Double lives: transfer of fungal endophytes from leaves to woody substrates

Culture methods

Twelve evergreen leaves of a randomly selected tree (Lauraceae; N. lineatifolia (Ruiz & Pav.) Mez) were collected in an Ecuadorian cloud forest. The tree was within Reserva Los Cedros, which is on the western slope of the Andes in northwestern Ecuador (00°18031.000 N, 78°46044.600 W), at 1,200 m above sea level. Eight 2-cm² sections were cut from each leaf and surface-sterilized by successive immersion in 70% ethanol for 1 min, 5% sodium hypochlorite (equivalent to full strength bleach) for two min, then rinsed in sterile water. The leaf sections were placed onto twice-autoclaved white birch (Betula papyrifera Marshall) tongue depressors (Puritan, Guilford, ME, USA) as a standardized angiosperm woody substrate. The sections from each leaf were split between two tongue depressors (four sections each) resulting in a total of 24 tongue depressors. These were incubated in three 95% EtOH-sterilized Ziploc storage boxes (eight in each box) at the field station in ambient temperature for 6 weeks. Each box contained an open container of twice-autoclaved water to maintain humidity. The incubation period provided opportunity for the endophytic fungi in the leaves to colonize the wood. After incubation, the sticks were placed into airtight, sterile bags and brought to the University of Oregon.

Fungal cultures were isolated from the inoculated wood by breaking 15 small fragments (~5 mm² each) of wood from each tongue depressor using flame-sterilized tools and dispersing them evenly among five 100 mm water agar plates. The ends of growing hyphae were excised from the agar using a dissecting microscope and a scalpel and transferred onto nutrient plates (MEA, 2% maltose) over a 2-month period. Cultures were also made from several fruiting structures that grew directly from the birch substrate fragments. After a growth period of seven or more days the isolates were grouped into morphotypes (Lacap, Hyde & Liew, 2003) at the genus level based on macro-and microscopic features.

All field work was done with the approval of the Ecuadorian Ministry of the Environment (Ministerio del Ambiente de Ecuador, Permit No. 03-2011-IC-FLO-DPAI/MA).

Identification of viaphytes

A single representative of each morphotype was subcultured in liquid media (2% malt extract) for DNA extraction using the Qiagen DNeasy Plant kit following the manufacturer’s instructions, and the ITS region (the standard “barcode” locus for fungi; Schoch et al., 2012) was amplified using the fungal-specific primer set ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (White et al., 1990), or in cases where those primers were ineffective, isolates were amplified with ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) and LR3 (5′-CCGTGTTTCAAGACGGG-3′) primers. DNA amplification was conducted with 12.5-µL reaction volumes (2.5 µL of template, 6.25 µL of Sigma Aldrich JumpstartTM Taq ReadymixTM, 2.75 µL sterile water, 0.5 µL 25 mM MgCl₂ and 0.25 µL of each primer at 10 µM). PCR amplification was performed with an MJ Research PTC-200 DNA Engine thermal cycler under the following parameters: initial denaturation at 95 °C for 2 min, five cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min; followed by 25 cycles of denaturation of 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min; a final extension at 72 °C for 10 min, and a final step of indefinite duration at 4 °C. PCR products were visualized on a 1% agarose gel. Samples were then frozen until shipping for sequencing at Functional Biosciences, Inc (Madison, WI, USA) on ABI 3730xl instruments using Big Dye V3.1. ITS amplicons were sequenced bi-directionally, then assembled into contigs, and manually edited in Geneious (v6.0.3; Biomatters Limited, Auckland, New Zealand) to remove priming sites and resolve mismatches. The consensus sequences were then compared to published sequences in the UNITE database (v8.0; Kõljalg et al., 2013) using the assign_taxonomy.py function from the Quantitative Insights into Microbial Ecology pipeline (Caporaso et al., 2010). Taxa that returned species assignments as “unidentified” were further examined using BLAST against the NCBI nr database. Taxonomic identities were assigned at genus level and lower if the hit with the lowest E-Value had greater than 97% sequence identity across the entire ITS region. Sequences whose hits did not match these criteria were categorized as “unidentified”. Putative Xylaria species were compared to our database of ITS sequences generated from authenticated material within that genus at the same site (Thomas et al., 2016) and assigned to a taxon if sequences had greater than 98% sequence identity. Taxa with greater than 99% sequence identity were assumed to be the same taxon (i.e., OTU). All taxa with identical assignments by UNITE met this criterion.

Functional guilds were assigned to each genus by using the FUNGuild online tool (Nguyen et al., 2016), which assigns functional information to taxa in DNA datasets. If functional guilds were not available in FUNGuild, they were determined based on the literature wherever possible (Table S3).

Statistical methods

Species richness per leaf was estimated using Chao2 and Jacknife1 estimators (Burnham & Overton, 1978; Chao, 1984; Colwell & Coddington, 1994). Diversity was estimated between all leaves, within leaves, and within boxes using Shannon’s index (log base e was used; Shannon, 1948) and Simpson’s index (1-D; Simpson, 1949), and community structure was visualized using non-metric multidimensional scaling and differences assessed with permutational multivariate analysis of variance (PerMANOVA). Data were analyzed using R Statistical Software, v. 3.1.0 (R Core Team, 2014), including the vegan package (Oksanen et al., 2013).

All scripts, data tables, and raw data (morphotype counts and sequence chromatograms) is available via an open FigShare repository (Nelson et al., 2019). Edited sequences have been uploaded to GenBank (accession numbers provided in Table S1).

Diversity and abundance of viaphytes

Numerous endophytes from surface-sterilized leaves of N. lineatifolia successfully colonized the wood substrate: 477 fungal cultures were isolated after making the initial transfer from leaves to wood. Isolates were grouped into 64 morphotypes, 62 of which were successfully identified to genus (59 by DNA, three by morphology; Table S1). DNA identification resulted in the consolidation of the morphotypes into 24 unique taxa at the genus level (Table S2). The number of isolates for each taxon varied widely, such that 57% of the isolates were represented by just two genera (i.e., Trichoderma and Penicillium), and seven of the taxa were isolated only a single time (Fig. 1). In addition to hyphal growth from the wood substrates, anamorphic fruiting structures were observed growing out of five stick fragments originating from two leaves (Fig. S1). These isolates were identified as Xylaria flabelliformis (Schwein.) Berk. & M.A. Curtis using DNA extracted from stromatic tissues. Including X. flabelliformis, we observed a total of 24 viaphytic taxa, which were identified to the genus level (Fig. 1). Additionally, we observed that the majority of the woody substrate fragments displayed a dramatic decrease in substrate volume that may be explained by high levels of cell wall degrading enzymes typical of white-rot fungi. However, we did not attempt to determine which taxa were responsible for this dramatic reduction in volume.

The species accumulation curve did not reach a saturation point, suggesting that the full richness of viaphytes from these leaves was not isolated (Fig. 2). Estimates of actual species richness ranged from 36.5 (first order jackknife, SE = 4.1) to 42.3 (chao2, SE = 13.8).

Viaphyte communities within incubation boxes were more similar to each other than to communities from other boxes (PerMANOVA: F_1,23 = 6.34, p = 0.001), whereas communities from sticks that were inoculated by the same leaves were not more similar to each other than to sticks inoculated from different leaves (PerMANOVA: F_1,23 = 1.04, p = 0.404; Fig. 3). Isolates representing the four most common taxa were concentrated in common boxes, with 100% of Neopestalotiopsis foedans in Box 1 (44 total isolates across all boxes), 96% of Paecilomyces formosus in Box 1 (75 total isolates), 87% of Trichoderma spp. in Box 2 (89 total isolates), and 61% of Penicillium spp. in Box 3 (179 total isolates).

Taxonomic distribution

The higher order taxonomic ranks in our samples included two phyla, five classes, 12 orders and 19 families (Table S2). Although Ascomycota was the dominant phylum, both in terms of number of taxa and total number of isolates (73% and 94%, respectively), isolates of Basidiomycota also were obtained in culture. Among Ascomycota fungi, Sordariomycetes were the most common class in terms of number of taxa (38.4% of total taxa), whereas fungi in the Eurotiomycetes, driven by the frequency of Penicillium spp., represented more than half of the isolates (55.7%). At the ordinal level, the most common orders among all taxa were Xylariales (Sordariomycetes, Ascomycota) and Polyporales (Basidiomycta) (each representing 19.2% of all taxa). Isolates of Eurotiales (Eurotiomycetes, Ascomycota), again driven by Penicillium spp., represented the most isolates (55.1% of all isolates).

Functional guilds

The FUNGuild database contained putative functional guilds for all but two of the genera we isolated as viaphytes. The first unassigned genus, Alloconiothyrium, is newly described and presently represented by a single species, A. aptrootii, which was isolated from a soil sample in Papua New Guinea (Verkley et al., 2014). We therefore did not assign it to a functional guild since so little information is available. The second, Neopestalotiopsis, we classified as a “plant pathogen/saprotroph” based on substrates listed in species descriptions (Maharachchikumbura et al., 2014). The viaphyte genera of our study fit into three distinct functional guilds: saprotroph, plant pathogen and plant pathogen/saprotroph. Saprotroph was the dominant functional guild in terms of number of genera (70.8%; 17 out of 24) and number of isolates (82.3%, 389 out of 467). Four of the genera were classified as plant pathogens (16.7%) and three genera were classified as plant pathogen/saprotrophs (12.5%). Of the isolates, 64 were classified as plant pathogen/saprotrophs (13.7%) and fourteen were classified as plant pathogens (3.0%).

Viaphyte prevalence

Here, we demonstrate for the first time that a diverse array of tropical leaf endophytes can colonize woody substrates through direct contact with leaves, thus representing an ability to alternate between endophytic and saprotrophic life stages. Our results show that viaphytes are commonplace and multiple fungal species have a potential for viaphytic dispersal from within each leaf, even though it is likely that we underestimate richness due to the biases of culture-based studies (Schmit & Lodge, 2005) and the incompleteness of our sequencing efforts. The high frequency of viaphytic colonization suggests that the underlying mechanisms are likely mechanistically straightforward (i.e., as simple as hyphae extending from one substrate into the other), although the enzymatic potential to successfully colonize woody substrates may be taxon-dependent.

While the present viaphyte survey examined only a single tree of N. lineatifolia, it seems unlikely that this host is unique in allowing the transfer of endophytes to woody substrates, or that the viaphytes observed within its tissues are only able to transfer from this particular host. In other words, if the host tree and its endophytic symbionts are taken to represent what is typical for a broad-leaved tropical tree, it follows that viaphytes are likely commonplace symbionts in the leaves of tropical forests. Other studies that have demonstrated the high abundance of endophytes in tropical forests corroborate this potential (Arnold & Lutzoni, 2007; Rodriguez et al., 2009; Thomas et al., 2016; Del Olmo-Ruiz & Arnold, 2017; Roy & Banerjee, 2018).

Yet even if fungi with viaphytic abilities are common, the extent to which viaphytic colonization events occur in natural systems is unknown. While we placed leaves containing endophytes on sterile wood substrates, viaphytes in nature would face competition from other sources of colonization, such as spores or saprotrophs already present in the wood (Thomas et al., 2016). Future experiments should empirically test the ability of viaphytic fungi to successfully colonize such diverse woody substrates in the face of competition. It is likely that viaphytism and direct spore colonization each have their own set of advantages. For instance, it is possible that the carbon and water supplies inherent in leaf tissues give an advantage to viaphytic dispersal as compared to spores, especially if conditions are dry or otherwise unsuitable for spore germination. In addition, leaves could trap moisture between the leaf and substrate, and may act as barriers that exclude competing spores from being deposited on the woody substrate surfaces (Thomas et al., 2016). Certainly, direct spore dispersal has its own advantages in the form of reduced complexity (i.e., no intermediate colonization stage is required), increased potential travel distance via air currents (McCartney & West, 2007; Calhim et al., 2018), and much greater abundances compared to leaf-born colonies. These ideas were previously explored by (Thomas, Vandegrift & Roy, 2020) using a simple agent-based model. As predicted by Thomas et al. (2016), in these simulations viaphytism is advantageous under adverse conditions given retention of endophyte infections and at least some trees on the landscape.

The viaphyte community of N. lineatifolia was characterized by a few taxa with high abundances and a large number of taxa with low abundances (Fig. 2). While this pattern is typical for culturable studies of leaf endophytes (Arnold et al., 2000, 2007; Vega et al., 2010; Gazis & Chaverri, 2010; Ikeda et al., 2014; Del Olmo-Ruiz & Arnold, 2017), some patterns in the data suggest that they are partly due to methodological biases. For instance, Penicillium spp. and Trichoderma spp. were both observed to be fast growing in culture in this study, and culture-based studies are known to be biased for faster-growing taxa (Kirk et al., 2004). Also, given that each of the four most dominant taxa had a disproportionately high number of isolates concentrated in a single box, these dominant taxa likely colonized the sticks within their respective boxes via sporulation during the inoculation period (Fig. 3). All four of these dominant taxa readily produced a high quantity of conidia in culture. Therefore, the number of isolates for these abundant taxa should be interpreted with caution as they likely do not reflect the actual abundance in host leaves, but rather comparatively fast growth and within-box contamination. It is also notable that our experiment did not have a true negative control, without an inoculation source, to account for true contaminants (i.e., taxa that may have originated outside of the leaves). While it is possible that some taxa detected may have been contaminants, there are several factors which suggest relatively low rates of outside contamination: (1) the thorough sterilization procedures we employed; (2) the high endophyte load in the tropics (Arnold et al., 2000; Arnold & Lutzoni, 2007); (3) the near ubiquity of detected taxa being found in tropical endophyte datasets; and (4) the restriction of common taxa to single boxes.

Ecological strategies

It is well documented that many endophytes have a much broader host range in the endophytic state than as saprotrophs—for example, Xylariaceae, the majority of which do not typically reproduce in the litter (Davis et al., 2003; Peršoh et al., 2010; U’Ren et al., 2016). It is, in fact, apparently common for such endophytes to be present in the leaves of hosts upon whose wood they never fruit (Carroll & Carroll, 1978; Peršoh et al., 2010; Unterseher, Peršoh & Schnittler, 2013). This is evidence for a FA ecology, since latent saprotrophism is excluded as a strategy for species which are incapable of fruiting out of leaves (Thomas et al., 2016). It is interesting that many fungi that are not typically observed fruiting on litter, such as members of the Xylariaceae, are well known as highly competitive litter decay organisms (Koide, Osono & Takeda, 2005; Osono, 2007; Osono et al., 2011). It is logical that increased substrate utilization in the litter, and therefore increased resource accumulation, translates to increased ability to compete for substrates external to the litter (Boddy, 2000).

Latent saprotrophism is a well-documented strategy of some leaf endophytes (Osono, 2006; Parfitt et al., 2010; Voříšková & Baldrian, 2013). An excellent example of this ecological strategy is the fungus Rhabdocline parkeri (Sherwood-Pike, Stone & Carroll, 1986), which spends most of its lifecycle as an endophyte in the needles of Pseudotsuga menziesii, waiting for the needles to die (typically 4–5 years). After needle senescence, the fungus rapidly invades the surrounding needle tissues (often before they are even shed), and then produces its conidial state, followed by a small perithecial teleomorph early in the winter, soon after the leaves are shed (Stone, 1987). The host specificity of R. parkerii, and other fungi like it, is explained by the role of priority effects (Chase, 2003) in the latent saprotrophic habit: while priority effects may work to benefit viaphytic fungi somewhat, they serve as a strong evolutionary filter for fungi utilizing a latent saprotrophic strategy. Future studies examining viaphytic ecological strategies should focus on exploring the boundaries between viaphytic and latent saprotrophic ecologies.

Taxonomic distribution

The viaphytes in this study belong to a wide taxonomic breadth, consisting of both Basidiomycota and Ascomycota. This implies that the benefits described by the FA hypothesis are available to members of the Basidiomycota as well, though the original idea concerned only the Ascomycota (Carroll, 1999). The taxonomic distribution of viaphytes from this study resemble those of general tropical leaf-endophytes described in other work (Arnold & Lutzoni, 2007; Thomas et al., 2016; Roy & Banerjee, 2018). In particular, Arnold et al. (2007) reported a similar pattern and proportion of Eurotiomycetes, Dothideomycetes and Sordariomycetes, also noting the dominance of Ascomycota.

The wide taxonomic distribution of viaphytes suggests that viaphytic dispersal may be a deeply ancestral trait. This would parallel endophytes in general, which appear to have associated with plants since at least 400 mya (Krings et al., 2007). Future taxonomic and paleontological work may help inform when viaphytism emerged as a dispersal strategy within the Fungi.

Functional guilds

Most of the viaphytic taxa in our study (17 of 24 taxa) were classified by FUNGuild as having saprotrophic abilities (Table S3). Many of these saprotrophic taxa are known wood-decay fungi, including Xylaria spp. and Phanerochaete spp. (Nguyen et al., 2016). In addition, our host leaves were harboring at least some species capable of physiological white-rot fungi, as evidenced by bleaching of the wood and a substantial decrease in size in several of our substrate fragments. Even some ascomyceteous molds are known to be degraders of lignin, including some Penicillium spp., Trichoderma spp., and Fusarium oxysporum, all of which were present among our isolates (Rodriguez et al., 1996; Ryazanova, Chuprova & Luneva, 2015). While the prevailing explanation for the occurrence of saprotrophic fungi as endophytes is that they are latent saprotrophs waiting to consume leaves upon senescence (Peršoh, 2013), many taxa we observed here, and others commonly isolated as endophytes, are not known to reproduce on dead leaves. Alternately, such endophytic saprotrophs may represent an evolutionary “dead-end” if they are unable to escape that state (Bayman et al., 1998), but our data suggests that it may be the norm for such fungi to transfer out of an endophytic state. Additionally, the presence of several taxa classified as primarily pathotrophs suggests that the facultative ability to access saprotrophic lifestyles may serve as a functional bridge for certain biotrophic species. One might expect that if biotrophs are cultivated on any given substrate, the resulting community would be dominated by fungi that were typically biotrophic, but with facultative saprotrophic abilities. This, however, is not what we find here, indicating that it is likely that a large proportion of endophytes isolated here are not transitioning to saprotrophy in a facultative manner, but as a transition back to their primary nutritional mode.

We observed several instances of fungi apparently thriving after colonizing wood. For example, despite the fact that only very few, generally host-specific, Xylaria are capable of fruiting from leaves (Rogers, 2000), Xylaria flabelliformis was observed fruiting directly from the woody substrates after transfer from an endophytic state. Interestingly, this taxon was found to be a common endophyte of forests in Taiwan (Vandegrift et al., 2019). Previously, we found five Xylaria species both as endophytes and as stromata on woody substrates at Los Cedros (Thomas et al., 2016). Emigration from leaves to wood is likely necessary for such endophytic individuals to regain reproductive potential.