Samantha Fernández-Brime

Implementing a cumulative supermatrix approach for a comprehensive phylogenetic study of the Teloschistales (Pezizomycotina, Ascomycota)

The resolution of the phylogenetic relationships within the order Teloschistales (Ascomycota, lichen-forming-fungi), with nearly 2000 known species and outstanding phenotypic diversity, has been hindered by the limitation in the resolving power that single-locus or two-locus phylogenetic studies have provided to date. In this context, an extensive taxon sampling within the Teloschistales with more loci (especially nuclear protein-coding genes) was needed to confront the current taxonomic delimitations and to understand evolutionary trends within this order. Comprehensive maximum likelihood and bayesian analyses were performed based on seven loci using a cumulative supermatrix approach, including protein-coding genes RPB1 and RPB2 in addition to nuclear and mitochondrial ribosomal RNA-coding genes. We included 167 taxa representing 12 of the 15 genera recognized within the currently accepted Teloschistineae, 22 of the 43 genera within the Physciineae, 49 genera of the closely related orders Lecanorales, Lecideales, and Peltigerales, and the dubiously placed family Brigantiaeaceae and genus Sipmaniella. Although the progressive addition of taxa (cumulative supermatrix approach) with increasing amounts of missing data did not dramatically affect the loss of support and resolution, the monophyly of the Teloschistales in the current sense was inconsistent, depending on the loci-taxa combination analyzed. Therefore, we propose a new, but provisional, classification for the re-circumscribed orders Caliciales and Teloschistales (previously referred to as Physciineae and Teloschistineae, respectively). We report here that the family Brigantiaeaceae, previously regarded as incertae sedis within the subclass Lecanoromycetidae, and Sipmaniella, are members of the Teloschistales in a strict sense. Within this order, one lineage led to the diversification of the mostly epiphytic crustose Brigantiaeaceae and Letrouitiaceae, with a circumpacific center of diversity and found mostly in the tropics. The other main lineage led to another epiphytic crustose family, mostly tropical, and with an Australasian center of diversity--the Megalosporaceae--which is sister to the mainly rock-inhabiting, cosmopolitan, and species rich Teloschistaceae, with a diversity of growth habits ranging from crustose to fruticose. Our results confirm the use of a cumulative supermatrix approach as a viable method to generate comprehensive phylogenies summarizing relationships of taxa with multi-locus to single locus data.

Expansion of the Stictidaceae by the addition of the saxicolous lichen-forming genus Ingvariella

The monotypic, lichen-forming genus Ingvariella originally was segregated from Diploschistes and placed within the Thelotremataceae (Ostropales) based on aspects of exciple morphology. However, the I+ hymenium and amyloid ascus wall suggest affinities to families other than the Thelotremataceae. To assess the identity of Ingvariella and to investigate its placement within the Ostropales, we inferred phylogenetic relationships of I. bispora by comparison of mtSSU rDNA and nuLSU rDNA sequences for 59 species encompassing a broad array of ostropalean fungi by means of Bayesian, maximum likelihood and weighted maximum parsimony methods. Here we report that Ingvariella is a member of the Stictidaceae, sister to the mainly saprotrophic genus Cryptodiscus. The inclusion of the first saxicolous lichen-forming fungus within this family expands the broad ecological diversity of the Stictidaceae, where saprotrophic fungi, corticicolous lichen-forming fungi and lichenized and non-lichenized conspecific taxa have been described previously. We also present new insights into the relationships among other families within the Ostropales.

Microbiome change by symbiotic invasion in lichens

Lichens are obligate symbioses between fungi and green algae or cyanobacteria. Most lichens resynthesize their symbiotic thalli from propagules, but some develop within the structures of already existing lichen symbioses. Diploschistes muscorum starts as a parasite infecting the lichen Cladonia symphycarpa and gradually develops an independent Diploschistes lichen thallus. Here we studied how this process influences lichen-associated microbiomes and photobionts by sampling four transitional stages, at sites in Sweden and Germany, and characterizing their microbial communities using high-throughput 16S rRNA gene and photobiont-specific ITS rDNA sequencing, and fluorescence in situ hybridization. A gradual microbiome shift occurred during the transition, but fractions of Cladonia-associated bacteria were retained during the process of symbiotic reorganization. Consistent changes observed across sites included a notable decrease in the relative abundance of Alphaproteobacteria with a concomitant increase in Betaproteobacteria. Armatimonadia, Spartobacteria and Acidobacteria also decreased during the infection of Cladonia by Diploschistes. The lichens differed in photobiont specificity. Cladonia symphycarpa was associated with the same algal species at all sites, but Diploschistes muscorum had a flexible strategy with different photobiont combinations at each site. This symbiotic invasion system suggests that partners can be reorganized and selected for maintaining potential roles rather than depending on particular species.

The adaptive radiation of lichen-forming Teloschistaceae is associated with sunscreening pigments and a bark-to-rock substrate shift

Significance The tempo of diversification of life can be accelerated by fortuitous ecological opportunity or by phenotypic innovation. In this study, we document how both factors are likely to have played a role in the origin and success of a major fungal lineage, the Teloschistaceae (comprising ∼1% of all fungi). Anthraquinone pigments are found in a widespread, but scattered, range of fungi and plants, but are particularly abundant in the lichen-forming Teloschistaceae, where they provide sunlight protection, especially needed when growing in arid deserts of the world. We found that anthraquinones evolved in these lichens, in conjunction with an ecological switch to exposed, rocky environments, allowing them to colonize swathes of unexploited habitats worldwide and sparking an acceleration in diversification. Adaptive radiations play key roles in the generation of biodiversity and biological novelty, and therefore understanding the factors that drive them remains one of the most important challenges of evolutionary biology. Although both intrinsic innovations and extrinsic ecological opportunities contribute to diversification bursts, few studies have looked at the synergistic effect of such factors. Here we investigate the Teloschistales (Ascomycota), a group of >1,000 lichenized species with variation in species richness and phenotypic traits that hinted at a potential adaptive radiation. We found evidence for a dramatic increase in diversification rate for one of four families within this order—Teloschistaceae—which occurred ∼100 Mya (Late Cretaceous) and was associated with a switch from bark to rock and from shady to sun-exposed habitats. This adaptation to sunny habitats is likely to have been enabled by a contemporaneous key novel phenotypic innovation: the production in both vegetative structure (thallus) and fruiting body (apothecia) of anthraquinones, secondary metabolites known to protect against UV light. We found that the two ecological factors (sun exposure and rock substrate) and the phenotypic innovation (anthraquinones in the thallus) were all significant when testing for state-dependent shifts in diversification rates, and together they seem likely to be responsible for the success of the Teloschistaceae, one of the largest lichen-forming fungal lineages. Our results support the idea that adaptive radiations are driven not by a single factor or key innovation, but require a serendipitous combination of both intrinsic biotic and extrinsic abiotic and ecological factors.

Phylogenetic study of Diploschistes (lichen-forming Ascomycota: Ostropales: Graphidaceae), based on morphological, chemical, and molecular data

The genus Diploschistes includes crustose lichen-forming fungi with a carbonized proper excipulum with lateral paraphyses, and a chemistry dominated by orcinol depsides. However, the taxon D. ocellatus lacks these excipular characters and has β-orcinol depsidones, raising doubts about its inclusion within this genus. Using a two-locus dataset (mtSSU, nuLSU), our phylogenetic analyses confirm the classification of D. ocellatus within Diploschistes. Three different groups have been recognized within this genus, based on ascomatal morphology: Actinostomus (perithecioid), Scruposus (urceolate), and Ocel- latus (lecanoroid). These groups have been widely used in monographic studies and keys, but their taxonomic value has not been confirmed yet. Here we inferred phylogenetic relationships within Diploschistes, with a special emphasis on the D. scruposus complex, using a combined dataset consisting of morphological, chemical, nrITS, and mtSSU data in order to determine if these species groups and phenotypically based species delimitations were monophyletic. Based on our results, a new subgeneric treatment for Diploschistes is proposed, and the taxonomic value of fruiting body types is confirmed. The clade correspond- ing to D. ocellatus consists of two well-supported subclades, one of them grouping specimens without ascomata, having only pycnidia. It is also remarkable that the clade containing specimens of D. diacapsis subsp. neutrophilus appears distantly related to the clade containing all other accessions of D. diacapsis. Our analysis revealed that for some taxa, such as D. scruposus and D. interpediens, molecular variability did not correlate with either morphological or chemical diversity.

Lichenostigma rupicolae ( Lichenotheliaceae ), a new lichenicolous species growing on Pertusaria rupicola

The lichenicolous fungus Lichenostigma rupicolae Fdez.-Brime & Nav.-Ros., collected in France, Spain and Turkey, is described as new to science. It is found growing on the surface of the thalli and apothecia of Pertusaria rupicola , where it forms black patches of radiating plurihyphal strands connecting the fertile stromata. An updated key of the species of subgenus Lichenogramma is included.

Collolechia revisited and a re-assessment of ascus characteristics in Placynthiaceae (Peltigerales, Ascomycota)

Collolechia revisited and a re-assessment of ascus characteristics in Placynthiaceae (Peltigerales, Ascomycota)

Using multi-locus sequence data for addressing species boundaries in commonly accepted lichen-forming fungal species

Accurate species delimitations are of great importance for effectively characterizing biological diversity. Our criteria for delimiting species have changed dramatically over the last decades with the increasing availability of molecular data and improvement of analytical methods to evaluate these data. Whereas reciprocal monophyly is often seen as an indicator for the presence of distinct lineages, recently diverged species often fail to form monophyletic groups. At the same time, cryptic species have repeatedly been detected in numerous organismal groups. In this study, we addressed the species delimitation in the crustose lichen-forming fungal genus Diploschistes using multilocus sequence data from specimens representing 16 currently accepted species. Our results indicate the presence of previously undetected, cryptic species-level lineages in the subgenus Limborina. In the subgenus Limborina, samples from different continents currently classified under the same species were shown to be only distantly related. At the same time, in parts of subgen. Diploschistes characterized by short branches, none of the currently accepted species formed monophyletic groups. In spite of the lack of monophyly in phylogenetic reconstructions, a multispecies coalescent method provided support for eight of the nine accepted species in subgen. Diploschistes as distinct lineages. We propose to reduce D. neutrophilus to synonymy with D. diacapsis and point out that additional sampling will be necessary before accepting additional species in subgen. Limborina.

Schizoxylon as an experimental model for studying interkingdom symbiosis

Experiments to re-synthesise lichens so far focused on co-cultures of fungal and algal partners. However, recent studies have revealed that bacterial communities colonise lichens in a stable and host-specific manner. We were therefore interested in testing how lichenised fungi and algae interact with selected bacteria in an experimental setup. We selected the symbiotic system of Schizoxylon albescens and the algal genera Coccomyxa and Trebouxia as a suitable model. We isolated bacterial strains from the naturally occurring bacterial fraction of freshly collected specimens and established tripartite associations under mixed culture experiments. The bacteria belong to Actinobacteria, Firmicutes and Proteobacteria and corresponded to groups already found associated with fungi including lichens. In mixed cultures with Coccomyxa, the fungus formed a characteristic filamentous matrix and tightly contacted the algae; the bacteria distributed in small patches between the algal cells and attached to the cell walls. In mixed cultures with Trebouxia, the fungus did not develop the filamentous matrix, but bacterial cells were observed to be tightly adhering to the fungal hyphae. Our experiments show that this tripartite fungal-algal-bacterial model system can be maintained in culture and can offer multiple opportunities for functional studies based on experiments under controlled conditions in the laboratory.

Some new and interesting taxa of Cortinarius subgenus Phlegmacium from the European Mediterranean Basin.

We clarify the taxonomy and nomenclature of several taxa of the genus Cortinarius subgenus Phlegmacium. To this aim, we have used a combination of morphological and molecular data. The evolutionary relationships of the species were inferred by comparison of the nuITS by means of weighted maximum parsimony, maximum likelihood and two different types of Bayesian methods (with and without a priori alignments). Phylogenetic resolution and support of all or most of the species included in this study and their relationships were possible only when including the phylogenetic signal from ambiguously aligned regions in weighted maximum parsimony analyses (recoded INAASE characters) and when the analysis simultaneously optimized alignment and phylogeny (with BAli-phy). Three species are described as new, Cortinarius mediterraneensis, C. cistoglaucopus and C. palazonianus, and C. olivaecodionysae is proposed for C. dionysae f. olivaceus. Descriptions are provided for these taxonomic and nomenclatural novelties, along with discussions of morphological and phylogenetic affinities to closely related taxa. Scanning microphotographs of the basidiospores are provided for the discussed taxa, and color pictures of the basidiomes in their natural habitat are provided for C. cistoglaucopus, C. mediterraneensis and C. palazonianus.