Jürgen Otte

Coalescent-Based Species Delimitation Approach Uncovers High Cryptic Diversity in the Cosmopolitan Lichen-Forming Fungal Genus Protoparmelia (Lecanorales, Ascomycota)

Species recognition in lichen-forming fungi has been a challenge because of unsettled species concepts, few taxonomically relevant traits, and limitations of traditionally used morphological and chemical characters for identifying closely related species. Here we analyze species diversity in the cosmopolitan genus Protoparmelia s.l. The ~25 described species in this group occur across diverse habitats from the boreal -arctic/alpine to the tropics, but their relationship to each other remains unexplored. In this study, we inferred the phylogeny of 18 species currently assigned to this genus based on 160 specimens and six markers: mtSSU, nuLSU, ITS, RPB1, MCM7, and TSR1. We assessed the circumscription of species-level lineages in Protoparmelia s. str. using two coalescent-based species delimitation methods – BP&P and spedeSTEM. Our results suggest the presence of a tropical and an extra-tropical lineage, and eleven previously unrecognized distinct species-level lineages in Protoparmelia s. str. Several cryptic lineages were discovered as compared to phenotype-based species delimitation. Many of the putative species are supported by geographic evidence.

Integrating coalescent and phylogenetic approaches to delimit species in the lichen photobiont Trebouxia

The accurate assessment of species boundaries in symbiotic systems is a prerequisite for the study of speciation, co-evolution and selectivity. Many studies have shown the high genetic diversity of green algae from the genus Trebouxia, the most common photobiont of lichen-forming fungi. However, the phylogenetic relationships, and the amount of cryptic diversity of these algae are still poorly understood, and an adequate species concept for trebouxiophycean algae is still missing. In this study we used a multifaceted approach based on coalescence (GMYC, STEM) and phylogenetic relationships to assess species boundaries in the trebouxioid photobionts of the lichen-forming fungus Lasallia pustulata. We further investigated whether putative species of Trebouxia found in L. pustulata are shared with other lichen-forming fungi. We found that L. pustulata is associated with at least five species of Trebouxia and most of them are shared with other lichen-forming fungi, showing different patterns of species-to-species and species-to-community interactions. We also show that one of the putative Trebouxia species is found exclusively in association with L. pustulata and is restricted to thalli from localities with Mediterranean microclimate. We suggest that the species delimitation method presented in this study is a promising tool to address species boundaries within the heterogeneous genus Trebouxia.

Fungal–algal association patterns in lichen symbiosis linked to macroclimate

Both macroclimate and evolutionary events may influence symbiont association and diversity patterns. Here we assess how climatic factors and evolutionary events shape fungal-algal association patterns in the widely distributed lichen-forming fungal genus Protoparmelia. Multilocus phylogenies of fungal and algal partners were generated using 174 specimens. Coalescent-based species delimitation analysis suggested that 23 fungal hosts are associating with 20 algal species. Principal component analysis (PCA) was performed to infer how fungal-algal association patterns varied with climate. Fungi associated with one to three algal partners whereas algae accepted one to five fungal partners. Both fungi and algae were more specific, associating with fewer partners, in the warmer climates. Interaction with more than one partner was more frequent in cooler climates for both the partners. Cophylogenetic analyses suggest congruent fungal-algal phylogenies. Host switch was a more common event in warm climates, whereas failure of the photobiont to diverge with its fungal host was more frequent in cooler climates. We conclude that both environmental factors and evolutionary events drive fungal and algal evolution in Protoparmelia. The processes leading to phylogenetic congruence of fungi and algae are different in different macrohabitats in our study system. Hence, closely related species inhabiting diverse habitats may follow different evolutionary pathways.

Adaptive differentiation coincides with local bioclimatic conditions along an elevational cline in populations of a lichen-forming fungus

BackgroundMany fungal species occur across a variety of habitats. Particularly lichens, fungi forming symbioses with photosynthetic partners, have evolved remarkable tolerances for environmental extremes. Despite their ecological importance and ubiquity, little is known about the genetic basis of adaption in lichen populations. Here we studied patterns of genome-wide differentiation in the lichen-forming fungus Lasallia pustulata along an altitudinal gradient in the Mediterranean region. We resequenced six populations as pools and identified highly differentiated genomic regions. We then detected gene-environment correlations while controlling for shared population history and pooled sequencing bias, and performed ecophysiological experiments to assess fitness differences of individuals from different environments.ResultsWe detected two strongly differentiated genetic clusters linked to Mediterranean and temperate-oceanic climate, and an admixture zone, which coincided with the transition between the two bioclimates. High altitude individuals showed ecophysiological adaptations to wetter and more shaded conditions. Highly differentiated genome regions contained a number of genes associated with stress response, local environmental adaptation, and sexual reproduction.ConclusionsTaken together our results provide evidence for a complex interplay between demographic history and spatially varying selection acting on a number of key biological processes, suggesting a scenario of ecological speciation.

Sequencing genomes from mixed DNA samples - evaluating the metagenome skimming approach in lichenized fungi

The metagenome skimming approach, i.e. low coverage shotgun sequencing of multi-species assemblages and subsequent reconstruction of individual genomes, is increasingly used for in-depth genomic characterization of ecological communities. This approach is a promising tool for reconstructing genomes of facultative symbionts, such as lichen-forming fungi, from metagenomic reads. However, no study has so far tested accuracy and completeness of assemblies based on metagenomic sequences compared to assemblies based on pure culture strains of lichenized fungi. Here we assembled the genomes of Evernia prunastri and Pseudevernia furfuracea based on metagenomic sequences derived from whole lichen thalli. We extracted fungal contigs using two different taxonomic binning methods, and performed gene prediction on the fungal contig subsets. We then assessed quality and completeness of the metagenome-based assemblies using genome assemblies as reference which are based on pure culture strains of the two fungal species. Our comparison showed that we were able to reconstruct fungal genomes from uncultured lichen thalli, and also cover most of the gene space (86–90%). Metagenome skimming will facilitate genome mining, comparative (phylo)genomics, and population genetics of lichen-forming fungi by circumventing the time-consuming, sometimes unfeasible, step of aposymbiotic cultivation.

Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist-mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad-scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub-species-level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen-forming fungi to broaden their ecological niche. The extent and direction of photobiont-mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub-species-level resolution across the mycobiont’s Europe-wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiontphotobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub-species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiontmediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.

Comparing Sanger sequencing and high-throughput metabarcoding for inferring photobiont diversity in lichens

The implementation of HTS (high-throughput sequencing) approaches is rapidly changing our understanding of the lichen symbiosis, by uncovering high bacterial and fungal diversity, which is often host-specific. Recently, HTS methods revealed the presence of multiple photobionts inside a single thallus in several lichen species. This differs from Sanger technology, which typically yields a single, unambiguous algal sequence per individual. Here we compared HTS and Sanger methods for estimating the diversity of green algal symbionts within lichen thalli using 240 lichen individuals belonging to two species of lichen-forming fungi. According to HTS data, Sanger technology consistently yielded the most abundant photobiont sequence in the sample. However, if the second most abundant photobiont exceeded 30% of the total HTS reads in a sample, Sanger sequencing generally failed. Our results suggest that most lichen individuals in the two analyzed species, Lasallia hispanica and L. pustulata, indeed contain a single, predominant green algal photobiont. We conclude that Sanger sequencing is a valid approach to detect the dominant photobionts in lichen individuals and populations. We discuss which research areas in lichen ecology and evolution will continue to benefit from Sanger sequencing, and which areas will profit from HTS approaches to assessing symbiont diversity.

Circumscription of the genus Lepra, a recently resurrected genus to accommodate the "Variolaria"-group of Pertusaria sensu lato (Pertusariales, Ascomycota)

Pertusarialean lichens include more than 300 species belonging to several independent phylogenetic lineages. Only some of these phylogenetic clades have been comprehensively sampled for molecular data, and formally described as genera. Here we present a taxonomic treatment of a group of pertusarialean lichens formerly known as “Pertusaria amara-group”, “Monomurata-group”, or “Variolaria-group”, which includes widespread and well-known taxa such as P. amara, P. albescens, or P. ophthalmiza. We generated a 6-locus data set with 79 OTUs representing 75 species. The distinction of the Variolaria clade is supported and consequently, the resurrection of the genus Lepra is followed. Thirty-five new combinations into Lepra are proposed and the new species Lepra austropacifica is described from mangroves in the South Pacific. Lepra is circumscribed to include species with disciform ascomata, a weakly to non-amyloid hymenial gel, strongly amyloid asci without clear apical amyloid structures, containing 1 or 2, single-layered, thin-walled ascospores. Chlorinated xanthones are not present, but thamnolic and picrolichenic acids occur frequently, as well as orcinol depsides. Seventy-one species are accepted in the genus. Although the distinction of the genus from Pertusaria is strongly supported, the relationships of Lepra remain unresolved and the genus is tentatively placed in Pertusariales incertae sedis.