Antioxidant and cytotoxic activities of quinones from Cetraria laevigata.

Chemical investigation of the extracts obtained from the red thallus tips from Cetraria laevigata resulted in the isolation of five known quinoid pigments identified by FT-IR, UV, NMR, MS methods and by comparison with literature data (skyrin (1), 3-ethyl-2,7-dihydroxynaphthazarin (2), graciliformin (3), cuculoquinone (4) and islandoquinone (5)). An antioxidant capacity of compounds 1-5 were evaluated and compared with quercetin using a lipid peroxidation inhibitory assay and superoxide radical (SOR), nitric oxide radical (NOR), 1,1-diphenyl-2-picrylhydrazine (DPPH), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) scavenging assays. Compounds 2, 4 and 5 were far more active: they demonstrated the antioxidant capacity in various test assays with the IC50 values 5-409 µM comparable to the flavonoid quercetin. While, the isolated quinones (1-5) exhibited weak cytotoxicity in human cancer cell line A549 assessed by MTT assay.

Anti-Influenza Activity of Cetraria islandica Lichen Extracts in In Vitro Experiments.

The toxicity and antiviral activity of extracts obtained by the methods of aqueous and ethanol extraction of bioactive substances from Cetraria islandica lichen as a raw material were studied. Aqueous and ethanol extracts of lichen were characterized by low toxicity with respect to the passaged MDCK cell culture and exhibited antiviral activity. The ethanol extract showed more potent in vitro antiviral activity against human A/H3N2 and avian A/H5N1 influenza viruses: in a concentration of 50 µg/ml, it suppressed replication of these viruses by 3.5 and 4 log10, respectively, while the aqueous extract inhibited replication of viruses by 2 and 6 log10, respectively, when taken in a concentration of 500 µg/ml that was 10-fold higher than the concentration of the ethanol extract.

Ultrastructural, Energy-Dispersive X-ray Spectroscopy, Chemical Study and LC-DAD-QToF Chemical Characterization of Cetraria islandica (L.) Ach.

The lichen Cetraria islandica (L.) Ach. has been used in traditional and modern medicines for its many biological properties such as immunological, immunomodulating, antioxidant, antimicrobial, and anti-inflammatory activities. This species is gaining popularity in the market, with interest from many industries for selling as medicines, dietary supplements, and daily herbal drinks. This study profiled the morpho-anatomical features by light, fluorescence, and scanning electron microscopy; conducted an elemental analysis using energy-dispersive X-ray spectroscopy; and phytochemical analysis was performed using high-resolution mass spectrometry combined with a liquid chromatography system (LC-DAD-QToF) of C. islandica. In total, 37 compounds were identified and characterized based on comparisons with the literature data, retention times, and their mass fragmentation mechanism/s. The identified compounds were classified under five different classes, i.e., depsidones, depsides, dibenzofurans, aliphatic acids, and others that contain simple organic acids in majority. Two major compounds (fumaroprotocetraric acid and cetraric acid) were identified in the aqueous ethanolic and ethanolic extracts of C. islandica lichen. This detailed morpho-anatomical, EDS spectroscopy, and the developed LC-DAD-QToF approach for C. islandica will be important for correct species identification and can serve as a useful tool for taxonomical validation and chemical characterization. Additionally, chemical study of the extract of C. islandica led to isolation and structural elucidation of nine compounds, namely cetraric acid (1), 9'-(O-methyl)protocetraric acid (2), usnic acid (3), ergosterol peroxide (4), oleic acid (5), palmitic acid (6), stearic acid (7), sucrose (8), and arabinitol (9).

A new depsidone from the neotricone-rich chemotype of the lichenised fungus Usnea fulvoreagens.

Individuals of Usnea fulvoreagens (Parmeliaceae, lichenised Ascomycota), a shrubby corticolous species that is widespread in Europe, East Asia and North America, produce medullary lichen acids in several distinct chemotypic patterns. One such chemotype reportedly contains an unidentified substance as the major secondary metabolite. We isolated this compound from Californian specimens of U. fulvoreagens and identified it as the rare depsidone neotricone. A co-occurring compound, conneotricone, was identified as 4,10-dihydroxy-5-(hydroxymethyl)-8-methyl-3,7-dioxo-1,3-dihydro-7H-isobenzofuro[4,5-b][1,4]benzodioxepine-11-carboxylic acid by NMR and HPLC-UV-MSn comparison with the material synthesised from salazinic acid.

The Genus Cetraria s. str.-A Review of Its Botany, Phytochemistry, Traditional Uses and Pharmacology.

The genus Cetraria s. str. (Parmeliaceae family, Cetrarioid clade) consists of 15 species of mostly erect brown or greenish yellow fruticose or subfoliose thallus. These Cetraria species have a cosmopolitan distribution, being primarily located in the Northern Hemisphere, in North America and in the Eurasia area. Phytochemical analysis has demonstrated the presence of dibenzofuran derivatives (usnic acid), depsidones (fumarprotocetraric and protocetraric acids) and fatty acids (lichesterinic and protolichesterinic acids). The species of Cetraria, and more particularly Cetraria islandica, has been widely employed in folk medicine for the treatment of digestive and respiratory diseases as decoctions, tinctures, aqueous extract, and infusions. Moreover, Cetraria islandica has had an important nutritional and cosmetic value. These traditional uses have been validated in in vitro and in vivo pharmacological studies. Additionally, new therapeutic activities are being investigated, such as antioxidant, immunomodulatory, cytotoxic, genotoxic and antigenotoxic. Among all Cetraria species, the most investigated by far has been Cetraria islandica, followed by Cetraria pinastri and Cetraria aculeata. The aim of the current review is to update all the knowledge about the genus Cetraria covering aspects that include taxonomy and phylogeny, morphology and distribution, ecological and environmental interest, phytochemistry, traditional uses and pharmacological properties.

Phytochemical Composition of Lichen Parmotrema hypoleucinum (J. Steiner) Hale from Algeria.

In this work, we carried out studies of the chemical composition of hexane, chloroform and ethanol extracts from two samples of the lichen Parmotrema hypoleucinum collected in Algeria. Each sample of the lichen P. hypoleucinum was collected on two different supports: Olea europaea and Quercus coccifera. Hexane extracts were prepared, in Soxhlet; each hexane extract was fractionated by its solubility in methanol; the products soluble in methanol were separated (cold): 1-Hexane, 2-Hexane; and the products insoluble in methanol (cold): 1-Cires, 2-Cires. A diazomethane esterified sample of 1-Hexane, 2-Hexane, 1-Cires and 2-Cires was analyzed by GC-MS, and the components were identified as methyl esters. In the 1-Hexane and 2-Hexane fractions, the methyl esters of the predominant fatty acids in the lichen were identified: palmitic acid, linoleic acid, oleic acid and stearic acid; a hydrocarbon was also identified: 13-methyl-17-norkaur-15-ene and several derivatives of orsellinic acid. In the 1-Cires and 2-Cires fractions, the previous fatty acids were no longer observed, and only the derivatives of orsellinic acid were found. The analysis of the 1-Hexane, 2-Hexane fractions by HPLC-MS/MS allows us to identify different chemical components, and the most characteristic products of the lichen were identified, such as Atranol, Chloroatranol, Atranorin and Chloroatranorin. In the fractions of 1-Cires and 2-Cires, the HPLC-MS/MS analysis reveals that they are very similar in their chemical components; the characteristic products of this lichen in this fraction are Atranorin and Chloroatranorin. In the extracts of chloroform, 1-Chloroform and 2-Chloroform, the analysis carried out by HPLC-MS/MS shows small differences in their chemical composition at the level of secondary products; among the products to be highlighted for this work, we have chloroatranorin, the stictic acid, norstictic acid and other derivatives. In the analysis of the most polar extracts carried out in ethanol: 1-Ethanol and 2-Ethanol, HPLC-MS/MS analysis shows very similar chemical compositions in these two extracts with small differences. In these extracts, the following acids were identified as characteristic compounds of this lichen: constictic acid, stictic acid, substictic acid and methylstictic acid. In the HPLC-MS/MS analysis of all these extracts, alectoronic acid was not found.

Platismatia glauca-Lichen species with suppressive properties on migration and invasiveness of two different colorectal carcinoma cell lines.

Platismatia glauca is a popular lichen traditionally used as a spice and possesses significant anti-cancer potential, whose anti-migratory/anti-invasive properties were mostly disregarded. Migration/invasion of cancer cells is processed in cancer metastasis and targeting their markers is an important strategy in anti-cancer treatment. We examined the anti-migratory/anti-invasive properties of P. glauca extract on two colorectal carcinoma cell lines (HCT-116 and SW-480) and elucidated possible mechanisms underlying these properties. Cell migration was evaluated by wound healing and RTCA methods. Immunofluorescent assay was used for the analysis of protein, while qRT-PCR for gene expression of migratory/invasive markers. ELISA assay was applied for the determination of MMP-9 concentration. P. glauca extract inhibited the motility of tested cells, by reducing pro-migratory/pro-invasive markers and potentially retaining intercellular connections. Treatment showed cell-selective effects, and HCT-116 cells were more responsive. Our study presents important scientific novelty, thus these lichen properties should be furtherly examined regarding the amelioration of anti-cancer treatment. PRACTICAL APPLICATIONS: Based on the evidence we provided in the present study, we have demonstrated that lichen species Platismatia glauca possess important biological activity, which has not been sufficiently investigated so far. It is of great importance to explore its anti-cancer potential, not only from a cytotoxic point of view but especially anti-migratory and anti-invasive. Herein, we showed that this species expresses significant suppressive effects on migration and invasiveness of colorectal carcinoma cells. This tested lichen has the potential to be used as a natural complementary anti-cancer treatment, with special reference on the dose applied and type of carcinoma. Our study represents a significant novelty in the field of scientific investigation of lichens and natural products, and further detailed studies are needed on in vitro and in vivo model systems.

A new depsidone from the lichen Usnea ceratina Arch.

Chemical investigation of the lichen Usnea ceratina Arch led to the isolation of five depsidones, including one new compound ceratinalone (1) along with four known compounds bailesidone (2), stictic acid (3), 8'-O-methylstictic acid (4) and 8'-O-ethylstictic acid (5). The structures were determined by analysis of their MS and NMR data as well as by comparison with literature values. Compounds 1 and 4 were evaluated the cytotoxic activity against HeLa (human epithelial carcinoma), NCI-H460 (human lung cancer), HepG2 (liver hepatocellular carcinoma), and MCF-7 (human breast cancer) cell lines, showing the moderate activity.

Usneaceratins A and B, two new secondary metabolites from the lichen Usnea ceratina.

Two new compounds, comprising one dibenzofuran, named usneaceratin A (1), and one phenolic acid, named usneaceratin B (2), together with one known dibenzofuran, isousnic acid (3), and two known phenolics, orsellinic acid (4) and methyl orsellinate (5) were clarified from the lichen Usnea ceratina using variously chromatographic methods. Their structures were testified by comprehensive HR-ESI-MS, and NMR spectroscopic analysis, and comparison with published data. Their α-glucosidase inhibitory activity of all compounds was measured. Usneaceratin B (2) possessed better inhibition against α-glucosidase enzyme (IC50 value of 41.8 µM) than the standard drug acarbose (IC50 value of 214.50 µM).

Potential human health risks of toxic/harmful elements by consumption of Pseudevernia furfuracea.

The potential human health risks of some toxic/harmful elements related to the consumption of Pseudevernia furfuracea (L.) Zopf. were investigated. The toxic/harmful elements (cadmium (Cd), chromium (Cr), copper (Cu), lead (Pb), manganese (Mn), nickel (Ni), and zinc (Zn)) were determined in P. furfuracea. According to the analysis result, the maximum (max.) toxic/harmful element value was 62 ± 3.1 mg/kg for Mn and minimum (min.) value was 0.19 ± 0.01 mg/kg for Cd. The estimated daily exposure doses (EDEXDs) for men, women and children were dietary (bread) > dietary (tea) > dermal. For dietary (bread) and dietary (tea) non-carcinogenic (HQ) risk was children > women > men. For dermal, HQ risk was women > children > men. Hazard index (HI) value for men was >1 for Cr. HI value for men was 1.36 for Cr. HI value for women was >1 for Cr and Mn. HI values for women were 1.54 for Cr and 1.01 for Mn. Also, the HI value for children was >1 for Cr, Mn, and Pb. HI values for children were 3.44 for Cr, 2.24 for Mn, and 1.66 for Pb. This situation showed that there was a non-carcinogenic risk. Carcinogenic risk values were dietary (bread) > dietary (tea) > dermal. The total max. carcinogenic value was 1.97E-03 for Cr while the total min. carcinogenic value was 1.31E-05 for Pb. As a result, it has been determined that there may be a risk of cancer due to the consumption of lichen as bread and this situation may adversely affect human health.

Isolation of bioactive compounds from lichen Parmelia sulcata and evaluation of antimicrobial property.

BACKGROUND: Lichens were used as an ailment in the traditional medicine for treating various disorders for centuries. Since there is less evidence in the literature about the medicinal property of Parmelia sulcata (P. sulcata), we made a pioneer attempt to explore the antioxidant and antimicrobial properties of lichens. METHODS: In the present study, the three Samples were collected by using the column chromatography by elucidating the ethyl acetate extract of P. sulcata, and the samples were subjected to DPPH and ABTS assays to find the free radical scavenging activity, total phenols and flavonoids were estimated. The minimum inhibitory concentration was evaluated against the bacterial species (Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae) and fungal species (Candida albicans and Aspergillus fumigatus) by the microdilution method. The best activity sample was analyzed using the Gas Chromatography-Mass Spectrometry (GC-MS), Fourier Transmission Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR). RESULTS: The results shown that all the samples contain phenols and flavonoids which are responsible for antioxidants, antibacterial and antifungal activities. Among that sample-3 shown best antimicrobial activity and it was analyzed and identified as 7-hydroxy-3-(2-methylbut-3-en2-yl)-chromen-2-one. CONCLUSION: The outcome of the study suggests that sample-3 shown good antimicrobial activity and identified as 7-hydroxy-3-(2-methylbut-3-en2-yl)-chromen-2-one. It can be a resource for further studies.

Antimicrobial and antioxidant activity of Evernia prunastri extracts and their isolates.

Lichens are symbiotic organisms formed by a fungus and one or more photosynthetic partners which are usually alga or cyanobacterium. Their diverse and scarcely studied metabolites facilitate adaptability to extreme living conditions. We investigated Evernia prunastri (L.) Ach., a widely distributed lichen, for its antimicrobial and antioxidant potential. E. prunastri was sequentially extracted by hexane (Hex), dichloromethane (DCM) and acetonitrile (ACN) that were screened for their antioxidant and antimicrobial (against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans) activities. The Hex extract possessed the highest antioxidant capacity (87 mg ascorbic acid/g extract) corresponding to the highest content of phenols (73 mg gallic acid/g extract). The DCM and Hex extracts were both active against S. aureus (MICs of 4 and 21 µg/ml, respectively) but were less active against Gram-negative bacteria and yeast. The ACN extract exhibited activity on both S. aureus (MIC 14 µg/ml) and C. albicans (MIC 38 µg/ml) and was therefore further fractionated by silica gel column chromatography. The active compound of the most potent fraction was subsequently characterized by 1H and 13C-NMR spectroscopy and identified as evernic acid. Structural similarity analyses were performed between compounds from E. prunastri and known antibiotics from different classes. The structural similarity was not present. Antioxidant and antimicrobial activities of E. prunastri extracts originate from multiple chemical compounds; besides usnic acid, most notably evernic acid and derivatives thereof. Evernic acid and its derivatives represent possible candidates for a new class of antibiotics.

Nanosilver reinforced Parmelia sulcata extract efficiently induces apoptosis and inhibits proliferative signalling in MCF-7 cells.

The Lichen, Parmelia sulcata synthesizes various secondary metabolites, in which phenolic based compounds received much attention due to their importance in biomedical application. Especially the phenolic compound was effective against the cancer treatment. An effective administration of such plant natural product can represent a significant conventional management of cancer in terms of chemoprevention. The nanomedicines are group of agents that selectively interfere the cancer cells which leads to reduction of side effect thereby reducing the doses. Silver nanoparticles is a promising antitumor agent, however, the conventional production of silver nanoparticles have many drawbacks which led to increase in need of eco-friendly biological production methods. In this study, we made an attempt to synthesise a nano silver (Ps-AgNPs) from phenolic extract of lichen Parmelia sulcata extract. The Ps-AgNps was applied for anticancer activity using MCF-7 cells and the effect was characterised by western blotting method. The FTIR, XRD, UV and TEM results confirms the presence of silver nanoparticles in phenolic extract of lichen Parmelia sulcata. The cytotoxicity assay shows that the Ps-AgNPs is toxic against cancer cells (MCF-7) but not to normal cells (NIH3T3), which confirm the selective induction of cell death (apoptosis) against cancer cells. The Western blot analysis also clearly indicates the down regulation of inflammatory genes (TNF-alpha and IL-6) and cell cycle genes (PCNA and Cyclin-D1) thus promoting intrinsic apoptotic pathway. The results suggest that Ps-AgNPs can effectively kill cancer cells and can be used as an alternative therapeutic agent for cancer treatment.

Usnic acid suppresses cervical cancer cell proliferation by inhibiting PD-L1 expression and enhancing T-lymphocyte tumor-killing activity.

The programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway is abnormally expressed in cervical cancer cells. Moreover, PD-1/PD-L1 blockade reduces the apoptosis and exhaustion of T cells and inhibits the development of malignant tumors. Usnic acid is a dibenzofuran compound originating from Usnea diffracta Vain and has anti-inflammatory, antifungal, and anticancer activities. However, the molecular mechanism of its antitumor effects has not been fully elucidated. In this work, we first observed that usnic acid decreased the expression of PD-L1 in HeLa cells and enhanced the cytotoxicity of co-cultured T cells toward tumor cells. Usnic acid inhibited PD-L1 protein synthesis by reducing STAT3 and RAS pathways cooperatively. It was subsequently shown that usnic acid induced MiT/TFE nuclear translocation through the suppression of mTOR signaling pathways, and promoted the biogenesis of lysosomes and the translocation of PD-L1 to the lysosomes for proteolysis. Furthermore, usnic acid inhibited cell proliferation, angiogenesis, migration, and invasion, respectively, by downregulating PD-L1, thereby inhibiting tumor growth. Taken together, our results show that usnic acid is an effective inhibitor of PD-L1 and our study provide novel insights into the mechanism of its anticancer targeted therapy.

Potential Effect of Pseudevernia furfuracea (L.) Zopf Extract and Metabolite Physodic Acid on Tumour Microenvironment Modulation in MCF-10A Cells.

Lichens comprise a number of unique secondary metabolites with remarkable biological activities and have become an interesting research topic for cancer therapy. However, only a few of these metabolites have been assessed for their effectiveness against various in vitro models. Therefore, the aim of the present study was to assess the effect of extract Pseudevernia furfuracea (L.) Zopf (PSE) and its metabolite physodic acid (Phy) on tumour microenvironment (TME) modulation, focusing on epithelial-mesenchymal transition (EMT), cancer-associated fibroblasts (CAFs) transformation and angiogenesis. Here, we demonstrate, by using flow cytometry, Western blot and immunofluorescence microscopy, that tested compounds inhibited the EMT process in MCF-10A breast cells through decreasing the level of different mesenchymal markers in a time- and dose-dependent manner. By the same mechanisms, PSE and Phy suppressed the function of Transforming growth factor beta (TGF-ß)-stimulated fibroblasts. Moreover, PSE and Phy resulted in a decreasing level of the TGF-ß canonical pathway Smad2/3, which is essential for tumour growth. Furthermore, PSE and Phy inhibited angiogenesis ex ovo in a quail embryo chorioallantoic model, which indicates their potential anti-angiogenic activity. These results also provided the first evidence of the modulation of TME by these substances.

Phytochemical constituents, antioxidant and antistaphylococcal activities of Evernia prunastri (L.) Ach., Pseudevernia furfuracea (L.) Zopf. and Ramalina farinacea (L.) Ach. from Morocco.

The purpose of this work was to assess chemical composition, antibacterial activity against Staphylococcus aureus isolates from catheter-associated infections and antioxidant activity of methanol extracts of three lichens collected from Morocco. The phytochemical analysis of the methanol extracts of these lichens was performed by HPLC-UV method, the predominant phenolic compounds were evernic acid, physodalic acid and usnic acid for Evernia prunastri, Pseudevernia furfuracea and Ramalina farinacea, respectively. Total phenolic compounds and total flavonoid content of all extracts were also determined. As a result, Pseudevernia furfuracea extract had the strongest effect and the highest phenolic compounds content. All extracts showed antibacterial activity against all tested strains (MIC values ranging from 0.078 to 0.625 mg/mL), the strongest inhibition was obtained with the extract of Evernia prunastri.

Biological activity of selected lichens and lichen-based Ag nanoparticles prepared by a green solid-state mechanochemical approach.

Lichens dispose a wide spectrum of bioactive compounds known as secondary metabolites. Their biological effects like antioxidant and antibacterial activities are widely studied. Green synthesis of silver nanoparticles (AgNPs) is a method where the compounds/substances present in plants are used for reduction of AgNO3instead of toxic chemicals. However, this methodology is usually a two-step process (extract preparation step and the synthesis step) performed under the elevated temperatures nad in the case of lichens, the redicing compounds are insoluble in water. These disadvantages can be overcome by a solid-state mechanochemical synthesis applied in the present study. As microorganisms are becoming more resistant to commercial antibiotics, AgNPs prepared in an environmentally friendly way represent an interesting alternative. In the present study, we compared the processing of lichen material of Pseudevernia furfuracea and Lobaria pulmonaria for extraction as well as for synthesis of AgNPs, and tested the antibacterial and antioxidant activity of the extracts. Both selected lichen species could be successfully used as reducing agents to produce AgNPs. Six different bacterial strains were tested for antibacterial activity of AgNPs-containing products and it was highly effective on all strains. However, the antioxidant activity of lichen extracts showed the lowest effect even if AgNPs are present which positively correlated with the content of total phenols and flavonoids. Both phenols and flavonoids are natural antioxidants and react with silver nitrate. Due to this fact, we observed a decrease of total phenols, total flavonoids as well as antioxidant activity when processing of lichen extracts with silver nitrate was used. We demonstrated that the formation of AgNPs increased the antibacterial activity but on the other hand reduced the antioxidant activity. Thus, antibacterial and antioxidant effects have to be treated differentially.

Aqueous extract of oakmoss produces antihypertensive activity in L-NAME-induced hypertensive rats through sGC-cGMP pathway.

BACKGROUND: Lichens are a symbiotic association of a fungus with a green alga or cyanobacterium. They are widely used in traditional medicine as a treatment against skin disorders, diabetes and hypertension. THE AIM OF THE STUDY: The goal of this paper was to assess the possible antihypertensive and vasorelaxant capacity of the aqueous extract of a lichen species called Oakmoss or Evernia prunastri (L.). MATERIAL AND METHODS: In the present study, the aqueous extract of Oakmoss was prepared, its antihypertensive activity was examined in N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats, and its vasorelaxant ability was performed in rat isolated thoracic aorta. RESULTS: The results proved that Oakmoss reduced the systolic, diastolic, mean arterial blood pressure, and heart rate in hypertensive rats but not in normotensive rats. Besides, the data showed that Oakmoss exerts its antihypertensive effect through vasorelaxant ability. CONCLUSION: The present study presents the favorable action of Oakmoss as an antihypertensive agent.

In vitro resynthesis of lichenization reveals the genetic background of symbiosis-specific fungal-algal interaction in Usnea hakonensis.

BACKGROUND: Symbiosis is central to ecosystems and has been an important driving force of the diversity of life. Close and long-term interactions are known to develop cooperative molecular mechanisms between the symbiotic partners and have often given them new functions as symbiotic entities. In lichen symbiosis, mutualistic relationships between lichen-forming fungi and algae and/or cyanobacteria produce unique features that make lichens adaptive to a wide range of environments. Although the morphological, physiological, and ecological uniqueness of lichens has been described for more than a century, the genetic mechanisms underlying this symbiosis are still poorly known. RESULTS: This study investigated the fungal-algal interaction specific to the lichen symbiosis using Usnea hakonensis as a model system. The whole genome of U. hakonensis, the fungal partner, was sequenced by using a culture isolated from a natural lichen thallus. Isolated cultures of the fungal and the algal partners were co-cultured in vitro for 3 months, and thalli were successfully resynthesized as visible protrusions. Transcriptomes of resynthesized and natural thalli (symbiotic states) were compared to that of isolated cultures (non-symbiotic state). Sets of fungal and algal genes up-regulated in both symbiotic states were identified as symbiosis-related genes. CONCLUSION: From predicted functions of these genes, we identified genetic association with two key features fundamental to the symbiotic lifestyle in lichens. The first is establishment of a fungal symbiotic interface: (a) modification of cell walls at fungal-algal contact sites; and (b) production of a hydrophobic layer that ensheaths fungal and algal cells;. The second is symbiosis-specific nutrient flow: (a) the algal supply of photosynthetic product to the fungus; and (b) the fungal supply of phosphorous and nitrogen compounds to the alga. Since both features are widespread among lichens, our result may indicate important facets of the genetic basis of the lichen symbiosis.

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.