Rock-inhabiting fungi originated during periods of dry climate in the late Devonian and middle Triassic

Non-lichenized rock-inhabiting fungi (RIF) are slow-growing melanized ascomycetes colonizing rock surfaces in arid environments. They possess adaptations, which allow them to tolerate extreme abiotic conditions, such as high UV radiations and extreme temperatures. They belong to two separate lineages, one consisting in the sister classes Dothideomycetes and Arthoniomycetes (Dothideomyceta), and the other consisting in the order Chaetothyriales (Eurotiomycetes). Because RIF often form early diverging groups in Chaetothyriales and Dothideomyceta, the ancestors of these two lineages were suggested to most likely be rock-inhabitants. The lineage of RIF related to the Chaetothyriales shows a much narrower phylogenetic spectrum than the lineage of RIF related to Dothideomyceta, suggesting a much more ancient origin for the latter. Our study aims at investigating the times of origin of RIF using a relaxed clock model and several fossil and secondary calibrations. Our results show that the RIF in Dothideomyceta evolved in the late Devonian, much earlier than the RIF in Chaetothyriales, which originated in the middle Triassic. The origin of the chaetothyrialean RIF correlates well with a period of recovery after the Permian-Triassic mass extinction and an expansion of arid landmasses. The period preceding the diversification of the RIF related to Dothideomyceta (Silurian--Devonian) is also characterized by large arid landmasses, but temperatures were much cooler than during the Triassic. The paleoclimate record provides a good explanation for the diversification of fungi subjected to abiotic stresses and adapted to life on rock surfaces in nutrient-poor habitats.     

Three common bryophilous fungi with meristematic anamorphs and phylogenetic alliance to Teratosphaeriaceae,Capnodiales

Bryophilous ascomycetes are an overlooked and poorly known fungal group. In this study, the extreme and small-sized niche of Polytrichum piliferum hyaline leaf tips was screened for the presence of these fungi in Finland. Three closely related species were found. Bryochiton perpusillus and Bryochiton monascus were identified from several samples, and DNA isolations revealed a third closely related species, Bryochiton sp. In addition, melanised hyphae, typical to the Bryochiton species, were present in all the samples. According to phylogenetic analyses consisting of combined small subunit (SSU), large subunit (LSU), and 5.8S rDNA sequences, and internal transcribed spacer (ITS) rDNA sequences, the species showed affinity with Teratosphaeriaceae within Capnodiales, and especially with black, meristematic species often inhabiting rock substrate in extreme environments. The connection was supported by meristematic growth of the Bryochiton species in culture. Bryochiton is the second sexual genus associated within the family Teratosphaeriaceae, and B. perpusillus, and B. monascus constitute examples of teleomorphs within a group of meristematic anamorphs. These findings emphasize the multiform diversity underlying poorly researched fungal groups, such as the bryophilous fungi.     

Hypersaline conditions induce changes in cell-wall melanization and colony structure in a halophilic and a xerophilic black yeast species of the genus Trimmatostroma

Melanized yeast-like meristematic fungi are characteristic inhabitants of highly stressed environments and are rare eukaryotic extremophiles. Therefore, they are attractive organisms for studies of adaptations. In this study we compared two meristematic species of the genus Trimmatostroma on media of differing water potentials isolated from distinct water-stressed environments: T. salinum from the hypersaline water of a solar saltern, and T. abietis from a marble monument in Crimea. The morphology and melanization of both isolates in response to sodium chloride-induced water stress were investigated by means of light and electron microscopy. We describe and compare the colony form and structure, ultrastructure, and degree of cell-wall melanization of both species in reaction to salinity and to inhibited melanin synthesis. The halophilic T. salinum responded to changed salinity conditions on the level of individual cell ultrastructure and degree of cell wall melanization, whereas the xerophilic rock-inhabiting T. abietis responded with modification of its colony structure. Surprisingly, both the halophilic and the xerophilic Trimmatostroma species were able to adapt to hypersaline growth conditions, although their growth patterns show distinct adaptation of each species to their natural ecological niches.     

The yeast lichenosphere: high diversity of basidiomycetes from the lichens Tephromela atra and Rhizoplaca melanophthalma

Lichens are well-known examples of complex symbiotic associations between organisms from different Kingdoms. Microfungi in particular, establish diverse associations with the hosting lichen thallus, as species-specific parasites or transient co-inhabitants. The whole community of lichen-associated fungi constitute the ‘lichen mycobiome’ comprising both ascomycetes and basidiomycetes, including filamentous and yeast taxa. Metabarcoding results and microscopy analyses show that in some thalli, basidiomycetes are frequent lichen-associated fungi but still only a few species could be axenically isolated and morphologically characterized. Within a broad project aiming at characterizing the mycobiome diversity by culture-dependent and independent approaches in two lichen species selected as reference models – Rhizoplaca melanophthalma and Tephromela atra, we succeed in isolating and culturing 76 new strains of basidiomycetous yeasts. The lichen thalli were collected in different mountain regions worldwide and at relatively high elevation. The yeast strains were isolated on different growth media and were studied for their morphological and genetic diversity. Nuclear internal transcribed spacer (ITS) and ribosomal large subunit (LSU) sequence analyses identified them to belong to ten families within the orders Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes. The yeasts here detected showed patterns of host-preference in a few cases and they are potentially related to the ecological conditions.     

New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata

Isolation of meristematic tissue of the red alga Palmaria palmata by a freezing-thawing method and further maintenance of the tissue in culture showed the existence of groups of meristematic cells in superficial cortical layers of thallus forming wart-like outgrowths. For the first time, proliferations (plantlets) were obtained from meristematic tissue of sporophytic and male gametophytic fronds and tetraspores from submeristematic tissue of sporophytic fronds within a short period (6 weeks). Tissue fragments (1 × 1 mm²) from upper margins of fresh thalli and cell aggregates (10−100,000 cells) from marginal meristem and meristematic warts of fresh thalli and thalli after the freezing-thawing procedure were cultured for getting plantlets. Tissue fragments (TF) and cell aggregates (CA) from submeristematic tissue of fresh thalli were cultured for obtaining tetraspores. For mass getting proliferations (plantlets) and tetraspores we recommend to use CA from marginal tissue of fresh fronds because of fast growth, high numbers of proliferations and simple techniques of the method. The freezing-thawing method allows also to identify meristematic tissue and to obtain plantlets of red algae with apical meristem (e.g., Gelidium spp.).     

Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Background and Aims

The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear how this variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, >300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts.

Methods

Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains.

Key Results

Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineages were also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed.

Conclusions

Low selectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi.     

Two new Cladophialophora species,C. tumbae sp. nov. and C. tumulicola sp. nov., and chaetothyrialean fungi from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.     

Meristem clusters in cortical layer of thallus cells of the cultured red alga Palmaria palmata

For the first time somatic spotlike formations of 1.5 mm in diameter and height up to 0.5 mm were revealed in thalli of the red alga Palmaria palmata reared in an aerated stirred culture. It was determined that some cortical cells of the thallus are able to divide in the periclinal and anticlinal directions forming spots. Deep freezing and subsequent thawing of thalli showed that the cortical cells of spots were meristematic cells. In certain conditions they enabled the formation of prolifications, which germinated plantlets. These clusters of meristem tissue in the cortical layer of cells of the thallus of P. palmata, which were formed in the culture as in nature, function as growth cells facilitating growth of thalli in thick and natural “planting material” formed upon thalli fragmenting after their freezing in the winter season.     

New molecular marker for phylogenetic reconstruction of black yeast-like fungi (Chaetothyriales) with hypothetical EIF2AK2 kinase gene

In eukaryotes, phosphorylation of the α-subunit of eIF2 is a mechanism to adjust cellular gene expression profiles in response to specific signals. The eIF2α kinases are a group of serine–threonine kinases that perform important functions in response to infection, proteotoxicity, and nutrient scavenging. The conserved nature of eIF2α kinases among fungi makes them potential evolutionary markers, which may contribute to deeper understanding of taxonomy and evolution. To date, only few studies are available of eIF2α kinases in black yeasts, which are members of Chaetothyriales containing potential agents of a gamut of major human diseases, such as chromoblastomycosis, phaeohyphomycosis and mycetoma. To establish the phylogenetic validity of sequences of eIF2α kinases hypothetical genes, we compared these genes between members of different classes of fungi, including black yeasts and allies, aiming at evaluation of the phylogeny of this group using an alternative molecular marker, compared to standard ribosomal genes. Trees generated with eIF2α kinase sequences of fungi were compared with those generated by ribosomal internal transcribed spacers (ITS rDNA) sequences from the same species. Sequences used were obtained from the protein Non-redundant database of NCBI, were aligned using CLUSTALX v1.8 and alignments were analyzed with RAxML v8.2.9 on the CIPRES Science Gateway portal. The trees generated had similar topologies, demonstrating that eIF2α kinases hypothetical gene sequences present a coherent reflection of evolution among fungi, compared to trees reconstructed by the use of ribosomal sequences. Our preliminary findings with a limited dataset strongly suggest that the evolution of kinases among black yeasts follows a similar path as revealed by ribosomal data, which underlines the validity of current taxonomy of black yeasts and relatives.     

Microcolonial Fungi on Rocks: A Life in Constant Drought?

Black microcolonial fungi (MCF) and black yeasts are among the most stress-resistant eukaryotic organisms known on Earth. They mainly inhabit bare rock surfaces in hot and cold deserts of all regions of the Earth, but some of them have a close phylogenetic relation to human pathogenic black fungi which makes them important model organisms also with respect to clinical mycology. The environment of those fungi is especially characterized by extreme changes from humidity to long periods of desiccation and extreme temperature differences. A key to the understanding of MCF ecology is the question about metabolic activity versus dormancy in the natural environments. In this study, the time lag from the desiccated state to rehydration and full metabolic activity and growth was measured and defined in accordance with simulated environmental conditions. The ability to survive after desiccation and the speed of rehydration as well as changes of the whole cell protein pattern are demonstrated. Whereas both mesophilic strains—Exophiala jeanselmei and Knufia perforans (=Coniosporium perforans)—show a clear reaction toward desiccation by production of small proteins, Cryomyces antarcticus—the extremotolerant MCF—does not show any response to desiccation but seems just to down-regulate its metabolism. Data on intracellular sugar suggest that both trehalose and mannitol might play a cell protective role in those fungi.     

Isolation and Screening of Black Fungi as Degraders of Volatile Aromatic Hydrocarbons

Black fungi reported as degraders of volatile aromatic compounds were isolated from hydrocarbon-polluted sites and indoor environments. Several of the species encountered are known opportunistic pathogens or are closely related to pathogenic species causing severe mycoses, among which are neurological infections in immunocompetent individuals. Given the scale of the problem of environmental pollution and the phylogenetic relation of aromate-degrading black fungi with pathogenic siblings, it is of great interest to select strains able to mineralize these substrates efficiently without any risk for public health. Fifty-six black strains were obtained from human-made environments rich in hydrocarbons (gasoline car tanks, washing machine soap dispensers) after enrichment with some phenolic intermediates of toluene and styrene fungal metabolism. Based on ITS sequencing identification, the majority of the obtained isolates were members of the genus Exophiala. Exophiala xenobiotica was found to be the dominant black yeast present in the car gasoline tanks. A higher biodiversity, with three Exophiala species, was found in soap dispensers of washing machines. Strains obtained were screened using a 2,6-dichlorophenol-indophenol (DCPIP) assay, optimized for black fungi, to assess their potential ability to degrade toluene. Seven out of twenty strains tested were able to use toluene as carbon source.     

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogens Cladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu >61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation.     

Adaptive traits of bark and ambrosia beetle-associated fungi

A phenotype is the expression of interactions between species genotype and environment. We quantified the contributions of ecological and phylogenetic associations to phenotypic variation in Geosmithia fungi. Geosmithia are symbiotic beetle-associated saprotrophs with a range of life histories and host specificities, including obligate nutritional beetle mutualists (ambrosia fungi) and phytopathogens. We hypothesized that: (1) species phenotypes are better explained by their ecology than by their phylogenetic relationships; (2) niche specialization was accompanied by enzymatic capability losses; and (3) ambrosia Geosmithia species have higher nutritional quality and antibiotic capabilities than species with facultative symbioses. Our results confirmed that long-term co-evolved specialists have reduced metabolic breadth in comparison to generalists. Phytopathogenic G. morbida produces unique enzyme suites with affinity to ligno-cellulose. Mycelia of ambrosia fungi contain large amounts of oleic fatty acid with nutritive and possibly allelopathic function. Overall, our results indicate that Geosmithia ecology have greater effect on species phenotype than their phylogenetic relationships.     

Detection and Identification of Fungi Intimately Associated with the Brown Seaweed Fucus serratus

The filamentous fungi associated with healthy and decaying Fucus serratus thalli were studied over a 1-year period using isolation methods and molecular techniques such as 28S rRNA gene PCR-denaturing gradient gel electrophoresis (DGGE) and phylogenetic and real-time PCR analyses. The predominant DGGE bands obtained from healthy algal thalli belonged to the Lindra, Lulworthia, Engyodontium, Sigmoidea/Corollospora complex, and Emericellopsis/Acremonium-like ribotypes. In the culture-based analysis the incidence of recovery was highest for Sigmoidea marina isolates. In general, the environmental sequences retrieved could be matched unambiguously to isolates recovered from the seaweed except for the Emericellopsis/Acremonium-like ribotype, which showed 99% homology with the sequences of four different isolates, including that of Acremonium fuci. To estimate the extent of colonization of A. fuci, we used a TaqMan real-time quantitative PCR assay for intron 3 of the beta-tubulin gene, the probe for which proved to be species specific even when it was used in amplifications with high background concentrations of other eukaryotic DNAs. The A. fuci sequence was detected with both healthy and decaying thalli, but the signal was stronger for the latter. Additional sequence types, representing members from the Dothideomycetes, were recovered from the decaying thallus DNA, which suggested that a change in fungal community structure had occurred. Phylogenetic analysis of these environmental sequences and the sequences of isolates and type species indicated that the environmental sequences were novel in the Dothideomycetes.     

Plant-fungal association in trees: Insights into changes in ecological strategies of Peroneutypa scoparia (Diatrypaceae)

Fungal endophytes comprise a highly diverse group of particular interest for their relevant implications to the ecosystems they inhabit. The objective of this study was to infer the phylogenetic affinity between strains of Peroneutypa scoparia exhibiting different lifestyles to elucidate possible shifts in ecological roles. Specimens and living cultures used in the present study were obtained from decaying wood and from live stem tissues of the invasive host species Broussonetia papyrifera. The similarity between the fungal strains was studied through molecular analyses. The results showed a close phylogenetic link and high genetic similarity between endophytic and saprotrophic strains. The main findings suggest that P. scoparia has primary access to the substrate as an endophyte and then this organism may change its use of the available resources presenting a saprotrophic growth. These results provide valuable information about the roles that diatrypaceous fungi play as endophytes or as decaying wood inhabitants and contributes to evaluate the ecological significance of this group.     

INTRASPECIFIC INTERACTIONS AND PARASITISM IN AN ASSOCIATION OFRHIZOCARPON LECANORINUMandR. GEOGRAPHICUM

Intraspecific thallus interactions in mixed populations ofRhizocarpon lecanorinumR. geographicumin North Wales are described. In contrast to the most commonly occurring morphotype ofR. geographicumwhich forms mosaics comprised of sharply delimited individual thalli,R. lecanorinumthalli consistently merge with one another at intraspecific contacts, leaving no visible trace of their initial boundaries. On the analogy of mycelial interactions in populations of wood-rotting and other non-lichen-forming fungi, it is postulated that these differences are the outcome, inR. geographicumof somatic incompatibility between distinct genotypes, and inR. lecanorinumof somatic compatibility among genetically identical or very closely related thalli. Possible mechanisms for the evolution of clonal population structure inR. lecanorinumand links between somatic interactions and reproductive output in these species are discussed. Facultative parasitism ofR. lecanorinumbySchaereria fuscocinereaand resistance to such parasitism by co-occurringR. geographicumare also documented.     

Molecular identifications uncover diverse fungal symbionts of Pleione (Orchidaceae)

Fungal mutualisms are essential for the evolution and diversification of Orchidaceae, yet the fungal symbionts of Pleione orchids are poorly understood because molecular data are unavailable for this genus. Based on ITS-rDNA sequencing for mycobionts of 15 Pleione species (both wild and cultivated plants were included), we conducted phylogenetic analyses for the most dominant mycobionts, and compared the operational taxonomic units (OTUs) of mycorrhizal fungi among species within Pleione. Tulasnellaceae, Ceratobasidiaceae, Serendipitaceae (Sebacinales), Atractiellales, and Auriculariales were reported as putative mycobionts of Pleione. In particular, the mycorrhizal associations between subtropical orchids and Atractiellales have not been observed before. For the dominant mycobionts in the roots of Pleione and its related genera, Bletilla and Coelogyne, we detected no fungal OTU that was shared. Within Pleione, species with a sympatric distribution showed preferences for different fungi. Epiphytic and lithophytic individuals of Pleione albiflora shared OTUs of Tulasnellaceae but harbored different OTUs of Sebacinales, indicating some degree of fungal specificity toward certain habitats. These findings provide new insights into the ecological adaptation and evolution of orchids, and will contribute to the conservation and utilization of species resources.     

Variation in the ribosomal ITS-sequence of the lichens Buellia frigida and Xanthoria elegans from the Vestfold Hills,eastern Antarctica

Abstract:Thalli of the lichens Buellia frigida and Xanthoria elegans were collected from five different locations each 5–15 km apart in the Vestfold Hills, Princess Elizabeth Land, eastern Antarctica. A further collection was made from Mawson Station, Mac Robertson Land, eastern Antarctica, 660 km away. DNA was extracted from whole thalli and the ribosomal ITS region amplified by PCR using fungal specific primers. Resulting products were sequenced to gain an indication of whether or not variation was present within populations of lichen-forming fungi from continental Antarctica, and therefore of the availability of genetic resources to react to pressures such as climate change. Three genotypes ofB. frigida and two of X. elegans were detected in the Vestfold Hill collections. However, these differed by only one nucleotide position suggesting the presence of relatively little genetic variation, if the ITS region is indicative of the overall genome. Buellia frigida collected from Mawson Station had an identical ITS region sequence to the most common Vestfold Hills genotype, indicating that this species may have a low level of genetic variation across much of eastern Antarctica. In contrast, X. elegans collected from Mawson showed considerable genetic variation from the Vestfolds thalli, differing at 14·2% of nucleotide positions and had an identical ITS region sequence to an isolate from maritime Antarctica 4960 km away. Samples from the Vestfold Hills formed a distinct cluster in a phylogenetic analysis of ITS sequences from a worldwide collection of X. elegans isolates.     

Lichen-associated fungi of the Letharietum vulpinae

In the present study, a well-defined lichen community was screened for associated fungi for the first time. The photophilous lichen community Letharietum vulpinae was chosen because its character species, Letharia vulpina, was expected to host rather specialized fungi due to the presence of antimycotic secondary compounds. A considerable number of the associated fungi that were isolated were probably selective for lichens, because they appeared to be distantly related to fungi known from other substrates. The majority of these obligatory, lichen-associated fungi were only isolated in the course of the present study and represent hitherto unknown phylogenetic lineages. Parts of the lichen-associated fungi overlapped those colonizing rock surfaces or were closely related to endophytic fungi, but the lichen-associated and endophytic fungi still represented separate lineages.