ITS1 metabarcoding highlights low specificity of lichen mycobiomes at a local scale.

As self-supporting and long-living symbiotic structures, lichens provide a habitat for many other organisms beside the traditionally considered lichen symbionts-the myco- and the photobionts. The lichen-inhabiting fungi either develop diagnostic phenotypes or occur asymptomatically. Because the degree of specificity towards the lichen host is poorly known, we studied the diversity of these fungi among neighbouring lichens on rocks in an alpine habitat. Using a sequencing metabarcoding approach, we show that lichen mycobiomes clearly reflect the overlap of multiple ecological sets of taxa, which differ in their trophic association with lichen thalli. The lack of specificity to the lichen mycobiome is further supported by the lack of community structure observed using clustering and ordination methods. The communities encountered across samples largely result from the subsampling of a shared species pool, in which we identify three major ecological components: (i) a generalist environmental pool, (ii) a lichenicolous/endolichenic pool and (iii) a pool of transient species. These taxa majorly belong to the fungal classes Dothideomycetes, Eurotiomycetes and Tremellomycetes with close relatives in adjacent ecological niches. We found no significant evidence that the phenotypically recognized lichenicolous fungi influence the occurrence of the other asymptomatic fungi in the host thalli. We claim that lichens work as suboptimal habitats or as a complex spore and mycelium bank, which modulate and allow the regeneration of local fungal communities. By performing an approach that minimizes ambiguities in the taxonomic assignments of fungi, we present how lichen mycobiomes are also suitable targets for improving bioinformatic analyses of fungal metabarcoding.

Effects of Growth Media on the Diversity of Culturable Fungi from Lichens.

Microscopic and molecular studies suggest that lichen symbioses contain a plethora of associated fungi. These are potential producers of novel bioactive compounds, but strains isolated on standard media usually represent only a minor subset of these fungi. By using various in vitro growth conditions we are able to modulate and extend the fraction of culturable lichen-associated fungi. We observed that the presence of iron, glucose, magnesium and potassium in growth media is essential for the successful isolation of members from different taxonomic groups. According to sequence data, most isolates besides the lichen mycobionts belong to the classes Dothideomycetes and Eurotiomycetes. With our approach we can further explore the hidden fungal diversity in lichens to assist in the search of novel compounds.

Extremotolerant fungi from alpine rock lichens and their phylogenetic relationships.

Fungi other than the lichen mycobiont frequently co-occur within lichen thalli and on the same rock in harsh environments. In these situations dark-pigmented mycelial structures are commonly observed on lichen thalli, where they persist under the same stressful conditions as their hosts. Here we used a comprehensive sampling of lichen-associated fungi from an alpine habitat to assess their phylogenetic relationships with fungi previously known from other niches. The multilocus phylogenetic analyses suggest that most of the 248 isolates belong to the Chaetothyriomycetes and Dothideomycetes, while a minor fraction represents Sordariomycetes and Leotiomycetes. As many lichens also were infected by phenotypically distinct lichenicolous fungi of diverse lineages, it remains difficult to assess whether the culture isolates represent these fungi or are from additional cryptic, extremotolerant fungi within the thalli. Some of these strains represent yet undescribed lineages within Chaethothyriomycetes and Dothideomycetes, whereas other strains belong to genera of fungi, that are known as lichen colonizers, plant and human pathogens, rock-inhabiting fungi, parasites and saprotrophs. The symbiotic structures of the lichen thalli appear to be a shared habitat of phylogenetically diverse stress-tolerant fungi, which potentially benefit from the lichen niche in otherwise hostile habitats.

Phylogenetic placement of the lichenicolous, anamorphic genus Lichenodiplis and its connection to Muellerella-like teleomorphs.

Lichenicolous fungi are a specialized group of taxa which inhabit lichens and develop diverse degrees of specificity and parasitic behaviour towards their hosts. They are recognized only by their phenotypic symptoms and sexual or asexual spore-producing structures on the lichen thalli. Only recently, molecular data and culture dependent approaches have helped in uncovering the species diversity and in verifying the phylogenetic position and anamorph-teleomorph relationships of some taxa. Here, we studied the phylogenetic placement of representative taxa of two lichenicolous genera, the coelomycete Lichenodiplis and the ascomycete Muellerella. We obtained molecular data for three nuclear and mitochondrial loci (28S, 18S, and 16S), both from fresh collected specimens and culture isolates. Our multilocus phylogeny places Lichenodiplis and Muellerella samples in one monophyletic, fully supported clade, sister to Epibryon (Epibryaceae) in Chaetothyriales (Eurotiomycetes). Morphological analyses of axenically cultured fungi show the formation of conidiomata and conidiospores in both Lichenodiplis and Muellerella isolates. We suggest that the species Lichenodiplis lecanorae and Muellerella atricola represent, respectively, the anamorphic and teleomorphic stages of the same fungus and discuss their relationships with the other fungal families in Chaetothyriomycetidae.

Community Analyses Uncover High Diversity of Lichenicolous Fungi in Alpine Habitats.

Lichens are frequently colonized by specialized, lichenicolous fungi. Symptomatic lichenicolous fungi usually display typical phenotypes and reproductive structures on the lichen hosts. The classification based on these structures revealed different host specificity patterns. Other fungi occur asymptomatically in the lichen thalli and are much less known. We aimed at studying the diversity of lichen-associated fungi in specific, lichen-rich communities on rocks in the Alps. We tested whether lichenicolous fungi developing symptomatically on their known hosts also occur asymptomatically in other thalli of the same or of different host species. We collected lichen thalli according to a uniform sampling design comprising individuals adjacent to thalli that showed symptoms of lichenicolous fungal infections. The total fungal communities in the selected lichen thalli were further studied by single-strand conformation polymorphism (SSCP) fingerprinting analyses and sequencing of internal transcribed spacer (ITS) fragments. The systematic, stratified sampling strategy helped to recover 17 previously undocumented lichenicolous fungi and almost exhaustively the species diversity of symptomatic lichenicolous fungi in the studied region. The results from SSCP and the sequencing analyses did not reveal asymptomatic occurrence of normally symptomatic lichenicolous fungi in thalli of both the same and different lichen host species. The fungal diversity did not correlate with the species diversity of the symptomatic lichenicolous fungus-lichen host associations. The complex fingerprint patterns recovered here for fungal communities, in associations of well-delimited lichen thalli, suggest lichen symbiosis as suitable subjects for fungal metacommunity studies.

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.

Genetic variation in brown trout Salmo trutta across the Danube, Rhine, and Elbe headwaters: a failure of the phylogeographic paradigm?

BACKGROUND: Brown trout Salmo trutta have been described in terms of five major mtDNA lineages, four of which correspond to major ocean basins, and one, according to some authors, to a distinct taxon, marbled trout Salmo marmoratus. The Atlantic and Danubian lineages of brown trout meet in a poorly documented contact zone in Central Europe. The natural versus human mediated origin of the Atlantic lineage in the upper Danube is a question of both theoretical and practical importance with respect to conservation management. We provide a comprehensive population genetic analysis of brown trout in the region with the aim of evaluating the geographic distribution and genetic integrity of these two lineages in and around their contact zone. RESULTS: Genetic screening of 114 populations of brown trout across the Danube/Rhine/Elbe catchments revealed a counter-intuitive phylogeographic structure with near fixation of the Atlantic lineage in the sampled portions of the Bavarian Danube. Along the Austrian Danube, phylogeographic informative markers revealed increasing percentages of Danube-specific alleles with downstream distance. Pure Danube lineage populations were restricted to peri-alpine isolates within previously glaciated regions. Both empirical data and simulated hybrid comparisons support that trout in non-glaciated regions north and northeast of the Alps have an admixed origin largely based on natural colonization. In contrast, the presence of Atlantic basin alleles south and southeast of the Alps stems from hatchery introductions and subsequent introgression. Despite extensive stocking of the Atlantic lineage, little evidence of first generation stocked fish or F1 hybrids were found implying that admixture has been established over time. CONCLUSIONS: A purely phylogeographic paradigm fails to describe the distribution of genetic lineages of Salmo in Central Europe. The distribution pattern of the Atlantic and Danube lineages is extremely difficult to explain without invoking very strong biological mechanisms.The peri-alpine distribution of relict populations of pure Danubian lineage brown trout implies that they colonized headwater river courses post-glacially ahead of the expansion of the Atlantic lineage. The recognition of natural as opposed to anthropogenic introgression of the Atlantic lineage into Danubian gene pools is of fundamental importance to management strategies.