al., 2012). As a whole, these fungi include diverse species whose life cycles often include pathogenic or saprobic phases (Porras-Alfaro & Bayman, 2011; Selosse, 2018; Terhonen et al., 2019). Endophytes and endolichenic fungi occur from polar regions to the tropics (Arnold et al., 2009), with most species transmitted horizontally (see Rodriguez et al., 2009). Although most endophyte–host interactions have not been examined, some have positive impacts on the physiology, growth or stress tolerance of their hosts (e.g. Arnold et al., 2003; Rodriguez et al., 2009; Porras-Alfaro & Bayman, 2011). Endophytic and endolichenic fungi are especially common and diverse among the largest nonlichenized lineages of the subphylum Pezizomycotina (Ascomycota), with variation among host lineages and biomes in the relative abundance of the five most common classes in which endophytism is known (Sordariomycetes, Dothideomycetes, Leotiomycetes, Eurotiomycetes and Pezizomycetes; Arnold et al., 2009).

Among these, fungi in the class Pezizomycetes are of special interest because the biology of many taxa is incompletely known. This class consists of one order (Pezizales), 23 families and an estimated 2000 species (Pfister & Healy, 2021). Species of Pezizomycetes include well-documented plant pathogens (Marek et al., 2009), ectomycorrhizal (ECM) fungi (Tedersoo et al., 2006) and saprobes (Hobbie et al., 2001; Hansen & Pfister, 2006). Some species colonize specific substrates, acting as parasites of bryophytes (Döbbeler, 1997) or as specialized saprobes of dung (Pfister, 2015; Richardson, 2019) or postfire materials (Egger, 1986). Over the past decade, studies have shown that some species of Pezizomycetes are common as endophytes within bryophytes and occur frequently in lichen thalli (U’Ren et al., 2010, 2019). The life cycles and trophic ecology of some Pezizomycete species are unclear or controversial, and the endophytic habit has not been considered an important ecological strategy across the class (Pfister, 2015). Yet evidence suggests a more complex story. For example, stable isotope analyses indicate that members of the genus Morchella (morels) are able to access dead organic matter (Hobbie et al., 2001, 2016), but they also can live endophytically in roots (Baynes et al., 2012) and in conifer needles (Baroni et al., 2018). Likewise, the esteemed black truffles Tuber melanosporum and T. aestivum are ECM fungi, but in an interesting twist to truffle ecology, they have been shown to live as endophytes in roots of non-ECM plants (Schneider-Maunoury et al., 2020).

While studying Pezizomycetes from an evolutionary and functional perspective, we noted that rDNA sequences from many Pezizomycete endophytes were available in GenBank, but that their phylogenetic affinities were not defined. Many sequences were generated from living cultures isolated from diverse plants and lichens sampled across the globe by Arnold and collaborators at the University of Arizona (UA) and maintained there as part of the Robert L. Gilbertson Mycological Herbarium (Myco-ARIZ; e.g. Hoffman & Arnold, 2010; U’Ren et al., 2010, 2012, 2014, 2019; Lau et al., 2013; Sandberg et al., 2014; Massimo et al., 2015; Huang et al., 2016, 2018a; U’Ren & Arnold, 2016; Bowman & Arnold, 2018; Oita et al., 2021a,b).

The many DNA barcode matches among endophytes in GenBank and our unpublished sequences from fruit bodies suggested that endophytism and endolichenism might be more common, phylogenetically dispersed and ecologically important among Pezizomycetes than documented previously. Therefore, we assembled the available ecological and phylogenetic data on endophytic and endolichenic Pezizomycete species to ask: of the estimated 2000 Pezizomycete species known to date, how many occur as endophytes or endolichenic fungi?; in which lineages does endophytism or endolichenism occur across the Pezizomycetes?; and what are the main nutritional modes of endophytic or endolichenic Pezizomycete species when they are outside their hosts?

We generated comprehensive phylogenies of Pezizomycetes based on 3315 sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS1-5.8s-ITS2 nrDNA; hereafter ITS) and 1102 sequences of the large subunit nrDNA (28S) from fruit bodies, endophytes, and environmental sequences. We used maximum-likelihood (ML) phylogenetic analysis to determine operational taxonomic units (OTUs) (Table 1), but also compared our phylogeny-based OTUs with those recovered from a clustering approach based on 97% sequence similarity (Supporting Information Table S1). We included representatives of 3784 ITS sequences from the UA endophyte collection as well as new and reference sequences from Pezizomycete fruit bodies (Tables 1, S2). We preferentially incorporated available sequences from type specimens and used representative sequences in combination with Blast to obtain additional sequences of endophytic and endolichenic species (see flow chart in Fig. S1 for graphic of the methods).

In order to examine placement of endophytes in Pezizomycetes, we first assembled a single 28S rDNA alignment with representatives of Pezizomycete lineages and ecological modes, including 266 endophytic and endolichenic sequences, and performed ML analyses via RAxML with 1000 bootstraps. The 50% majority rule tree (Figs 1, S2) was used in conjunction with Hansen & Pfister (2006) and Pfister (2015) as a guide for placement of endophytic and endolichenic taxa (Table 1). The ITS locus often is useful for species-level identification (Schoch et al., 2012; Kõljalg et al., 2013) and many endophyte studies generate only ITS sequences. Accordingly, we compiled 35 separate ITS alignments for individual families or lineages within families of Pezizomycetes, including 1046 endophytic or endolichenic sequences (Figs S3–S37). Details of methods, sequence alignments and accession numbers are provided in Notes S1, Fig. S1 and Tables S1 & S2.

We detected endophytic and endolichenic species in 50 Pezizomycete genera and in 14 lineages that could not be assigned confidently to a genus. Together these represented ≥ 160 OTUs distributed across ≥ 16 families (Table 1; Figs 1, S2–S37). Some families had endophytism or endolichenism represented in only one or two genera (e.g. Desmazierella in Chorioactidaceae, Pseudombrophila in Pseudombrophilaceae), whereas others had species with these lifestyles in many genera, including Pezizaceae (27 OTUs in nine genera) and Pyronemataceae (45 OTUs in 17 genera; see also Tedersoo et al., 2013). Our conservative OTU delimitations based on ITS phylogenies yielded 160 OTUs whereas a clustering-based approach yielded 216 OTUs. However, the two methods yielded similar inferences regarding the ecology and phylogenetic distribution of Pezizomycete endophytes and endolichenic fungi (Table S1). The results presented here are based on the phylogenetic approach.

Notably, we detected endophytism or endolichenism in two lineages for which this mode was previously undocumented: the ECM genus Otidea (Otideaceae) (Fig. S10) and dung saprobe genus Coprotus (Coprotaceae) (Fig. S6). In both genera we detected endophytic and endolichenic isolates from multiple hosts, suggesting that endophytism is a regular feature of their biology. Although a few Pezizomycete lineages with endophytic or endolichenic members were detected in only a limited range of hosts (e.g. Ascodesmidaceae was only isolated from vascular plants), most Pezizomycetes have a broad endophytic host range (as reported in detail by U’Ren et al., 2019). Endophytic and endolichenic Pezizomycetes are from lineages with a variety of trophic strategies, including saprobes, plant pathogens and mycorrhizal fungi (Tables S2, S4), but also include taxa for which the trophic strategies remain unresolved or unknown (Table S5).

One particularly striking finding was an OTU that could not be assigned to a known family. This OTU is represented by five isolates from angiosperm leaves and lichens in an Alaskan boreal forest (USA) (U’Ren et al., 2019). It represents a unique lineage nested between Tuberaceae and Geomoriaceae, both of which consist exclusively of ECM species (Fig. 2; Notes S1; Tables S1–S3).

The vast majority of Pezizomycete isolates in the culture-based UA dataset came from lichens (78%). Only one family (Ascodesmidaceae) was never detected in lichens, suggesting that lichens are important hosts for Pezizomycete species (Table S4). However, despite the importance of lichens as hosts, no Pezizomycete families were restricted to lichens alone and OTUs from most Pezizomycete families were detected in many photosynthetic hosts. For example, the most frequently isolated species were from Sarcosomataceae (Table S4), a family of largely saprobic taxa. At the genus level Pseudoplectania (Sarcoscomataceae) was especially well represented, comprising nearly 40% of Pezizomycete isolates in the UA dataset (Table S5).

More generally, one conspicuous and recurrent phylogenetic pattern is that both endophytes and endolichenic fungi are common in clades containing well-characterized saprobes from wood, dung or postfire substrates, but infrequent in closely related ECM or plant pathogenic clades, as noted for Pyronemataceae by Tedersoo et al. (2013). For example, endophytism and endolichenism were commonly detected for Geopyxis, a genus of Tarzettaceae with putatively biotrophic, weakly parasitic and/or pyrophilous species (Egger, 1986; Vrålstad et al., 1998), but not detected for the ECM sister genus Tarzetta (Fig. S35). Likewise, endophytism and endolichenism was commonly detected for species of saprobic Pezizaceae (e.g. Peziza s.s., Plicaria and Geoscypha) (Figs S13, S17, S18) but rarely or never detected for ECM species such as those in Legaliana or Ruhlandiella (Fig. S11). In the Discinaceae, endophytism and endolichenism was common for species of Gyromitra, a genus of putative saprobes, but absent in species of the ECM sister genus Hydnotrya (Hobbie et al., 2001) (Fig. S7).

Although endophytes were documented only rarely among ECM and pathogenic clades of Pezizomycetes, we detected endophytism and endolichenism in four lineages known previously only for ECM lifestyles (Otidea and several ECM Pezizaceae), and endophytism was also reported in species of the ECM genera Sphaerosporella (Hughes et al., 2020) and Tuber (Schneider-Maunoury et al., 2018). Although many ECM fungi are difficult or impossible to culture (Tedersoo et al., 2010), these endophytic or endolichenic isolates grow well in pure culture and thus enable future research in genomics, experimental manipulation and secondary metabolites. There is evidence from Sphaerosporella that endophytes of needles and colonization of ECM roots may rarely occur in the same individual host, but that the mode of infection for these two organs is different (Hughes et al., 2020). Additional studies are needed, perhaps employing culturing, resynthesis, inoculation and isotopic methods, to substantiate whether Pezizomycete endophytes with identical ITS sequences to those on their ECM host roots are actually the same genotype and are playing similar ecological roles. New approaches that allow visualization of the extent and morphology of fungal colonization, such as fluorescence in situ hybridization (Schneider-Maunoury et al., 2020), will be especially helpful in future work.

By contrast, other ECM Pezizomycetes (e.g. Otidea, some ECM Pezizaceae) were not detected as endophytes in their ECM hosts but were instead found in bryophytes and lichens, suggesting possible compartmentalization of different trophic modes on different hosts. A similar pattern was also found among the two lineages of plant pathogens where endophytism was detected. The endophytic state of Pithya cupressina is putatively a dormant pathogen because this fungus is considered the cause of twig die-back on Juniperus, but it was also found as an endophyte in healthy tissue of the same Juniperus species. By contrast, Rhizina undulata, a root pathogen of Pinaceae, was endophytic only in lycopods and ferns. Interestingly, no bryophyte parasites (such as Octospora or Lamprospora) were detected as endophytic or endolichenic in any sampled host, including mosses. These patterns suggest that host preferences, compartmentalization and the ability to colonize different hosts or host organs may be species- or lineage-specific.

Notably, endophytic and endolichenic species were particularly common among clades of fire-adapted and pyrophilous Pezizomycetes, including 24 OTUs from 19 genera (Table S6). Raudabaugh et al. (2020) found that pyrophilous taxa such as Anthracobia melaloma, Ascorhizoctonia praecox, Pyronema omphalodes and R. undulata are common as endophytes but appear to be rare to absent in soil. After wildfires, however, these fungi fruit prolifically on soil and burnt plant debris (e.g. Petersen, 1970; Reazin et al., 2016; Bruns et al., 2020). Likewise, in a study of endophytes of Bromus tectorum (cheatgrass), Baynes et al. (2012) identified several pyrophilous Pezizomycetes (e.g. Peziza ostracoderma, Pyronema domesticum, Morchella eximia and M. snyderi). Subsequent experiments showed that endophytic Morchella species increased B. tectorum growth and enhanced seed survival following fire, highlighting a previously unstudied benefit of this symbiosis. U’Ren et al. (2012) and Huang et al. (2016) reported the dominance of Pezizomycete endophytic and endolichenic species in Arizona forests where fire is common, indicating that this may be a widespread phenomenon. Our analysis revealed additional pyrophilous fungi that can be endophytic or endolichenic, including Geoscypha tenacella and Pyropyxis rubra. Available evidence suggests hidden roles of some pyrophilous Pezizomycetes as plant symbionts, setting the stage for studies of ecological effects of endophytism on host plants and endolichenism on host lichens.

These and other hypotheses will be testable in the future due to living fungal libraries such as the UA culture collection. Metagenomics and other culture-free tools have been, and will continue to be, critical for elucidating plant–fungi interactions, especially because these methods typically detect a far greater diversity of fungi than culture-based methods alone (U’Ren et al., 2019). However, studies such as ours highlight the importance of maintaining living endophytic and endolichenic fungus cultures and generating ITS and 28S DNA to identify them (see U’Ren et al., 2019 for benefits and drawbacks of molecular vs culture-based detection). Fungal cultures can be used for diverse purposes: to test nutritional requirements, characterize novel metabolites, sequence genomes and transcriptomes, and inoculate plants to study the effects on plant and fungal fitness (e.g. Wijeratne et al., 2012; Sarmiento et al., 2017; Torres-Cruz et al., 2017; Huang et al., 2018b; Harrington et al., 2019).

In this study, we identified endophytic and endolichenic species from an impressive 16 of 23 recognized Pezizomycete families, as well as a lineage that likely represents a new family (undetermined lineage in Fig. 2). Our results suggest that endophytism and endolichenism may indeed be the rule rather than the exception across families of Pezizomycetes. Our conservative phylogenetic approach to OTU delimitation detected a minimum of 160 OTUs of endophytic and endolichenic Pezizomycetes. This is equivalent to c. 8% of the estimated 2000 species in this class, but these are spread across c. 70% of the families. Given that only a small fraction of potential hosts and geographical areas have been sampled for endophytes or endolichenic fungi, the number of OTUs is probably a marked underestimate. Notably, relatively few studies have broadly sampled lichens wherein Pezizomycete species are dominant (but see U’Ren et al., 2012, 2019). It seems probable that more sampling will detect additional Pezizomycete species as cryptic residents in plants and lichens from biomes ranging from tropical forests to polar deserts, as illustrated by culture-based and culture-free studies (e.g. Higgins et al., 2007; U’Ren et al., 2019; Oita et al., 2021a).

The ecologies of many rare or understudied species of Pezizomycetes in genera such as Carbomyces, Eremiomyces, Glaziella, Hydnocystis, Kalaharituber, Pseudotricharina and Sowerbyella remain mysterious and currently unclear (Læssøe & Hansen, 2007; Tedersoo et al., 2010; Tedersoo & Smith, 2013). Although the trophic nature for most endophytic Pezizomycetes outside their hosts is putatively saprobic, endophytism and endolichenism also appear to be a normal part of the life history in many ECM and pathogenic species. Functional roles during their endophytic or endolichenic phase are unknown for any of these trophic groups and require further investigation. Our results suggest that future studies to elucidate the lifestyles of these poorly known Pezizomycetes should look first to the nearest plants and lichens to see what fungi might be living inside.