Abstract

Introduction

Bare rock surfaces provide little comfort to life. They are poor sources of nutrients and are constantly exposed to a variety of extremes in abiotic conditions. Variations in surface temperatures and water availability can occur at very short time spans and be the source of diverse stresses (Zakharova et al. 2013; Sterflinger et al. 2012), and with enormous amplitudes. In addition, direct exposure to full sunlight includes a threatening level of energy-rich ultraviolet wavelengths. Not many organisms can cope with such surfaces at this “edge of life”, thus these surfaces are colonized by specialists with particular adaptations (Selbmann et al. 2005, 2013; Onofri et al. 2007; Marzban et al. 2013). In fact, some fungal lineages, which are known as “black fungi” or “microcolonial fungi” are among the most stress-resistant eukaryotic organisms on Earth and can occur at considerable diversity on rocks (Ruibal et al. 2005, 2009). The adaptations of these rock-inhabiting fungi (RIF) include pleomorphic growth, efficient osmolyte management, melanin production, biofilm formation, and survival in cryptobiotic stage (Gostincar et al. 2010, 2011).

Black fungi do not form a monophyletic lineage but are members of Dothideomycetes and Chaetothyriomycetidae (Gueidan et al. 2008; Ruibal et al. 2009) which evolved during periods of dry climate in the late Devonian and middle Triassic, respectively (Gueidan et al. 2011). At approximately the same time scale, the symbiotic association whereby a fungus shelters microscopic algae or cyanobacteria in exchange for fixed carbon and nitrogen helped to ameliorate nutrient deficiencies on rocks. The lichen symbiosis was this key innovation in the evolution of fungi and lichenized mycobionts have since evolved and diversified (Lutzoni et al. 2001; Hawksworth 2015). Particularly, in alpine altitudes where conditions prevent the development of higher plants, lichen thalli express their phenotypic and phylogenetic diversity and shape the landscapes with colorful mosaics on rock surfaces.

In such variably stressed situations, black fungi and lichens can occur side by side on rock, and black fungi also colonize asymptomatic lichens, especially in arid situations (Harutyunyan et al. 2008). Harutyunyan et al. (2008) showed that black fungi may opportunistically infect lichens, but do not cause damage to their host thalli. Some of the fungi resemble hyphomycetous lichenicolous fungi. However, most lichenicolous fungi have a host specific occurrence and are recognized by their phenotypic symptoms and their sexual or asexual spore-producing structures (Hawksworth 1979; Lawrey and Diederich 2003). It is not known whether black fungi, cryptically colonizing lichen thalli, are directly in contact with the photobiont to obtain nutrients. Some studies, however, suggest that black fungi indeed have some affinity to microscopic algae (Brunauer et al. 2007; Gorbushina et al. 2005). Arnold et al. (2009) also used micro-dissection followed by surface sterilization to show that more culturable fungi were associated with the algal layer compared to the medulla and cortex.

In this study, we conducted a comprehensive sampling of saxicolous lichen species (as reported in Fleischhacker et al. 2015), including samples infected by symptoms-developing lichenicolous fungi from different sites of an alpine range, above the tree-line. We prepared culture isolates of the fungi and produced sequence data for phylogenetic analyses. With these we aimed at answering the following questions: i) are there patterns of co-occurrence among cryptic, black extremotolerant fungi, symptomatic lichenicolous fungi and lichen mycobionts?; ii) are lichen-associated fungal communities structured by mycobiont host? ; iii) what is the phylogenetic placement of the isolated strains?

Material and methods

Results

Phylogenetic and morphological analysis of Chaetothyriomycetidae (Fig. ​(Fig.1,1, Fig. ​Fig.2,2, Table ​Table11 and Table S1)

Open in a separate window

Open in a separate window

Fig. 1

Multilocus phylogenetic inference of Eurotiomycetes. The ML and the Bayesian phylogenetic hypotheses were inferred from the combined dataset of nucLSU, nucSSU and mtSSU loci and corresponded in their topologies; the ML analysis is shown. Bayesian posterior probabilities (PP ≥ 95 %) and ML bootstrap support values (≥ 70 %) are reported above branches (PP/bootstrap value). Newly identified clades of isolated fungi obtained from this study are highlighted in bold and are labelled as clade I to VII. Symbols indicate the different lichen host-lichenicolous fungal associations which represent the original thallus from where the fungal strains were isolated. A symbol reported multiple times for a clade indicates the number of different original thalli sharing the same lichen host-lichenicolous fungal association. Fungal life-styles are reported in parenthesis. Samples labelled with an asterisk (*) are those photographed in Fig. ​Fig.22

Open in a separate window

Open in a separate window

Fig. 2

Habitus of one year old, representative, cultured fungal strains belonging to Eurotiomycetes and included in the phylogenetic analysis of Fig. ​Fig.1.1. Anatomical structures were photographed from squashed sections mounted in water. Samples are reported with their number and the clade to which they belong as in Fig. ​Fig.1.1. A1-A4) A922 (clade I) - A1, A2 habitus of the mycelium; A3, A4 fine, hyaline hyphae. B1-B4) A1022 (clade I) - B1, B2 habitus of the mycelium; B3, B4 fine, hyaline hyphae, gathering in entangled, plectenchymatous strands. C1-C7) A993 (clade II) - C1, C2 habitus of the mycelium; C3-C7 hyaline hyphae with branching and globose cells intercalating with cylindrical cells. D1-D5) A528 (clade III) - D1, D2 habitus of the mycelium; D3, D4 brown cell structures containing conidia-like cells (arrow in D4); D5, hyaline hyphae. E1-E5) A1026 (Epibryaceae) - E1, E2 habitus of the mycelium; E3-E5 dense aggregate of roundish, melanised cells containing inclusions, filamentous hyphae rarely present (E5). F1-F5) A1016 (Sclerococcum) – F1, F2 habitus of the mycelium; F3-F5 hyaline hyphae with branching and cylindrical, more or less elongated cells. G1-G7) A1053 (single branch, basal to clade VI) – G1, G2 habit of the melanized mycelium; G3-G7 hyphae composed by melanised, single or 1-septate cells, with numerous apical and lateral buds. H1-H5) A514 (basal to clade VI) – H1, H2 habitus of the mycelium; H3-H5 melanized hyphae with cylindrical cells, apical bud with roundish cells (H4), infrequent branching. I1-I5) A986 (clade VI) – I1, I2 habitus of the melanized mycelium; I3-I5 hyphae composed by globose, roundish cells, sometimes 1-septate (I3 arrow), with thick cell wall. J1-J4) A971 (clade VI) – J1 habitus of the melanized mycelium; J2-J4 melanized hyphae with cylindrical cells intercalating with roundish cells (J3 arrow), ramifications originate both from the cylindrical and the roundish cells. K1-K5) A1033 (Herpotrichiellaceae) – K1, K2 habitus of the melanized mycelium; K3-K5 melanized hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa. L1-L3) A561 (basal to Chaetothyriaceae) melanized hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa, frequently laterally and apically budding. Scale bars =4 mm (D1, G1, H1, I1, K1), 3 mm (A1, B1, C1, E1, F1), 1 mm (B2, D2, I2, K2), 0.5 mm (A2, C2, E2, F2, G2, H2, J1), 50 μm (B3, D3, F3, I5), 20 μm (A3, A4, B4, C3-C7, D4, D5, F4, F5, G3-G7, H3-H5, I3, I4, J2-J4, K3, K4, L2, L3)

The phylogenetic relationships recovered in Chaetothyriomycetidae are highly congruent with previous studies of Gueidan et al. (Gueidan et al. 2008, Gueidan et al. 2011) and Diederich et al. (2013). There were no significant incongruences between single locus (not shown) and multilocus trees. The only exception is the clade of Sclerococcum sphaerale, which is placed in our multilocus reconstruction at the base of Chaetothyriomycetidae, possibly due to the availability of only the nucLSU marker (Fig. ​(Fig.1).1). In this Sclerococcum sphaerale clade we recovered the single isolate A1016. A1016 was isolated from a thallus of Pertusaria corallina infected by Sclerococcum sphaerale, and this placement seems to confirm the identity of the lichenicolous fungus. This isolated strain forms pale pinkish, compact mycelia with thin, hyaline hyphae (Fig. ​(Fig.22 F1-F5). Clade I is represented by six isolates (from three host species), which together with Celothelium cinchonarum are basal to the split between Verrucariales and Chaetothyriales. These isolates are similar in morphology, forming white mycelia composed by thin, hyaline hyphae, which occasionally gather in thick, plectenchymatous strands (Fig. ​(Fig.22 A1-A4, B1-B4). Clade II is represented by three isolates: they come from three different thalli of the same lichen host- lichenicolous fungus association (Rhizocarpon geographicum – Muellerella pygmaea-Rh). These strains also present a pale pinkish mycelium, but hyphae are formed by cylindrical to semi-elliptical cells which are occasionally intercalated by roundish cells (Fig. ​(Fig.22 C1-C7). Two samples, A579 and A1026, are nested within Epibryaceae, the lineage formed by Epibryon and two rock-inhabiting fungi. The mycelium of these isolates is a dense aggregate of roundish, melanised cells containing inclusions, and filamentous hyphae are rarely present (Fig. ​(Fig.22 E1-E5). Clade III represents the lichenicolous fungus Lichenodiplis lecanorae (Muggia et al. in prep.), which appears here basal to the split between the families Epibryaceae, Chaetothyriaceae and Herpotrichiellaceae. The identity of these isolates is also confirmed by the conidiomata-like structures and the conidia that are observed in the cultures (Fig. ​(Fig.22 D1-D4). Herpotrichiellaceae is here the most represented family of Chaetothyriales and comprises ecologically diverse fungi including human pathogens (Exophiala dermatitidis and Capronia semiimmersa), lichenicolous fungi (Capronia peltigerae and Cladophialophora parmeliae) and rock inhabiting fungi (Gueidan et al. 2008; Gueidan et al. 2011, Gueidan et al. 2014). Four newly cultured isolates are nested in this main Chaetothyriales lineage. A561 is nested in a clade with RIF and Phialophora europaea, and is morphologically similar to other previously isolated black RIFs (Fig. ​(Fig.22 L1-L3), having melanized hyphae frequently budding laterally and apically. Three other samples are nested in a clade with Cladophialophora parmeliae and Capronia semiimmersa. The isolates are characterized by melanized mycelia, with branching hyphae composed by elliptical, subcylindrical and subglobose cells constricted at the septa (Fig. ​(Fig.22 K1-K5).

In Chaetothyriales, the majority of the isolates group into subclades of a fully supported lineage sister to Chaetothyriales. Within this lineage we distinguished the main clades IV, V, VI and VII (as subclade of clade VI, Fig. ​Fig.1),1), each represented by more than four isolates. The other isolates are placed on separate smaller clades in this large assemblage of branches. Clade IV and clade V include isolates from six and seven, respectively, different lichen host- lichenicolous fungus associations. Clade VI contains the majority of the isolates which come from 16 different lichen host - lichenicolous fungus associations. These include lichens infected by known but unrelated lichenicolous fungi. With the exception of few isolates, such as A514 (Fig. ​(Fig.22 H1-H5) and A511, which lack melanized mycelium, all the fungal strains included in this big assemblage of lineages are characterized by melanized mycelia. However, two main morphologies are observed among the strains: i) mycelia with filamentous, branching hyphae composed by cylindrical cells, usually aseptate (rarely 1-septate), intercalating by roundish cells, ii) mycelia with hyphae composed exclusively by globose, roundish cells, sometimes 1-septate, forming dense assemblages and budding.

Except for the Sclerococcum clade and the clade III, we do not find clear evidence of correspondence of certain lineages with other lichenicolous fungi infecting the lichen samples.

Phylogenetic and morphological analysis of Dothideomycetes – (Fig. ​(Fig.3,3, Fig. ​Fig.4,4, Table ​Table22 and Table S2)

Open in a separate window

Open in a separate window

Fig. 3

Multilocus phylogenetic inference of Dothideomycetes. The ML and the Bayesian phylogenetic hypotheses were inferred from the combined dataset of nucLSU, nucSSU and mtSSU loci and corresponded in their topologies; the ML analysis is shown. ML bootstrap support values (≥ 70 %) and Bayesian posterior probabilities (PP ≥ 95 %) are reported above branches (bootstrap value/PP). Fungal isolates obtained from this study are highlighted in bold. Symbols indicate the different lichen host-lichenicolous fungal associations as reported in Fig. ​Fig.1.1. Fungal life-styles are reported in parenthesis. Samples labelled with an asterisk (*) are those photographed in Fig. ​Fig.44

Open in a separate window

Open in a separate window

Fig. 4

Habitus of one year old, representative, cultured fungal strains belonging to Dothideomycetes and included in the phylogenetic analysis of Fig. ​Fig.3.3. Anatomical structures were photographed from squashed sections mounted in water. Samples are reported with their number and the clade to which they belong as in Fig. ​Fig.3.3. A1-A5) A537 (Phoma) – A1, A2 habitus of the mycelium; A3-A5 hyaline hyphae with inclusions forming a dense aggregate. B1-B3) A542 (Phoma) – B1, B2 habitus of the mycelium, B3 dense aggregate of hyphae. C1-C5) A930 (Lichenostigmatales) – C1, C2 habitus of the mycelium; C3-C5 yeast-like melanised cells forming dense aggregates. D1-D6) A931 (clade sister to Lichenotheliales) – D1, D2 habitus of the mycelium; D3-D6 plectenchymatous structure of hyaline hyphae with cylindrical cells, round cells as buds at the apexes of the hyphae (arrows). E1-E3) A 567 (clade sister to Lichenotheliales) – E1 habitus of the mycelium; E2, E3 melanized hyphae composed by elliptical and subcylindrical cells constricted at the septa, laterally budding. F1-F4) A554 (Myriangiales) – F1, F2 habitus of the mycelium; F3, F4 thin, hyaline hyphae. G1-G6) A559 (Teratospaheriaceae I) – G1, G2 habitus of the mycelium; G3-G6 melanized hyphae, branching and composed by cylindrical to subglobose cells. H1-H6) A960 (clade nested in Teratosphaeriaceae) H1, H2 habitus of the mycelium, hyphae develop inside the growth medium; H3-H6 melanized hyphae, branching and composed by cylindrical to subglobose cells. Scale bars =4 mm (A1, C1, D1, E1, F1, G1), 3 mm (B1), 2 mm (B2, H1), 1.5 mm (C2), 1 mm (A2, D2, F2, G2), 0.4 mm (H2), 50 μm (G3), 40 μm (C3, D3), 20 μm (A3, C5, E2, E3, F3, G4, H3-H6), 10 μm (A5, B3, D4-D6, F4, G5, G6)

The phylogenetic relationships recovered in Dothideomycetes are highly congruent with previous studies of Ruibal et al. (2009), Lawrey et al. (Lawrey et al. 2011) Lawrey Diederich et al. 2012, Muggia et al. (2013); Hyde et al. (2013). Topological congruence was recovered between the Bayesian and the maximum likelihood analyses and among the single locus analyses. Also in Dothideomycetes the isolates are nested in clades together with fungi of diverse ecological niches and presenting different lifestyles (Fig. ​(Fig.33).

The isolate A930 is recovered within Lichenostigmatales, which includes lichenicolous fungi and RIFs. A930 is morphologically identical to the Lichenostigma cultures isolated by Ertz et al. (2014), presenting yeast-like, budding, melanized cells. Four isolates form a fully supported clade nested in Pleosporales. Also in Pleosporales, four further isolates group together with lichenicolous species of the genus Phoma; however they were isolated from thalli of four different lichen host-lichenicolous fungi associations and none of them showed the symptomatic presence of Phoma species. These isolates form whitish to pale pinkish mycelia, composed by hyaline hyphae distributed to form a dense aggregate (Fig. ​(Fig.44 A1-A5, B1-B3). Seven isolates represent a lineage sister to Lichenotheliales; these isolates originate also from four thalli representing different lichen hosts infected by different lichenicolous fungi. The isolates comprise both melanized and non-melanized fungi (Fig. ​(Fig.44 D1-D6 and E1-E3). Three isolates are recovered in Myriangiales, a lineage of saprobic fungi; they present white mycelium of very thin hyaline hyphae (Fig. ​(Fig.44 F1-F4). The single isolate A559 is recovered as a member of Teratosphaeriaceae I. The remaining isolates group as a single lineage in Capnodiales, being nested among the clades Teratosphaeriaceae I, Teratosphaeriaceae II and Mycosphaerellaceae. In this lineage we identify three subclades, even though all isolates have a similar morphology, with dark, melanized mycelia composed by suglobose to cylindrical cells with rough cell wall and sometimes constricted at the septa (Fig. ​(Fig.44 G1-G6, H1-H6).

Discussion

Rock-inhabiting alpine lichens are exposed to harsh environmental conditions, with drastic and sometimes sudden changes in temperature and hydration, as well as UV radiation. Conceivably, only fungi that tolerate such fluctuating conditions can persist or grow in lichens. In addition, these fungi must cope with the diverse and highly concentrated extracellular secondary products of their host species. We already found a surprising number of lichenicolous fungi in lichens (Fleischhacker et al. 2015), and evidence for a high number of additional, cryptically occurring fungi. Here we provided a comprehensive set of isolates of the culturable fungal fraction in lichens from an alpine habitat for a survey of their phylogenetic relationships, with special emphasis on members of Dothideomycetes and Chaetothyriomycetes.

Molecular data and the morphological analyses seem to confirm the identity of only two symptomatic lichenicolous fungal species with Eurotiomycetes. The isolates obtained from thalli infected by Sclerococcum sphaerale indeed group within the lineage Sclerococcum (Diederich et al. 2013). The formation of conidiomata and conidocells was observed in multiple cultured fungi from different thalli with infections of Lichenodiplis lecanorae. This proved the identity of the culture with the original infection of the lichenicolous hyphomycete.

Except for the above mentioned clades, we do not find clear evidence of correspondence of certain lineages with other lichenicolous fungal species infecting the lichen samples. Some of the observed lichenicolous fungi cannot be the origin of the sequenced cultures, since these belong to completely unrelated groups (e.g. Arthonia, Carbonea, Cecidonia, Opegrapha, Skyttea, Stigmidium; Ertz et al. 2009, 2014; Schmull et al. 2011; Suija et al. 2015). It is likely that the clade IV and V, and the plenty of clades with few representatives, so far correspond to lineages of still unknown fungi which may occur widespread in lichen thalli from rocks, but are unapparent to the eye.

The present phylogenetic results also show that some of the detected fungi are closely related to lichenicolous fungi as well as to fungi known from diverse other ecological niches. Two isolates are closely related to the genus Epibryon, which was originally described as bryophilous (Döbbeler 1978). It is now emended by non-lichenized lichenicolous species (Zhurbenko and Hafellner 1999; Sérusiaux et al. 1999), which demonstrates cross-kingdom host switches in this monophyletic genus. The host lichens of the Epibryon strain were also visibly infected by the genera Carbonea and Muellerella, respectively. Also Muellerella comprises species on bryophytes and lichens (Döbbeler and Triebel 1985), but its relationship with Epibryon requires further study. Clearly Carbonea, as a member of Lecanoraceae, is unrelated. The results suggest that Epibryon could occur also as a non-symptomatic lichen inhabitant, which agrees with the previous results of U’Ren et al. (2010), who discovered a group of fungi capable to live cryptically in both lichens and mosses. The cryptic presence of otherwise symptomatic lichenicolous fungi is also demonstrated by isolates placed with the lichenicolous lineage of the anamorph genus Phoma, and those strains which are nested with lichenicolous species of Capronia and Cladophialophora. None of these isolates, however, originated from thalli which were visibly infected by either Phoma, Capronia or Cladophialopora. The high similarity (>95%) that the new sequences showed with the already available Phoma sequences suggests that the isolated strain could represent closely related Phoma species.

The majority of the isolates are melanized fungi, which closely resemble previously studied rock-inhabiting fungi (RIF) and in fact are closely related to them. The presence or absence of these fungi in hosts of the same area seems to be largely unpredictable, unspecific and facultative. Rather than indicating host specificity, they seem to be broadly tolerant species whose presence might depend more on physical parameters. Nonetheless, lineage clade VI (Chaetothyriales) seems to be rather ubiquitous in lichens. All selected fungi likely represent the same species occurring in many thalli and in combination with different lichenicolous fungi.

The finding of few isolates in Myriangiales, Xylariales, Hypocreales and Coniochaetales is quite interesting, as this is the first record for lichens from rocks; members of these groups are mainly biotrophic plant-associated fungi, endophytes, saprotrophs on wood and insect parasites. Fungi in Xylariales were, though, already isolated from lichen thalli from other ecological niches (Ding et al. 2009, U’Ren et al. 2012). However, no diagnostic structure hinting at these fungi have ever been observed under the microscope. Arnold et al. (2009) first suggested that fungi may live a symptomless life in lichens and coined the term ´endolichenic fungi´ for such organisms. Arnold and co-authors (Arnold et al. 2009, U’Ren et al. 2010, 2012) also have studied lichens from different habitats, such as tropical forest, temperate, boreal and arctic locations. Though some of these lichens are of the same mycobiont genera as the species included in this study, Arnold and colleagues found a higher proportion of fungi in Leotiomycetes and Sordariomycetes, more closely related to lineages of plant endophytes, rather than to the lineages predominantly found in this survey. It is likely that the taxonomic diversities recovered between the two surveys correlates with the local vegetation and geologic histories of the regions. The cryptic occurrence of fungi has been also found in different environments (Stergiopoulos and Gordon 2014), and even included plant pathogens (Malcolm et al. 2013).

We also isolated fungi which constitute two monophyletic lineages, both closely related to orders and families of lichenicolous and lichenized genera, RIF and pathogens in Dothideomycetes: the first closely related to Lichenotheliales, the second nested in Capnodiales. The first lineage is closely related to species of the genus Lichenothelia, which are known to share multiple lifestyles on rocks (Hyde et al. 2013; Muggia et al. 2013, 2015). They dwell on bare rock surfaces, but are often found associated with free living algae also present on the rocks. Some species specialize as lichen parasites and seem to associate with the lichen photobiont (Muggia et al. 2015). Some oligotrophic fungi apparently improve their carbon supply by attaching to microscopic algae. A direct involvement of black fungi in fungal-algal interactions was earlier described as a balanced algal parasitism (Turian 1977). Several rock-inhabiting and lichen-inhabiting microcolonial fungi develop into lichenoid structures within months when co-cultured with algae obtained from lichen thalli (Gorbushina et al. 2005; Brunauer et al. 2007). Gorbushina and Broughton (2009) showed an example with a co-culture of Nostoc and a rock-inhabiting fungus (Sarcinomyces). They observed a specific spatial arrangement of both organisms and growth alterations in the photosynthetic cyanobacteria suggested a specific interaction. Therefore black fungi that loosely associate with algae in nature might be interpreted as “lichenoids” and are considered prime forms of symbiosis (Muggia et al. 2013).

The apparent ability of black fungi to associate loosely with algae sheds an interesting light on the evolution of lichens. In fact some of the rock-inhabitants are basal to the large lichenized Ascomycete lineages Arthoniomycetes and Verrucariales (Gueidan et al. 2008; Ruibal et al. 2009). Otherwise, the lichenized life styles are scattered in various clades of Dothideomycetes (Muggia et al. 2008; Ruibal et al. 2009; Nelsen et al. 2009), where lichen thallus morphology remains generally simple. However, not all of the lineages do associate with algae or establish lichen symbioses. Some Dothideales have evolved into highly adaptable and versatile species -e.g. Aureobasidium pullulans commonly found on leaf surfaces of plant- but have not been found to be associated with lichens.

In our survey, fungi of unrelated lineages were recovered several times from individual lichen thalli. This may indicate that there is no competition between the different fungi, which complies with a concept of niche-sharing (Crous et al. 2009), and that the occurrence of certain lineages does not implicate the presence or absence of others. Lichen-associated fungi, which do not develop any diagnostic structure on the thallus host, use the host just for their own cryptic internal life, likely awaiting the most suitable substrate/host to propagate. Perhaps not all isolated fungi grow equally well in lichens, and we cannot exclude that some might be present as spores or small germlings, while others form mycelia networks in their hosts. We often see mycelia of melanized fungi on the lichens and expect their growth is well adapted to the poikilohydric lichen habitat. The symbiotic structures of the lichen thalli function as a shared habitat of phylogenetically diverse stress-tolerant fungi, some of which use their host as protection, while others use it as nutrition sources in otherwise hostile environments.

Electronic supplementary material

Acknowledgement

References