Low genetic differentiation between apotheciate Usnea florida and sorediate Usnea subfloridana (Parmeliaceae, Ascomycota) based on microsatellite data.

Accurate species delimitation has a pivotal role in conservation biology, and it is especially important for threatened species where decisions have political and economic consequences. Finding and applying appropriate character sets and analytical tools to resolve interspecific relationships remains challenging in lichenized fungi. The main aim of our study was to re-assess the species boundaries between Usnea subfloridana and Usnea florida, which have been phylogenetically indistinguishable until now, but are different in reproductive mode and ecological preferences, using fungal-specific simple sequence repeats (SSR), i.e. microsatellite markers. Bayesian clustering analysis, discriminant analysis of principal components (DAPC), minimal spanning network (MSN), and principal component analysis (PCA) failed to separate U. florida and U. subfloridana populations. However, a low significant differentiation between the two taxa was observed across all populations according to AMOVA results. Also, analysis of shared haplotypes and statistical difference in clonal diversity (M) supported the present-day isolation between the apotheciate U. florida and predominantly sorediate U. subfloridana. Our results do not provide a clear support either for the separation of species in this pair or the synonymization of U. florida and U. subfloridana. We suggest that genome-wide data could help resolve the taxonomic question in this species pair.

New insights into the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota): Molecular analysis reveals high genetic diversity correlated with chemistry.

Biological processes such as hybridization, incomplete lineage sorting and gene flow can obscure the recognition of distinct evolutionary lineages, particularly in groups of organisms that have recently diverged. Therefore, compiling pieces of evidence from diverse data sources is critical to accurately assess species boundaries in such groups. The increasing availability of DNA sequence data allows for a much deeper understanding of diversification and speciation processes and their consequences on biodiversity. In this study, we applied an integrative approach based on DNA sequence, chemical, geographic and morphological data to attempt to define species boundaries in the lichen-forming genus Usnea (Parmeliaceae), particularly the U. cornuta aggregate, a cosmopolitan species group. We provide the first species delimitation for this group in the neotropics based on the multispecies coalescent (MSC) model. Using ITS rDNA and two protein-coding genes, Mcm7 and RPB1, we estimated the species tree under the MSC model in a Bayesian framework using STACEY. Our results indicate that at least nine strongly supported distinct lineages coexist in the U. cornuta aggregate, which are well chemically characterized. Additionally, we found evidence for the polyphyly of three morphospecies, Usnea brasiliensis, U. cornuta and U. dasaea.

The use of microsatellite markers for species delimitation in Antarctic Usnea subgenus Neuropogon.

Lichens are symbiotic associations consisting of a fungal (mycobiont) and one or more photosynthetic (photobionts) partners and are the dominant component, and most important primary producers, of Antarctic terrestrial ecosystems. The most common lichens in the maritime Antarctic are Usnea antarctica and U. aurantiacoatra, a so-called "species pair" in which U. antarctica shows asexual reproduction and propagation via soredia and U. aurantiacoatra forms ascospores in apothecia. Previous molecular analyses were not able to unambiguously distinguish the two morphotypes as species. Therefore, the goal of this study was to find out whether fast-evolving SSR (single sequence repeat) markers are able to separate morphotypes more clearly and help to clarify their taxonomy. We investigate 190 individuals from five mixed stands of both morphotypes collected in King George Island and Elephant Island (South Shetland Islands, Antarctica). Based on 23 microsatellite markers designed from sequenced genomes, discriminant analysis of principal components (DAPC), Bayesian clustering analysis, and coalescent-based estimation of gene flow show clear evidence for the existence of two different species distinguishable by reproductive mode. We did not detect any statistical association between genetic clusters and three previously reported chemical races of each species.

Unconstrained gene flow between populations of a widespread epiphytic lichen Usnea subfloridana (Parmeliaceae, Ascomycota) in Estonia.

Few studies have investigated the genetic diversity of populations of common and widespread lichenized fungi using microsatellite markers, especially the relationships between different measures of genetic diversity and environmental heterogeneity. The main aim of our study was to investigate the population genetics of a widespread and mainly clonally reproducing Usnea subfloridana at the landscape scale, focusing on the comparison of lichen populations within hemiboreal forest stands. Particular attention has been paid to the genetic differentiation of lichen populations in two geographically distinct regions in Estonia and the relationships between forest characteristics and measures of genetic diversity. We genotyped 578 Usnea thalli from eleven lichen populations using seven specific fungal microsatellite markers. Measures of genetic diversity (allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of multilocus genotypes, the number of private alleles, and the minimum number of colonization events) were calculated and compared between Usnea populations. Shared haplotypes, gene flow and AMOVA analyses suggest that unconstrained gene flow and exchange of multilocus genotypes exist between the two geographically remote regions in Estonia. Stand age, mean circumference of the host tree, size of forest site and tree species composition did not show any significant influence on allelic richness, Shannon's information index, Nei's unbiased genetic diversity, clonal diversity, the number of private alleles, and the minimum number of colonization events of U. subfloridana populations. Therefore it was concluded that other factors of habitat heterogeneity could probably have a more significant effect on population genetics of U. subfloridana populations.

From GenBank to GBIF: Phylogeny-Based Predictive Niche Modeling Tests Accuracy of Taxonomic Identifications in Large Occurrence Data Repositories.

Accuracy of taxonomic identifications is crucial to data quality in online repositories of species occurrence data, such as the Global Biodiversity Information Facility (GBIF), which have accumulated several hundred million records over the past 15 years. These data serve as basis for large scale analyses of macroecological and biogeographic patterns and to document environmental changes over time. However, taxonomic identifications are often unreliable, especially for non-vascular plants and fungi including lichens, which may lack critical revisions of voucher specimens. Due to the scale of the problem, restudy of millions of collections is unrealistic and other strategies are needed. Here we propose to use verified, georeferenced occurrence data of a given species to apply predictive niche modeling that can then be used to evaluate unverified occurrences of that species. Selecting the charismatic lichen fungus, Usnea longissima, as a case study, we used georeferenced occurrence records based on sequenced specimens to model its predicted niche. Our results suggest that the target species is largely restricted to a narrow range of boreal and temperate forest in the Northern Hemisphere and that occurrence records in GBIF from tropical regions and the Southern Hemisphere do not represent this taxon, a prediction tested by comparison with taxonomic revisions of Usnea for these regions. As a novel approach, we employed Principal Component Analysis on the environmental grid data used for predictive modeling to visualize potential ecogeographical barriers for the target species; we found that tropical regions conform a strong barrier, explaining why potential niches in the Southern Hemisphere were not colonized by Usnea longissima and instead by morphologically similar species. This approach is an example of how data from two of the most important biodiversity repositories, GenBank and GBIF, can be effectively combined to remotely address the problem of inaccuracy of taxonomic identifications in occurrence data repositories and to provide a filtering mechanism which can considerably reduce the number of voucher specimens that need critical revision, in this case from 4,672 to about 100.

Lichensphere: a protected natural microhabitat of the non-lichenised fungal communities living in extreme environments of Antarctica.

We surveyed the diversity, distribution and ecology of non-lichenised fungal communities associated with the Antarctic lichens Usnea antarctica and Usnea aurantiaco-atra across Antarctica. The phylogenetic study of the 438 fungi isolates identified 74 taxa from 21 genera of Ascomycota, Basidiomycota and Zygomycota. The most abundant taxa were Pseudogymnoascus sp., Thelebolus sp., Antarctomyces psychrotrophicus and Cryptococcus victoriae, which are considered endemic and/or highly adapted to Antarctica. Thirty-five fungi may represent new and/or endemic species. The fungal communities displayed high diversity, richness and dominance indices; however, the similarity among the communities was variable. After discovering rich and diverse fungal communities composed of symbionts, decomposers, parasites and endemic and cold-adapted cosmopolitan taxa, we introduced the term "lichensphere". We hypothesised that the lichensphere may represent a protected natural microhabitat with favourable conditions able to help non-lichenised fungi and other Antarctic life forms survive and disperse in the extreme environments of Antarctica.

Host switching promotes diversity in host-specialized mycoparasitic fungi: uncoupled evolution in the Biatoropsis-usnea system.

Fungal mycoparasitism-fungi parasitizing other fungi-is a common lifestyle in some basal lineages of the basidiomycetes, particularly within the Tremellales. Relatively nonaggressive mycoparasitic fungi of this group are in general highly host specific, suggesting cospeciation as a plausible speciation mode in these associations. Species delimitation in the Tremellales is often challenging because morphological characters are scant. Host specificity is therefore a great aid to discriminate between species but appropriate species delimitation methods that account for actual diversity are needed to identify both specialist and generalist taxa and avoid inflating or underestimating diversity. We use the Biatoropsis-Usnea system to study factors inducing parasite diversification. We employ morphological, ecological, and molecular data-methods including genealogical concordance phylogenetic species recognition (GCPSR) and the general mixed Yule-coalescent (GMYC) model-to assess the diversity of fungi currently assigned to Biatoropsis usnearum. The degree of cospeciation in this association is assessed with two cophylogeny analysis tools (ParaFit and Jane 4.0). Biatoropsis constitutes a species complex formed by at least seven different independent lineages and host switching is a prominent force driving speciation, particularly in host specialists. Combining ITS and nLSU is recommended as barcode system in tremellalean fungi.

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima.

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with ß-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.

DNA barcoding of lichenized fungi demonstrates high identification success in a floristic context.

• Efforts are currently underway to establish a standard DNA barcode region for fungi; we tested the utility of the internal transcribed spacer (ITS) of nuclear ribosomal DNA for DNA barcoding in lichen-forming fungi by sampling diverse species across eight orders. • Amplification of the ITS region (ITS1-5.8S-ITS2) was conducted for 351 samples, encompassing 107, 55 and 28 species, genera and families, respectively, of lichenized fungi. We assessed the ability of the entire ITS vs the ITS2 alone to discriminate between species in a taxonomic dataset (members of the genus Usnea) and a floristic dataset. • In the floristic dataset, 96.3% of sequenced samples could be assigned to the correct species using ITS or ITS2; a barcode gap for ITS is present in 92.1% of species. Although fewer species have a barcode gap in the taxonomic dataset (73.3% with ITS and 68.8% with ITS2), up to 94.1% of samples were assigned to the correct species using BLAST. • While discrimination between the most closely related species will remain challenging, our results demonstrate the potential to identify a high percentage of specimens to the correct species, and the remainder to the correct genus, when using DNA barcoding in a floristic context.

The delimitation of Antarctic and bipolar species of neuropogonoid Usnea (Ascomycota, Lecanorales): a cohesion approach of species recognition for the Usnea perpusilla complex.

Species of the Neuropogon group in the lichen genus Usnea have their centre of distribution in polar regions of the Southern Hemisphere. Their morphological and chemical variability is poorly understood and several asexual taxa with uncertain relationships to fertile taxa occur in the group. The species concept is controversial. A phylogenetic analysis revealed three related complexes of mainly asexual lineages arranged around three fertile Usnea species: U. aurantiaco-atra, U. trachycarpa and U. perpusilla. In this study a dataset of 80 specimens was used to resolve species circumscriptions in the U. perpusilla complex. We used a phylogenetic and a haplotype network approach based on three gene fragments (ITS, IGS and RPB1) to detect distinct lineages. To support the hypothesis that these lineages represent different species, we tested for correlation of morphological and chemical characters with hierarchical nested haplotype groups, employing statistical tests of contingency tables and analysis of variance (ANOVA). This cohesion species recognition method detected three fertile U. perpusilla lineages. We could also delimit an undescribed fertile species with yellow apothecia and a new asexual species from the High Andes. Interestingly, there is an additional bipolar species, U. lambii, which was formerly confused with U. sphacelata. The fact that U. lambii shows a geographically disjunct distribution pattern, but the genetic distances among specimens are low, points to recent long-distance dispersal.