Seagrasses provide invaluable ecosystem services yet very little is known about their root mycobiont diversity and distribution. Here we focused on the dominant Mediterranean seagrass Posidonia oceanica and assessed its root mycobiome at 32 localities covering most of the ecoregions in the NW Mediterranean Sea using light and scanning electron microscopy and tag-encoded 454-pyrosequencing. Microscopy revealed that the recently discovered dark septate endophytic association specific for P. oceanica is present at all localities and pyrosequencing confirmed that the P. oceanica root mycobiome is dominated by a single undescribed pleosporalean fungus, hitherto unknown from other hosts and ecosystems. Its numerous slow-growing isolates were obtained from surface-sterilised root segments at one locality and after prolonged cultivation, several of them produced viable sterile mycelium. To infer their phylogenetic relationships we sequenced and analysed the large (LSU) and small (SSU) subunit nrDNA, the ITS nrDNA and the DNA-directed RNA polymerase II (RPB2). The fungus represents an independent marine biotrophic lineage in the Aigialaceae (Pleosporales) and is introduced here as Posidoniomyces atricolor gen. et sp. nov. Its closest relatives are typically plant-associated saprobes from marine, terrestrial and freshwater habitats in Southeast Asia and Central America. This study expands our knowledge and diversity of the Aigialaceae, adds a new symbiotic lifestyle to this family and provides a formal name for the dominant root mycobiont of the dominant Mediterranean seagrass.

dark septate endophytes, Dothideomycetes, marine fungi, root endophytes, seagrasses

Although the occurrence of marine saprobic and endophytic fungi on mangroves and salt marsh plants is well-documented (e.g. Jones 1963; Jones and Pang 2012; Kohlmeyer and Kohlmeyer 1971; Gessner and Kohlmeyer 1976; Kohlmeyer and Volkmann-Kohlmeyer 1991, 2001, 2002), the mycobiota of seagrasses is generally neglected and relatively little understood (e.g. Kohlmeyer 1963; Kohlmeyer and Kohlmeyer 1979; Cuomo et al. 1985; Alva et al. 2002; Gnavi et al. 2014). Seagrasses are perennial flowering plants represented by several genera inhabiting shore environments practically everywhere outside the Arctic and Antarctic, but mainly in temperate, subtropical and especially tropical littoral zones. All seagrass genera are accommodated in various families of a single order, the Alismatales (Monocotyledons). Unlike most terrestrial and many aquatic plants, seagrasses seem to be devoid of mycorrhizae (Nielsen et al. 1999) and a specific root-fungus association has been so far reported only for a single seagrass species (Vohník et al. 2015).

Posidonia (Posidoniaceae) is the evolutionary oldest seagrass genus with the earliest fossil record from the Cretaceous (den Hartog 1970). It has a uniquely discontinuous distribution with eight of its nine species occurring in the Southern Hemisphere along the coast of Australia (Green and Short 2003). In our study, we focused on root mycobionts of the only non-Australian species, i.e. the dominant and endemic Mediterranean seagrass Posidonia oceanica. In the Mediterranean Sea, P. oceanica forms extensive clonal meadows which can be hundreds to thousands of years old and spread over one to several (up to 15) kilometres (Arnaud-Haond et al. 2012). These vast meadows are the primary source of carbon for the coastal ecosystems and, additionally, they play an important role in defining the coastal line and supply biogenic detritus made of seagrass roots, rhizomes and leaf debris entangling other living organisms like molluscs, algae or foraminifera (De Falco et al. 2017). Unlike other seagrasses, P. oceanica typically forms extensive branched root systems (Figure 1a) which support formation of “matte” (Figure 1b, c), i.e. a peat-like seabed layer which is exceptionally resistant to microbiological decay and may be up to several metres thick (Hemminga and Duarte 2000; Serrano et al. 2012).

Figure 1. 

The dominant Mediterranean seagrass Posidonia oceanica. a Overall appearance, note dense branched root system of the seagrass (encircled) b Posidonia oceanica growing on an approx. 1.5 m thick layer of matte c typical habitat of the dominant Mediterranean seagrass, note the layer of shed seagrass leaves on the seabed.

The mycobiota of P. oceanica only recently gained appropriate attention; from the few available reports it seems to be predominated by fungi belonging to three classes and five orders of Ascomycota, i.e. Dothideomycetes (Pleosporales, Capnodiales), Leotiomycetes (Helotiales) and Sordariomycetes (Lulworthiales, Microascales and Papulaspora incertae sedis). These include obligate marine lignicolous fungi, ubiquitous surface-dwelling saprobes and endophytic fungi colonising roots, rhizomes and leaves, thus forming tighter (symbiotic) relationships with the host plant. Typically, they were growing on living or decaying plant parts (Kohlmeyer 1963; Cuomo et al. 1985) or were isolated as sterile mycelia and identified only by DNA sequence analysis (Panno et al. 2013; Gnavi et al. 2014; Vohník et al. 2016, 2017). They either belong to well-studied genera (e.g. Corollospora, Halotthia, Lulworthia and Papulaspora) or represent new marine lineages.

Our previous microscopic observations revealed that living terminal roots of P. oceanica, particularly their surface and the thick-walled hypodermis, are regularly colonised by an unknown fungus with dark septate hyphae (Vohník et al. 2015). The resulting association resembles colonisation by the so-called dark septate endophytes (DSE) which regularly occur in the roots of most terrestrial plants (e.g. Jumpponen and Trappe 1998; Vohník and Albrechtová 2011; Lukešová et al. 2015) but seemed to be absent in the marine environment. The association is characterised by the formation of sparse, dark pigmented hyphae, dense finger-like pseudoparenchymatous nets or loose hyphal sheaths on the root surface and melanised intracellular microsclerotia in the hypodermis. However, in contrast to typical terrestrial DSE, although the dark septate hyphae were also infrequently observed inside rhizodermal cells, they never colonised vascular tissues of the host roots. Interestingly, this association was absent in the roots of Cymodocea nodosa, a widely distributed seagrass in the Mediterranean Sea which sometimes accompanies P. oceanica (Vohník et al. 2015).

In our previous work focused on the diversity and distribution of P. oceanica root mycobionts, cultivations and 454-pyrosequencing of fungal DNA from surface-sterilised root segments from a few localities in the NW Mediterranean Sea revealed a relatively narrow fungal community lacking typical terrestrial and freshwater endophytes and mycorrhizal fungi (Vohník et al. 2016, 2017). This unusually limited fungal spectrum (cf. Kohout et al. 2012, 2013; Bruzone et al. 2017) was dominated by a single dark-pigmented mycobiont tentatively named “Pleosporales sp. MV-2012” (Vohník et al. 2016). Interestingly, this symbiotic fungus has not been documented in any of the other studies on P. oceanica mycobiota (see above) and to our knowledge it is not known from any other hosts and environments. Its extremely slow growth and characteristic colony morphology enable unequivocal identification already during the isolation stage but spore formation has never been observed (Vohník et al. 2016). Consequently, despite the striking DSE root colonisation pattern in vivo (Vohník et al. 2015), the absence of sexual characters and the lack of formation of conidia and conidiophores in axenic culture, either on agar media (standard or containing salt water) or on surface-sterilised root segments placed on nutrient media, pose a difficulty in estimating precise phylogenetic relationships of this dominant P. oceanica root mycobiont. Nevertheless, its preliminary position in the Aigialaceae (Pleosporales, Dothideomycetes), based on sequences of the partial nuclear large subunit (nucLSU) 28S rDNA gene, was discussed in Vohník et al. (2016).

The present study was motivated by the need to confirm the presence/dominance of the pleosporalean DSE fungus in the P. oceanica root mycobiota at a much larger scale than previously studied as well as the need for circumscription and precise phylogenetic placement of this mycobiont into the fungal system. Thus, we characterised P. oceanica root mycobionts using tag-encoded 454-pyrosequencing at 32 localities in the NW Mediterranean Sea (covering the distribution of P. oceanica from its westernmost localities to the boundary between the Western and Eastern Mediterranean basins). We also isolated and characterised P. oceanica root mycobionts at the locality where the specific DSE association has been observed for the first time (Vohník et al. 2015). Subsequently, characteristic strains of the Pleosporales sp. MV-2012 were selected for its circumscription based on morphological characters and an analysis of a molecular data set consisting of sequences of the following nuclear markers: nucLSU, nuclear small subunit (nucSSU) 18S rDNA gene and the second largest subunit of the RNA polymerase II (RPB2) gene. Additionally, an analysis of the unusually divergent ITS region of nuclear rDNA was performed to screen the possible geographical variability of the dominant P. oceanica root mycobiont.

The microscopic screening of Posidonia oceanica root fungal colonisation confirms that the recently described DSE association (Vohník et al. 2015) formed by the Pleosporales sp. MV-2012 (Vohník et al. 2016) and introduced as Posidoniomyces atricolor in this study, is present at all investigated localities. The tag-encoded 454-pyrosequencing of fungal DNA extracted from surface-sterilised P. oceanica root segments confirms the dominance of this fungus in the root mycobiota of the dominant seagrass in the NW Mediterranean Sea. Our analysis of combined DNA sequences of nuclear ribosomal and protein-coding loci confirms the placement of P. atricolor in the Aigialaceae (Pleosporales, Dothideomycetes) and suggests an independent marine biotrophic lineage.

The root-symbiotic Posidoniomyces is related to mostly saprobic lignicolous marine fungi from estuarine environments colonising wood and roots of mangroves growing in tropical regions of both Eastern and Western Hemispheres, a situation resembling, at least to some extent, the relationship of the ubiquitous terrestrial root-symbiotic Rhizoscyphus ericae aggregate to saprobic fungi from the genus Hyaloscypha (Fehrer et al. 2019). Because mycorrhizal fungi from the R. ericae aggregate have significant saprobic abilities (Martino et al. 2018), they can decompose recalcitrant peat and exchange mineral nutrients (especially nitrogen) for the host photosynthetically bound carbon. Since P. oceanica often grows on thick layers of recalcitrant peat-like matte (Figure 1b, c), which typically stores large amounts of organically-bound nutrients (Fourqurean et al. 2012) directly unavailable to plants (Read 1991), it is tempting to speculate about the possible role of P. atricolor in mineral nutrition of the dominant Mediterranean seagrass (also see Borovec and Vohník 2018; Kolátková and Vohník 2019). On the other hand, genomes of DSE fungi typically combine saprobic and pathogenic traits (Schlegel et al. 2016; Knapp et al. 2018) and effects of root endophytes on host plant fitness vary along the parasitism-mutualism continuum (Newsham 2011; Mayerhofer et al. 2013). Thus, although the specific association with P. atricolor is omnipresent in P. oceanica at all so far investigated localities, to date there is no solid proof that it is of any benefit to the seagrass.

The Aigialaceae (Suetrong et al. 2009) was erected for marine ascomycetes characterised by fisstunicate asci with a non-amyloid apex and a ring-like apical apparatus containing septate or muriform ascospores with a gelatinous sheath or cap, trabeculate hamathecium and non-stromatic, carbonatious to coriaceous, non-papillate ascomata. Additionally, Fissuroma and Neoastrosphaeriella occurring on bamboo, palms and flowering plants in terrestrial and freshwater environments were added to the family by Liu et al. (2011). The asexual morphs of marine species are generally unknown. The asexual morph of Fissuroma was reported as coelomycetous, pleurophomopsis-like (Tanaka and Harada 2005; Liu et al. 2011). Axenic cultures of P. atricolor remained sterile and two colonial morphotypes, named compact and mycelial, were consistently formed originating from the primary source. Although the presence of both morphotypes on PCA, a low sugar content medium, would suggest that the mode of nutrition does not influence the colony appearance, the absence of one or the other morphotype on MMN and PDA may indicate that the nutrition mode could play a role to some extent. When a compact colony was regularly washed with sterile tap water, a number of daughter colonies were formed all over the agar plate (Figure 6e), suggesting that liquid culture might be an efficient way for producing larger quantities of P. atricolor mycelium. These colonies usually assumed the form of a miniature rhizoidal-like colony (Figure 6f) formed mainly by submerged mycelium or they formed the well-distinguishable compact colonial morphotype directly. It is probable that the capitate swellings protruding above the surface of the compact colonies (Figure 6g, h) together with hyphal fragments act as propagules in the absence of conidia and ensure the dispersal of the fungus in a simulated environment. These terminal, intercalary and sometimes lateral mostly globose swellings resemble intercalary conidia of species of Knufia, e.g. K. perforans, formed on elongated and monilioid hyphae (Tsuneda et al. 2011). However, P. atricolor compact colonies have never been observed in vivo directly on P. oceanica roots and it is thus unknown whether the capitate swellings form and act as propagules also under natural conditions.

The Dothideomycetes include several marine genera that usually do not form an asexual state and are distributed in several orders, i.e. Capnodiales, Dothideales, Hysteriales, Jahnulales, Patellariales and Pleosporales, or incertae sedis lineages (Suetrong et al. 2009). They include mainly taxa thriving in intertidal zone on a variety of substrates of mangroves in tropics or less frequently on salt marsh plants in temperate regions. Other marine Dothideomycetes can occur as parasites or possible endophytes of seagrasses or marine macroalgae and are completely submerged. The omnipresence and dominance of P. atricolor in the roots of P. oceanica suggests a close symbiotic relationship with the dominant Mediterranean seagrass, a trait so far unparalleled in other Dothideomycetes. At the same time, to our knowledge, the characteristic DSE colonisation pattern of P. atricolor has never been observed in any other seagrass species, suggesting its specificity for P. oceanica (also see Discussion in Vohník et al. 2015).

The analysis of all available P. atricolor ITS sequences (Vohník et al. 2016, 2017; this study) revealed several aspects that may connect with their geographic distribution and possibly also the symbiotic lifestyle. The ITS1 region of P. atricolor contains ca. 294 nt long insertion near the 5’-end when compared to ITS1 of other members of the Aigialaceae. Only four species of the whole family have their ITS sequences available; the ITS1 varies between 151–168 nt in Fissuroma (F. maculans, F. neoaggregatum) and between 186–201 nt in Neoastrosphaeriella (N. aquatica, N. krabiensis), compared to 445 nt in P. atricolor. When the ITS1, 5.8S and ITS2 of P. atricolor were checked for closest hits by the BLAST search in GenBank, the closest relatives for the 5.8S region were members of the Aigialaceae and other taxa of the Pleosporales; however, no close hits were revealed for ITS1 and ITS2. Since the ITS region was amplified and sequenced as a part of the whole nuc18S region with several forward and reverse primers, it is unlikely that this divergence was caused by PCR or sequencing errors. ITS is a rapidly evolving region where numerous insertions and deletions occur. Considering the probably obligate symbiotic lifestyle of P. atricolor in the host roots, the long insertion in ITS1 and high divergence in ITS2 sequences may be a result of co-evolution of both partners, higher gene flow rate and possibly horizontal gene transfer resulting in genetic mismatches in the fungal partner (Saikkonen et al. 2004, 2010; also see Kolařík and Vohník 2018). However, outside the Aigialaceae, the ITS1 region can be much longer, for example in Astrosphaeriella bambusae, the outgroup, it is 445 nt long. On the other hand, the length of the ITS2 region is comparable between P. atricolor (188–192 nt) and other members of the Aigialaceae (156–163 nt).

Although the ITS sequences of all P. atricolor isolates are nearly identical (99.87–98.99% identity between the type strain BRK-21 and other isolates), they differ in up to six indels near the 5’-end of the ITS2. These site mutations can be used to some extent to characterise different populations of P. atricolor (Figure 4). Only strains which could be compared morphologically, i.e. those successfully derived from P. oceanica surface-sterilised root segments into axenic culture (Vohník et al. 2016; this study), were analysed. Their colony characters and colonisation pattern in the roots of the host were identical. At the ITS2 sequence level, we could distinguish populations from the north-west regions of the Mediterranean (France, Spain) and those from the north-central part of the Adriatic coast (Croatia). Moreover, the Croatian population from Borak (locality HR-1; Table 1) seems to be a source of several mutations. Further screening of P. oceanica root mycobiota outside the NW Mediterranean is apparently needed to fully elucidate the usefulness of ITS sequences for distinguishing geographically different populations of P. atricolor.

Although it is a significant producer of biomass and an important source of decomposing organic matter in the sea and adjacent habitats, the mycobiota of P. oceanica has been studied only by a few authors (e.g. Kohlmeyer 1963; Cuomo et al. 1985; Panno et al. 2013; Gnavi et al. 2014; Vohník et al. 2016, 2017; this study), with differing results. Most significantly, no study prior to Vohník et al. (2016) reported P. atricolor in the mycobiota of the dominant Mediterranean seagrass. This is probably due to the manner of material sampling and isolation procedure, i.e. direct isolation from decaying plant matter vs. serially washed or surface-sterilised parts of living plants, the former often leading to detection of fast-growing surface-dwelling saprobes in contrast to isolation of true endophytes (Sieber 2002; also see Discussion in Vohník et al. 2016, 2017). Indeed, apart from P. atricolor and the obligate marine Sordariomycetes (Corollospora marina and C. intermedia in Microascales, Lulwoana sp. and Lulworthia sp. in Lulworthiales and Papulaspora halima incertae sedis) and Dothideomycetes (Halotthia posidoniae, Pontoporeia biturbinata and several other genera in Pleosporales), majority of the fungi reported from P. oceanica are asexually reproducing ubiquitous fungi (Panno et al. 2013).

The distribution pattern of P. oceanica mycobiota in leaves, rhizomes, roots and matte is affected by various environmental parameters, presence of growth-inhibiting substances in leaves or antagonistic organisms and may be also influenced by the season (Cuomo et al. 1985; Panno et al. 2013; Gnavi et al. 2014). However, no detailed data are yet available for the dominant root mycobiont P. atricolor, except that it seems to be restricted to P. oceanica roots. Mycorrhizal fungi form often vigorous extraradical mycelium penetrating the substrate far beyond the rhizosphere, thus forming the mycorrhizosphere (i.e. a volume of soil under a combined influence of the root and the emerging fungal hyphae) (Linderman 1988). The mycorrhizosphere significantly enlarges the volume of the substrate available for mycorrhizal nutrient uptake and in a way defines individual mycorrhizal types. It would therefore be interesting to screen the volume and enzymatic activity of the P. atricolor extraradical mycelium (if existing) to decide more precisely about the mode of the interaction between the dominant Mediterranean seagrass and its dominant root mycobiont.

This study confirms at an unprecedented scale that the diversity of the root mycobiota of the dominant Mediterranean seagrass is relatively narrow and dominated by a single pleosporalean fungus so far not known from any other hosts or environments. This fungus is introduced here as a new genus and species Posidoniomyces atricolor and resides as an independent marine biotrophic lineage in the Aigialaceae. The characteristic colonisation pattern of P. atricolor in P. oceanica roots has not been reported in any other seagrass and resembles colonisation by DSE fungi which are ubiquitous in terrestrial roots. Further research is needed on the distribution and genetic variability (especially ITS sequences) of P. atricolor in the rest of the Mediterranean Sea (i.e. Eastern Mediterranean Basin, the coast of North Africa). Additionally, given the uniquely discontinuous distribution area of the genus Posidonia (Green and Short 2003), targeted research on the root mycobiota of its Australian species would be of a special evolutionary significance.

This study constitutes a part of long-term research projects of the Czech Academy of Sciences, Institute of Botany (RVO 67985939) and Charles University, Faculty of Science (MŠMT LO1417). Judith Fehrer stimulated the cooperation between MV and MR from the outset, Jiří Machač helped with the photo documentation, Mirka Opičková helped with the DNA isolations (all Inst. of Botany, Průhonice), David Vondrášek (Charles Uni., Prague, CZ) helped with the sampling in France, Italy and Spain, Miroslav Kolařík helped with preliminary phylogenetic analyses and Jana Voříšková performed the pyrosequencing (both Inst. of Microbiology, Prague, CZ) and Ivan Župan (Zadar Uni., Zadar, HR) helped with obtaining a sampling permit issued by the Croatian Ministry of Environmental and Nature Protection (UP/I-612-07/13-48/48//517-07-1-1-1-13-2) as well as with the sampling in Croatia; all these contributions are greatly appreciated. The authors thank Conrad L. Schoch and Kazuaki Tanaka (reviewers) and Huzefa Raja (editor) for the careful reading and suggestions which helped to improve this paper.