Paresordin A, a new diphenyl cyclic peroxide from the lichen Parmotrema praesorediosum.

From the lichen Parmotrema praesorediosum, one new diphenyl peroxide, named praesordin A (1), together with four depsidones, including virensic acid (2), protocetraric acid (3), 8'-O-methylprotocetraric acid (4), and furfuric acid (5) were purified. Their structures were chacracterized using extensive HR-ESI-MS and NMR spectroscopic methods. The isolated compounds (2-5) possessed stronger α-glucosidase inhibitory activity (IC50 = 43.7-110.1 µM) than the standard drug acarbose (IC50 = 214.5 µM).

Phylogeographic reconstructions can be biased by ancestral shared alleles: The case of the polymorphic lichen Bryoria fuscescens in Europe and North Africa.

Large phylogeographic studies on lichens are scarce, and none involves a single species within which different lineages show fixed alternative dispersal strategies. We investigated Bryoria fuscescens (including B. capillaris) in Europe and western North Africa by phenotypically characterizing 1400 specimens from 64 populations and genotyping them with 14 microsatellites. We studied population structure and genetic diversity at the local and continental scales, discussed the post-glacial phylogeography, and compared dispersal capacities of phenotypes with and without soralia. Our main hypothesis is that the estimated phylogeography, migration routes, and dispersal capacities may be strongly biased by ancestral shared alleles. Scandinavia is genetically the richest area, followed by the Iberian Peninsula, the Carpathians, and the Alps. Three gene pools were detected: two partially linked to phenotypic characteristics, and the third one genetically related to the American sister species B. pseudofuscescens. The comparison of one gene pool producing soredia and one not, suggested both as panmictic, with similar levels of isolation by distance (IBD). The migration routes were estimated to span from north to south, in disagreement with the assessed glacial refugia. The presence of ancestral shared alleles in distant populations can explain the similar IBD levels found in both gene pools while producing a false signal of panmixia, and also biasing the phylogeographic reconstruction. The incomplete lineage sorting recorded for DNA sequence loci also supports this hypothesis. Consequently, the high diversity in Scandinavia may rather come from recent immigration into northern populations than from an in situ diversification. Similar patterns of ancestral shared polymorphism may bias the phylogeographical reconstruction of other lichen species.

Comprehensive Analysis of Secondary Metabolites in Usnea longissima (Lichenized Ascomycetes, Parmeliaceae) Using UPLC-ESI-QTOF-MS/MS and Pro-Apoptotic Activity of Barbatic Acid.

Considering the importance of ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS) hyphenated techniques for analysis of secondary metabolites from crude extracts, the present study was aimed at identification of secondary metabolites in acetone extract of the lichen Usnea longissima. From our study, 19 compounds were tentatively identified through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and comparison with literature data. In addition, potent cytotoxic activity of U. longissima extract prompted us to isolate four compounds, 18R-hydroxy-dihydroalloprotolichesterinic acid (19), neuropogolic acid (20), barbatic acid (21), and usnic acid (22) from this extract which were adequately identified through mass spectrometry and NMR spectroscopy. All four compounds displayed cytotoxic activity. Barbatic acid (21) manifested doxorubicin equivalent activity against A549 lung cancer cell line with IC50 of 1.78 µM and strong G0/G1 accumulation of cells. Poly ADP-ribose polymerase (PARP) cleavage confirmed that it induced cytotoxic activity via apoptosis. Finally, our work has discerned the depside, barbatic acid (21) from crude extract as a candidate anti-cancer molecule, which induces cell death by stepping up apoptosis.

Evolution of complex symbiotic relationships in a morphologically derived family of lichen-forming fungi.

We studied the evolutionary history of the Parmeliaceae (Lecanoromycetes, Ascomycota), one of the largest families of lichen-forming fungi with complex and variable morphologies, also including several lichenicolous fungi. We assembled a six-locus data set including nuclear, mitochondrial and low-copy protein-coding genes from 293 operational taxonomic units (OTUs). The lichenicolous lifestyle originated independently three times in lichenized ancestors within Parmeliaceae, and a new generic name is introduced for one of these fungi. In all cases, the independent origins occurred c. 24 million yr ago. Further, we show that the Paleocene, Eocene and Oligocene were key periods when diversification of major lineages within Parmeliaceae occurred, with subsequent radiations occurring primarily during the Oligocene and Miocene. Our phylogenetic hypothesis supports the independent origin of lichenicolous fungi associated with climatic shifts at the Oligocene-Miocene boundary. Moreover, diversification bursts at different times may be crucial factors driving the diversification of Parmeliaceae. Additionally, our study provides novel insight into evolutionary relationships in this large and diverse family of lichen-forming ascomycetes.

Species delimitation in the lichenized fungal genus Vulpicida (Parmeliaceae, Ascomycota) using gene concatenation and coalescent-based species tree approaches.

UNLABELLED: • PREMISE OF THE STUDY: Species boundaries in many organism groups are still in a state of flux, and for empirical species delimitation, finding appropriate character sets and analytical tools are among the greatest challenges. In the lichenized fungal genus Vulpicida, six morphologically circumscribed species have been distinguished, but phenotypic characters partly overlap for three of these and intermediate forms occur. We used a combination of phylogenetic strategies to delimit the species in this genus.• METHODS: Five DNA loci were sequenced and analyzed. Single-locus gene trees and a five-locus concatenated phylogeny were constructed to assess current Vulpicida species. Species boundaries were inferred from molecular data using two coalescent-based species delimitation methods (BP&P and Brownie) and from species trees reconstructed with three different algorithms (*BEAST, BEST, and STEM).• KEY RESULTS: The two species restricted to North America, Vulpicida canadensis and V. viridis, are clearly distinct in all analyses. The four other traditionally accepted species form two strongly supported, closely related species-level lineages within the core group of the genus. On the basis of these results, we propose four instead of the current six species in the genus: V. canadensis, V. juniperinus, V. pinastri, and V. viridis, while V. tilesii and V. tubulosus are reduced to synonymy under V. juniperinus.• CONCLUSIONS: Coalescent species delimitation and tree inference give consistent results for fully distinct Vulpicida species but not for diverging populations. Even the inconsistent results were informative, revealing developing isolation despite a complex history of recombination and incomplete lineage sorting.

The First Miniature, Small Foliose, Brown Xanthoparmelia in the Northern Hemisphere.

The genus Xanthoparmelia includes several subcrustose, squamulose, small foliose, and small subfruticose species, primarily in the Southern Hemisphere. Here, we report on the first small foliose species lacking usnic acid in the genus occurring in the Holarctic. The species has been previously known as Lecanora olivascens Nyl., but subsequent studies of the morphology, secondary chemistry, and molecular data of the nuITS rDNA indicate that this species instead belongs to Xanthoparmelia. Consequently, the new combination Xanthoparmelia olivascens (Nyl.) V.J. Rico and G. Amo is proposed, and an epitype is designated here. We discuss the unique presence of a subcrustose Xanthoparmelia species lacking cortical usnic acid in the Northern Hemisphere. This species fits phylogenetically into a clade that was previously only known from the Southern Hemisphere, and hence represents another example of N-S disjunction in lichenized fungi.

Liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry characterization of depsides and depsidones from the Chilean lichen Parmotrema perlatum.

Lichens are recognized by their unique compounds and diverse applications in food, medicines, and cosmetics. Using ultra-high pressure liquid chromatography, coupled with a high-resolution mass spectrometer, metabolomic profiling of the lichen Parmotrema perlatum, from a methanolic extract, was performed. Based on characteristic fragmentation patterns, twenty-five lichenic substances were tentatively identified including 5 depsides, 12 depsidones, 2 diphenyl ethers, 1 aromatic considered as possible artifact, 1 dibenzofuran, 1 carbohydrate, 1 organic acid, and 2 undefined compounds. To the best of our knowledge, this is a more complete report of their phytochemistry from P perlatum. Our findings of the P perlatum profile may contribute and complement the current data of the Parmotrema genus.

Tinctoric acid A-B, two new hopan-type triterpenoids from the Vietnamese lichen, Parmotrema tinctorum (Despr. ex Nyl.) hale with α-glucosidase inhibitory activity.

Two new hopan-type triterpenoids, namely tinctoric acid A-B (1-2), were isolated from the lichen Parmotrema tinctorum (Despr. ex Nyl.) Hale. Their structures were elucidated by extensive spectroscopic analyses (1D and 2D NMR). The absolute configuration at C-22 of 1 was established through DP4 probability. Compounds 1-2 were evaluated for their inhibitory activity against α-glucosidase and found to be more potent than those of positive control (acarbose, IC50 168 µM) with values IC50 74.7 and 98.2 µM, respectively. Both of these compounds interacted well with enzyme α-glucosidase MAL32 through H-bonds and hydrophobic interaction.

Mycobiont-specific primers facilitate the amplification of mitochondrial small subunit ribosomal DNA: a focus on the lichenized fungal genus Melanelia (Ascomycota, Parmeliaceae) in Iceland.

The fungal mitochondrial small subunit (mtSSU) ribosomal DNA is one of the most commonly used loci for phylogenetic analysis of lichen-forming fungi, but their primer specificity to mycobionts has not been evaluated. The current study aimed to design mycobiont-specific mtSSU primers and highlights their utility with an example from the saxicolous lichen-forming fungal genus Melanelia Essl. in Iceland. The study found a 12.5% success rate (3 out of 24 specimens with good-quality mycobiont mtSSU sequences) using universal primers (i.e. mrSSU1 and mrSSU3R), not including off-target amplification of environmental fungi, e.g. Cladophialophoracarrionii and Lichenotheliaconvexa. New mycobiont-specific primers (mt-SSU-581-5' and mt-SSU-1345-3') were designed by targeting mycobiont-specific nucleotide sites in comparison with environmental fungal sequences, and assessed for mycobiont primer specificity using in silico PCR. The new mycobiont-specific mtSSU primers had a success rate of 91.7% (22 out of 24 specimens with good-quality mycobiont mtSSU sequences) on the studied Melanelia specimens. Additional testing confirmed the specificity and yielded amplicons from 79 specimens of other Parmeliaceae mycobiont lineages. This study highlights the effectiveness of designing mycobiont-specific primers for studies on lichen identification, barcoding and phylogenetics.

Parmoferone A, a new depsidone from the lichen Parmotrema cristiferum.

A new depsidone, parmoferone A (1), together with three known compounds, parmosidone K (2), albifolione (3), and 4-chloroorcinol (4) were isolated from the lichen Parmotrema cristiferum (Taylor) Hale (Parmeliaceae). The structures of isolated compounds were identified from its spectroscopic data and by comparison with the literature. Compounds 1-4 were evaluated for alpha-glucosidase inhibition. Compound 1 was determined to be a potent non-competitive inhibitor against alpha-glucosidase with an IC50 value of 18.1 µM.

Assessment of Cytotoxic/Antitumour Potential and in silico Study of Salazinic Acid Isolated from Parmotrema concurrens.

INTRODUCTION: Despite numerous scientific advances, cancer continues to be one of the main causes of death in the world. This situation has driven the search for promising molecules. Lichen substances have been widely described for their pharmacological potential. OBJECTIVE: The present study evaluated the antitumour potential of a depsidone isolated from Parmotrema concurrens- salazinic acid (SAL) - through in vitro, in vivo and in silico studies. METHODS: The molecule was isolated from the acetonic extract of the lichen and recrystallized in acetone. The macrophage J774, sarcoma-180 and MDA-MB-231 cell lines were used for the MTT cytotoxicity assay. The antitumor assay used a murine model (Swiss albino mice) with sarcoma-180. The animals were treated for seven consecutive days with doses of SAL (25 and 50 mg/kg) and 5-fluorouracil (20 mg/kg). RESULTS: Its purity was determined using high-performance liquid chromatography (94%), and its structure was confirmed by H1 and C13 nuclear magnetic resonance. SAL was not considered toxic to cancer cell lines, showing cell viability rates of 79.49 ± 4.15% and 86.88 ± 1.02% for sarcoma-180 and MDA-MB-231, respectively. The tumour inhibition rate was greater than 80% in the animals treated with SAL and 65% for those that received 5-fluorouracil. Simulations of molecular dynamics to estimate the flexibility of the interactions between human thymidylate synthase and derivatives of SAL and 5-fluorouracil revealed that SAL exhibited greater enzymatic interaction capacity, with highly favourable energy, compared to 5-fluorouracil. CONCLUSION: The present results demonstrate the potential of salazinic acid as a tumour inhibition agent.

Tinctorin, a new spiroterpenoid from the lichen Parmotrema tinctorum (Despr. ex Nyl.) Hale growing in Vietnam.

A new spiroterpenoid, namely tinctorin (1), along with one known compound, norreticulatin (2), were isolated from the lichen Parmotrema tinctorum (Despr. ex Nyl.) Hale. Their structures were elucidated by extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations. The absolute configuration of 2 was established for the first time. Compound 1 was evaluated for its inhibitory activity against α-glucosidase and found to be inactive.

Biosynthetic gene cluster synteny: Orthologous polyketide synthases in Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata.

Lichens are symbiotic associations consisting of a photobiont (algae or cyanobacteria) and a mycobiont (fungus), which together generate a variety of unique secondary metabolites. To access this biosynthetic potential for biotechnological applications, deeper insights into the biosynthetic pathways and corresponding gene clusters are necessary. Here, we provide a comparative view of the biosynthetic gene clusters of three lichen mycobionts derived from Hypogymnia physodes, Hypogymnia tubulosa, and Parmelia sulcata. In addition, we present a high-quality PacBio metagenome of Parmelia sulcata, from which we extracted the mycobiont bin containing 214 biosynthetic gene clusters. Most biosynthetic gene clusters in these genomes were associated with T1PKSs, followed by NRPSs and terpenes. This study focused on biosynthetic gene clusters related to polyketide synthesis. Based on ketosynthase homology, we identified nine highly syntenic clusters present in all three species. Among the four clusters belonging to nonreducing PKSs, two are putatively linked to lichen substances derived from orsellinic acid (orcinol depsides and depsidones, e.g., lecanoric acid, physodic acid, lobaric acid), one to compounds derived from methylated forms of orsellinic acid (beta orcinol depsides, e.g., atranorin), and one to melanins. Five clusters with orthologs in all three species are linked to reducing PKSs. Our study contributes to sorting and dereplicating the vast PKS diversity found in lichenized fungi. High-quality sequences of biosynthetic gene clusters of these three common species provide a foundation for further exploration into biotechnological applications and the molecular evolution of lichen substances.

Phytochemical composition, biological activity and anti-inflammatory potential of acetone extract from the lichen Platismatia glauca (L.) W.L. Culb. & C.F. Culb.

This investigation examined the antioxidant, antimicrobial, cytotoxic, and anti-inflammatory activities of the acetone extract of the lichen Platismatia glauca (L.) W.L. Culb. & C.F. Culb. (PGAE). The phytochemical study of PGAE showed presence of seven compounds: salazinic acid, ß-orcinol carboxylic acid, 3-hydroxyphysodalic acid, physodalic acid, physodic acid, atranorin, and chloroatranorin. The antimicrobial potential was determined by microdilution which showed that S. aureus was most sensitive to the effect of PGAE with MIC value 0.312 mg/ml. Antioxidant activity was evaluated by using DPPH method. The obtained IC50 value for PGAE was 194.30 ± 3.32 µg/ml. The cytotoxic effect of the extract was evaluated by MTT test and the strongest activity was towards human epithelial carcinoma cells with IC50 value of 59.10 ± 0.46 µg/ml. The findings revealed that the application of lichen extracts decreased the paw edoema in a dose-dependent manner at 1, 2, 3, and 4 h following carrageenan administration.

Three new diphenyl ethers from the lichen Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae).

Three new diphenyl ethers, named praesorethers E, F and G (1, 2 and 3), were isolated from the lichen Parmotrema praesorediosum. Their chemical structures were elucidated on the basis of extensively spectroscopic analysis including HR-ESI-MS and NMR as well as comparison with previously published data. These compounds were evaluated for the cytotoxicity against three human cancer cell lines (HeLa, NCI-H460 and MCF-7) using SRB assay. As results, 1 and 2 exhibited weak cytotoxic activity against three tested cancer cell lines with the inhibitive percentage of 64-79.9% at the concentration of 100 µg/mL while 3 was inactive.

Two new phenolic compounds from the lichen Parmotrema cristiferum growing in Vietnam.

Two new phenolic compounds, cristiferides A-B (1-2) together with six known compounds, 2,4-dihydroxyphthalide (3), lecanoric acid (4), orsellinic acid (5), 5-chloroorsellinic acid (6), methyl haematommate (7), and methyl ß-orsellinate (8) were isolated from the lichen Parmotrema cristiferum (Taylor) Hale (Parmeliaceae). The structures of isolated compounds were identified from its spectroscopic data and by comparison with the literatures. Compounds 1-3 and 6-8 were evaluated for alpha-glucosidase inhibition. Compounds 2 and 7 revealed potent activity with IC50 values of 72.66 µM and 48.73 µM, respectively.

Reticulatin, a novel C43-spiroterpenoid from the lichen Parmotrema reticulatum growing in Vietnam.

A novel C43-spiroterpenoid, reticulatin (1), was isolated from the lichen Parmotrema reticulatum (Taylor) M. Choisy (Parmeliaceae). Five previously-reported compounds were also isolated: zeorin (2), leucotylin (3), lupeol (4), betulinic acid (5), and dihydroreynosin (6). The structures were elucidated by 1D, 2D NMR, and HRESIMS spectroscopy and comparison with the literature. We propose that reticulatin is a biosynthetic product of fusicoccadiene and vinapraesorediosic acid A via Diels-Alder addition. Reticulatin is the first C43-spiroterpenoid identified from lichen metabolites. All compounds were evaluated for inhibition of α-glucosidase. Compound 1 showed the most potent inhibition, with an IC50 value of 3.90 µM, much lower than that of the acarbose positive control (IC50 165 µM).

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Infraspecific variation of some brown Parmeliae (in Poland) - a comparison of ITS rDNA and non-molecular characters.

Infraspecific variation of the ITS rDNA region of some brown Parmeliae occurring in Poland is studied and compared with non-molecular characters. Haplotype networks are used to illustrate the variability within the species. Both newly-produced sequences from Central Europe and from all over the world, downloaded from the GenBank, are used. The number of haplotypes found for each taxon ranged from five in Melaneliastygia to 12 in Melaneliahepatizon and Montaneliadisjuncta; however, their numbers correlate with the number of specimens tested. New haplotypes for Melaneliaagnata, M.hepatizon and Cetrariacommixta are found. Based on our 169-sample dataset, we could not infer any geographical correlation, either locally or world-wide. Many of the analysed haplotypes were widely distributed and the same haplotype was often shared between temperate and polar populations. A comparison of molecular, morphological, anatomical and chemical characters also shows no correlation.

Genome-Wide Analysis of Biosynthetic Gene Cluster Reveals Correlated Gene Loss with Absence of Usnic Acid in Lichen-Forming Fungi.

Lichen-forming fungi are known to produce a large number of secondary metabolites. Some metabolites are deposited in the cortical layer of the lichen thallus where they exert important ecological functions, such as UV filtering. The fact that closely related lineages of lichen-forming fungi can differ in cortical chemistry suggests that natural product biosynthesis in lichens can evolve independent from phylogenetic constraints. Usnic acid is one of the major cortical pigments in lichens. Here we used a comparative genomic approach on 46 lichen-forming fungal species of the Lecanoromycetes to elucidate the biosynthetic gene content and evolution of the gene cluster putatively responsible for the biosynthesis of usnic acid. Whole-genome sequences were gathered from taxa belonging to different orders and families of Lecanoromycetes, where Parmeliaceae is the most well-represented taxon, and analyzed with a variety of genomic tools. The highest number of biosynthetic gene clusters was found in Evernia prunastri, Pannoparmelia angustata, and Parmotrema austrosinense, respectively, and lowest in Canoparmelia nairobiensis, Bulbothrix sensibilis, and Hypotrachyna scytodes. We found that all studied species producing usnic acid contain the putative usnic acid biosynthetic gene cluster, whereas the cluster was absent in all genomes of species lacking usnic acid. The absence of the gene cluster was supported by an additional unsuccessful search for ß-ketoacylsynthase, the most conserved domain of the gene cluster, in the genomes of species lacking usnic acid. The domain architecture of this PKS cluster-homologous to the already known usnic acid PKS cluster (MPAS) and CYT450 (MPAO)-varies within the studied species, whereas the gene arrangement is highly similar in closely related taxa. We hypothesize that the ancestor of these lichen-forming fungi contained the putative usnic acid producing PKS cluster and that the gene cluster was lost repeatedly during the evolution of these groups. Our study provides insight into the genomic adaptations to the evolutionary success of these lichen-forming fungal species and sets a baseline for further exploration of biosynthetic gene content and its evolutionary significance.