Phylogeny of the genus Loxospora s.l. (Sarrameanales, Lecanoromycetes, Ascomycota), with Chicitaea gen. nov. and five new combinations in Chicitaea and Loxospora.

Loxospora is a genus of crustose lichens containing 13 accepted species that can be separated into two groups, based on differences in secondary chemistry that correlate with differences in characters of the sexual reproductive structures (asci and ascospores). Molecular phylogenetic analyses recovered these groups as monophyletic and support their recognition as distinct genera that differ in phenotypic characters. Species containing 2'-O-methylperlatolic acid are transferred to the new genus, Chicitaea Guzow-Krzem., Kukwa & Lendemer and four new combinations are proposed: C.assateaguensis (Lendemer) Guzow-Krzem., Kukwa & Lendemer, C.confusa (Lendemer) Guzow-Krzem., Kukwa & Lendemer, C.cristinae (Guzow-Krzem., Lubek, Kubiak & Kukwa) Guzow-Krzem., Kukwa & Lendemer and C.lecanoriformis (Lumbsch, A.W. Archer & Elix) Guzow-Krzem., Kukwa & Lendemer. The remaining species produce thamnolic acid and represent Loxospora s.str. Haplotype analyses recovered sequences of L.elatina in two distinct groups, one corresponding to L.elatina s.str. and one to Pertusariachloropolia, the latter being resurrected from synonymy of L.elatina and, thus, requiring the combination, L.chloropolia (Erichsen) Ptach-Styn, Guzow-Krzem., Tønsberg & Kukwa. Sequences of L.ochrophaea were found to be intermixed within the otherwise monophyletic L.elatina s.str. These two taxa, which differ in contrasting reproductive mode and overall geographic distributions, are maintained as distinct, pending further studies with additional molecular loci. Lectotypes are selected for Lecanoraelatina, Pertusariachloropolia and P.chloropoliaf.cana. The latter is a synonym of Loxosporachloropolia. New primers for the amplification of mtSSU are also presented.

Pseudolepraria, a new leprose genus revealed in Ramalinaceae (Ascomycota, Lecanoromycetes, Lecanorales) to accommodate Leprariastephaniana.

The new genus Pseudolepraria Kukwa, Jablonska, Kosecka & Guzow-Krzeminska is introduced to accommodate Leprariastephaniana Elix, Flakus & Kukwa. Phylogenetic analyses of nucITS, nucLSU, mtSSU and RPB2 markers recovered the new genus in the family Ramalinaceae with strong support. The genus is characterised by its thick, unstratified thallus composed entirely of soredia-like granules, the presence of 4-O-methylleprolomin, salazinic acid, zeorin and unknown terpenoid, and its phylogenetic position. The new combination, P.stephaniana (Elix, Flakus & Kukwa) Kukwa, Jablonska, Kosecka & Guzow-Krzeminska, is proposed.

Phylogeny and Ecology of Trebouxia Photobionts From Bolivian Lichens.

In the past few years, new phylogenetic lineages in Trebouxia were detected as a result of molecular approaches. These studies included symbiont selectivity in lichen communities, transects along altitudinal gradients at local and global scales and the photobiont diversity in local populations of lichen-forming fungal species. In most of these studies, phylogenetic and haplotype analyses based on the internal transcribed spacer (ITS) locus have continuously allowed the recognition of new monophyletic lineages, which suggests that still numerous undiscovered Trebouxia lineages can be hidden in lichens from unexplored areas, especially in the tropics. Here, we estimated the biodiversity of photobionts in Bolivian Andean vegetation and assessed their specificity. About 403 lichen samples representing 42 genera, e.g., Haematomma, Heterodermia, Hypotrachyna, Lecanora, Lepra, Leucodermia, Parmotrema, Pertusaria, Polyblastidium, and Usnea, containing Trebouxia photobionts, were analyzed. ITS ribosomal DNA (rDNA) and rbcL markers were used. We obtained Trebouxia sequences from Bolivian samples belonging to already described clades A, C, I, and S. Thirty-nine Trebouxia lineages were distinguished within these clades, while 16 were new. To reveal the structure of the community of Bolivian photobionts and their relationships with mycobionts, the comparative effects of climate, altitude, geographical distances, substrate, and habitat type, as well as functional traits of lichens such as growth forms, propagation mode and secondary metabolites, were analyzed. Furthermore, new Bolivian records were included in analysis on a global scale. In our study, the mycobiont genus or even species are the most important factors correlated with photobiont identity. Moreover, we revealed that the community of Bolivian photobionts is shaped by altitude.

The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

New lineages of photobionts in Bolivian lichens expand our knowledge on habitat preferences and distribution of Asterochloris algae.

We studied the biodiversity of Asterochloris photobionts found in Bolivian lichens to better understand their global spatial distribution and adaptation strategies in the context of a worldwide phylogeny of the genus. Based on nuclear ITS rDNA, the chloroplast rbcL gene and the actin type I gene we reconstructed a phylogenetic tree that recovered nine new Asterochloris lineages, while 32 Bolivian photobiont samples were assigned to 12 previously recognized Asterochloris lineages. We also show that some previously discovered Asterochloris photobiont species and lineages may occur in a broader spectrum of climatic conditions, and mycobiont species and photobionts may show different preferences along an altitude gradient. To reveal general patterns of of mycobiont specificity towards the photobiont in Asterochloris, we tested the influence of climate, altitude, geographical distance and effects of symbiotic partner (mycobiont) at the species level of three genera of lichen forming fungi: Stereocaulon, Cladonia and Lepraria. Further, we compared the specificity of mycobionts towards Asterochloris photobionts in cosmopolitan, Neotropical, and Pantropical lichen forming fungi. Interestingly, cosmopolitan species showed the lowest specificity to their photobionts, but also the lowest haplotype diversity. Neotropical and Paleotropical mycobionts, however, were more specific.

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.

Trentepohlialean Algae (Trentepohliales, Ulvophyceae) Show Preference to Selected Mycobiont Lineages in Lichen Symbioses.

The main aims of this work were to assess phylogenetic relationships of the trentepohlialean photobionts in tropical, mainly sterile, lichens collected in Bolivia, to examine their genetic diversity, host specificity, and the impact of habitat factors on the occurrence of Trentepohliales. Based on rbcL marker analysis, we constructed a phylogenetic tree with eight major clades of Trentepohliales, of which seven free-living species are intermingled with lichenized ones. Our analyses show that the studied photobionts are scattered across the phylogenetic tree and algae from temperate and tropical regions do not form monophyletic groups, except within one clade that seems to be restricted to the tropics. There is no significant occurrence pattern of lichenized Trentepohliaceae on a specific substratum, except Cephaleuros spp. and Phycopeltis spp., which are restricted to leaves, while some clades with lichenized algae may be specialized to tree bark and wood. Moreover, we found two patterns of associations: first, closely related algae can associate with distantly related mycobionts; second, some other trentepohlioid algae associate with selected lineages of fungi (e.g., Arthoniaceae or Graphidaceae). We also found that some lineages of photobionts are even more selective and associate exclusively with one species (e.g., Dichosporidium nigrocinctum, Diorygma antillarum) or closely related lichen-forming fungi (Herpothallon spp.). Concluding, we found that occurrence of some trentepohlialean photobionts may correlate with the particular type of the mycobiont.

Morphology and secondary chemistry in species recognition of Parmelia omphalodes group - evidence from molecular data with notes on the ecological niche modelling and genetic variability of photobionts.

To evaluate the importance of morphological and chemical characters used in the recognition of species within the Parmelia omphalodes group, we performed phylogenetic, morphological and chemical analyses of 335 specimens, of which 34 were used for molecular analyses. Phylogenetic analyses, based on ITS rDNA sequences, show that P. pinnatifida is distinct from P. omphalodes and the most important difference between those species is the development of pseudocyphellae. In P. pinnatifida, they are mostly marginal and form white rims along lobes margins, but laminal pseudocyphellae can develop in older parts of thalli and are predominantly connected with marginal pseudocyphellae. In contrast, in P. omphalodes laminal pseudocyphellae are common and are predominantly not connected to marginal pseudocyphellae. Chemical composition of secondary lichen metabolites in both analysed species is identical and therefore this feature is not diagnostic in species recognition. Few samples of P. discordans, species morphologically similar to P. omphalodes and P. pinnatifida, were also included in the analyses and they are nested within the clade of P. omphalodes, despite the different chemistry (protocetraric acid present versus salazinic acid in P. omphalodes). All taxa of the P. omphalodes group occupy similar niches, but their potential distributions are wider than those currently known. The absence of specimens in some localities may be limited by the photobiont availability. Parmelia omphalodes and P. pinnatifida are moderately selective in photobiont choice as they form associations with at least two or three lineages of Trebouxia clade S. Parmelia pinnatifida, as well as P. discordans are associated with Trebouxia OTU S02 which seems to have a broad ecological amplitude. Other lineages of Trebouxia seem to be rarer, especially Trebouxia sp. OTU S04, which is sometimes present in P. pinnatifida. This study indicates the importance of extensive research including morphology, chemistry and analysis of molecular markers of both bionts in taxonomical studies of lichens.

Understanding the evolution of phenotypical characters in the Micarea prasina group (Pilocarpaceae) and descriptions of six new species within the group.

Six new Micarea species are described from Europe. Phylogenetic analyses, based on three loci, i.e. mtSSU rDNA, Mcm7 and ITS rDNA and ancestral state reconstructions, were used to evaluate infra-group divisions and the role of secondary metabolites and selected morphological characters on the taxonomy in the M. prasina group. Two main lineages were found within the group. The Micarea micrococca clade consists of twelve species, including the long-known M. micrococca and the newly described M. microsorediata, M. nigra and M. pauli. Within this clade, most species produce methoxymicareic acid, with the exceptions of M. levicula and M. viridileprosa producing gyrophoric acid. The M. prasina clade includes the newly described M. azorica closely related to M. prasina s.str., M. aeruginoprasina sp. nov. and M. isidioprasina sp. nov. The species within this clade are characterised by the production of micareic acid, with the exception of M. herbarum which lacks any detectable substances and M. subviridescens that produces prasinic acid. Based on our reconstructions, it was concluded that the ancestor of the M. prasina group probably had a thallus consisting of goniocysts, which were lost several times during evolution, while isidia and soredia evolved independently at multiple times. Our research supported the view that the ancestor of M. prasina group did not produce any secondary substances, but they were gained independently in different lineages, such as methoxymicareic acid which is restricted to M. micrococca and allied species or micareic acid present in the M. prasina clade.

Synthesis of Usnic Acid Derivatives and Evaluation of Their Antiproliferative Activity against Cancer Cells.

Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC50 values after 48 h of treatment were ca. 3 µM for MCF-7 and PC-3 cells and 1 µM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells.

Phylogenetic placement of Leprariacryptovouauxii sp. nov. (Lecanorales, Lecanoromycetes, Ascomycota) with notes on other Lepraria species from South America.

Leprariacryptovouauxii is described as a new semicryptic species similar to L.vouauxii, from which it differs geographically (South America) and phylogenetically; both species differ in nucleotide position characters in nucITS barcoding marker. Leprariaharrisiana is reported as new to South America and L.nothofagi as new to Antarctica, Bolivia, and Peru. Leprariaincana (South American records are referred to L.aff.hodkinsoniana) and L.vouauxii (most South American records are referred to L.cryptovouauxii) should be excluded at least temporarily from the lichen list of South America. All records previously referred to as L.alpina from Bolivia and Peru belong to L.nothofagi. Most of Bolivian records of L.pallida belong to L.harrisiana. Leprariaborealis and L.caesioalba should be included in L.neglecta. Leprariaachariana, L.impossibilis, and L.sipmaniana are sequenced for the first time.

Photobiont switching causes changes in the reproduction strategy and phenotypic dimorphism in the Arthoniomycetes.

Phylogenetic analyses using mtSSU and nuITS sequences of Buellia violaceofusca (previously placed in Lecanoromycetes), a sterile, sorediate lichen having a trebouxioid photobiont, surprisingly prove that the species is conspecific with Lecanographa amylacea (Arthoniomycetes), a fertile, esorediate species with a trentepohlioid photobiont. These results suggest that L. amylacea and B. violaceofusca are photomorphs of the same mycobiont species, which, depending on the photobiont type, changes the morphology and the reproduction strategy. This is the first example of a lichenized fungus that can select between Trebouxia (Trebouxiophyceae) and trentepohlioid (Ulvophyceae) photobionts. Trebouxia photobionts from the sorediate morphotype belong to at least three different phylogenetic clades, and the results suggest that Lecanographa amylacea can capture the photobiont of other lichens such as Chrysothrix candelaris to form the sorediate morphotype. Phylogenetic analyses based on rbcL DNA data suggest that the trentepohlioid photobiont of L. amylacea is closely related to Trentepohlia isolated from fruticose lichens. The flexibility in the photobiont choice enables L. amylacea to use a larger range of tree hosts. This strategy helps the lichen to withstand changes of environmental conditions, to widen its distribution range and to increase its population size, which is particularly important for the survival of this rare species.

Antibacterial and anticancer activities of acetone extracts from in vitro cultured lichen-forming fungi.

BACKGROUND: Lichens that were used in traditional medicine for ages produce numerous secondary metabolites, however our knowledge about biological activities of substances secreted by separated bionts is scarce. The main objectives of this study were to isolate and find optimal conditions for the growth of mycelia from three common lichen-forming fungi, i.e. Caloplaca pusilla, Protoparmeliopsis muralis and Xanthoria parietina and to evaluate antibacterial and antiproliferative activities of their acetone extracts. METHODS: Agar disc diffusion and broth microdilution methods were used to test antimicrobial activity against six species of bacteria. MTT method, flow cytometry assay and DAPI staining were applied to test antiproliferative activity of selected extracts against MCF-7 (human breast adenocarcinoma), PC-3 (human prostate cancer) and HeLa (human cervix adenocarcinoma) cancer cells. RESULTS: P. muralis strongly inhibited the growth of Gram-positive bacteria, i.e. Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis (MICs from 6.67 to 100.00 µg mL-1). X. parietina grown on PDA and G-LBM media decreased HeLa or MCF-7 cancer cells viability with IC50 values of about 8 µg mL-1, while C. pusilla grown on G-LBM medium showed the highest potency in decreasing MCF-7 (7.29 µg mL-1), PC-3 (7.96 µg mL-1) and HeLa (6.57 µg mL-1) cancer cells viability. We also showed induction of apoptosis in HeLa, PC-3 and MCF-7 cell lines treated with increasing concentrations of C. pusilla extract. CONCLUSION: We showed that selected acetone extracts demonstrated a strong antimicrobial and anticancer effects that suggests that aposymbiotically cultured lichen-forming fungi can be a source of antibacterial and antiproliferative compounds.

Phylogenetic relationship of the stringent response-related genes of marine bacteria.

Bacteria living in marine environment encounter various challenges and limitations, thus in order to survive, they need to employ efficient stress-response mechanisms. One of these mechanisms is the stringent response, where unusual nucleotides, guanosine tetra- and pentaphosphates, herald starvation and physico-chemical stresses. All so far sequenced free-living bacteria contain the gene(s) responsible for (p)ppGpp synthesis - rsh (named after Escherichia coli genes, relA and spoT). Two similar genes were identified mostly in ß- and γ-proteobacteria while other bacteria have only one gene coding the dual function of (p)ppGpp synthesis and degradation. Although the presence of (p)ppGpp-mediated response to the stress conditions has been shown for a few, and predicted for some other marine microorganisms, the (p)ppGpp effects may vary among different organisms. Thus, in this work we asked whether marine bacteria could have evolved a genetic adaptation specifically suited to adapt to environment with limited resources. The phylogenetic analyses of SpoT, RelA and RSH proteins from organisms associated with marine environment showed, however, that the evolutionary correlations obtained for these proteins are congruent with those constructed for 16S rRNA sequences and reflect taxonomical relationships of these organisms. Likewise, the similarity of specific amino acid residues indispensable for catalytic activity of these enzymes is very high, and any observed changes parallel with the taxonomical and evolutionary relationships. However, potential homologs of Mesh1 enzyme (metazoan SpoT homologs) that occur in both eukaryotic and prokaryotic organisms and contain the hydrolytic domain orthologous to SpoT were identified in Cellulophaga, Erythrobacter and Flavobacterium genera for the first time, as well as in soil bacterium Cytophaga hutchinsonii and freshwater Rhodothermus marinus.

A rapidly progressing, deadly disease of Actias selene (Indianmoonmoth) larvae associated with a mixed bacterial and baculoviral infection.

The outbreak of an infectious disease in captive-bred Lepidoptera can cause death of all the caterpillars within days. A mixed baculoviral-bacterial infection observed among Actias selene (Hubner 1807), the Indian moon moth (Insecta: Lepidoptera: Saturniidae), larvae was characterized and followed by a photographic documentation of the disease progression. The etiological agents were determined using mass spectrometry and polymerase chain reaction (PCR). It appeared that the disease was caused by a mixed infection of larvae with a baculovirus and Morganella morganii. A molecular phylogenetic analysis of the virus and microbiological description of the pathogenic bacterium are presented.

Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.