DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least–squares to latent structures–discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.The study was financially supported by the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013/ under REA grant agreement No. 606895 (http://cordis.europa.eu/project/rcn/109122_en.html) as well as a minor contribution from Bergthora and Thorsteinn Scheving Thorsteinsson Fund. The funders provided support in the form of salaries for author [MX], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of this author is articulated in the ‘author contributions’ section. The commercial affiliation Arctic Mass has no financial or competing interests in this study but two of the authors MT and FFE are partially affiliated there. Arctic Mass had the role of providing access to instruments (mass spectrometers) used in the study.Peer Reviewe

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials

Phylogenetic diversity of the lichenized algal genus Trebouxia (Trebouxiophyceae, Chlorophyta): a new lineage and novel insights from fungal-algal association patterns of Icelandic cetrarioid lichens (Parmeliaceae, Ascomycota)

Publisher's version (útgefin grein)Lichens have high tolerance to harsh environmental conditions, where lichen symbiont interactions (e.g. myco- and photobionts) may play a crucial role. The characterization of fungal-algal association patterns is essential to understand their symbiotic interactions. This study investigated fungal-algal association patterns in Icelandic cetrarioid lichens using a multi-locus phylogenetic framework, including fungal nrITS, MCM7, mtSSU, RPB1 and RPB2 and algal nrITS, nrLSU, rbcL and mtCOXII data. Most Icelandic cetrarioid lichenized fungi were found to be specifically associated to the known Trebouxia clade “S” (Trebouxia simplex/suecica group), whereas the lichen-forming fungus Cetrariella delisei forms a symbiosis with a previously unrecognized lineage of Trebouxia, provisionally named as the “D” clade. This new Trebouxia lineage is supported by maximum likelihood and Bayesian phylogenetic analyses using all four included algal loci.This project was supported by the European Union’s Seventh Framework Programme for research, technological development and demonstration (grant agreement number 606895) to FP7-MCA-ITN MedPlant, “Phylogenetic Exploration of Medicinal Plant Diversity”. The Icelandic Research Fund (grant number 185442051) and the Bergthora and Thorsteinn Scheving Thorsteinsson Fund are also acknowledged for financial support.Peer Reviewe

Alkaloid fingerprinting resolves Huperzia selago genotypes in Iceland

Pre-print (óritrýnt handrit)The club moss family Lycopodiaceae produces a diverse array of bioactive lycopodium alkaloids (LAs). In particular, the alkaloid huperzine A (hupA) has grasped attention since it is a potent acetylcholinesterase inhibitor of medical interest in Alzheimer's disease. Although the structural diversity and bioactivities of LAs have been studied to some extent, their chemotaxonomic value is mostly unexplored, especially to a lower taxonomic unit (e.g. subspecies or genotypes). This study focused on previously reported Icelandic Huperzia selago genotypes, and aimed to evaluate the chemotaxonomic value of LAs in resolving them. Using liquid chromatography-mass spectrometry (LC-MS), alkaloid fingerprints of H. selago taxa were subjected to principal component analysis (PCA). Our results revealed that each genotype tends to have its own alkaloid profile. Genotype 1 and 3 form distinct groups in a PCA plot, where genotype 2 is an intermediate between the other two genotypes. HupA and its derivative, huperzine B, both contribute to the differentiation of genotype 3 from the others. Therefore, our study demonstrated the potential of alkaloid fingerprints in resolving deep taxonomic groups and selecting plant taxa of medicinal importance.This work was supported by The People Program (Marie Curie Actions) of the European Union’s Seventh Framework Program FP7/2007-2013 (grant number 606895); Icelandic Research Fund (grant number 152604051); and the Bergthora and Thorsteinn Scheving Thorsteinsson Fund

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex

Publisher's version (útgefin grein)The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimerʼs disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 – 679 µg/g) than genotype 1 (20 – 180 µg/g), where the former shows a typical green and reflexed “selago” morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 – 599 µg/g). The content of huperzine B in Icelandic taxa (6 – 13 µg/g) is much lower than that in Chinese H. serrata (79 – 207 µg/g).The People Programme (Marie Curie Actions) of the European Unionʼs Seventh Framework Programme FP7/2007–2013 (grant number 606895) and the Icelandic Research Fund (grant number 152604051) are acknowledged for financial support. This study was also funded from the Bergthora and Thorsteinn Scheving Thorsteinsson Fund.Peer Reviewe

Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells

To access Publisher full text version of this article. Please click on the hyperlink in Additional Links fieldMany naturally occurring polysaccharides from fungi and lichens have been found to have immunomodulating activity. However, the majority of these studies have focused on their effects on the innate arm of the immune system. Although dendritic cells (DCs) belong to the innate immune system, they play an important role as a bridge between the innate and the adaptive immune response. In this study, the effects of 11 chromatographically purified and well-characterised lichen polysaccharides (of different structural types) on the maturation of DCs were tested by analysing the secretion of IL-12p40 and IL-10 by human monocyte-derived dendritic cells in vitro. Four of the polysaccharides upregulated IL-10 secretion by the dendritic cells, as compared with unstimulated cells, the beta-glucans lichenan and Ths-2 and the heteroglycans Pc-4 and thamnolan. IL-12p40 secretion was significantly upregulated by the beta-glucan lichenan and the heteroglycans Pc-2, Pc-4, thamnolan and Ths-4, while the mature dendritic cells stimulated with the heteroglycan Pc-1 secreted significantly less IL-12p40 than the unstimulated cells. Proportional index (PI) was used to determine the relationship between the IL-12p40 and IL-10 secretion. The PI of all the beta-glucans, i.e. lichenan, pustulan and Ths-2, and the heteroglycan thamnolan was significantly lower than the PI observed for the unstimulated cells, which was mainly due to increased IL-10 secretion. Therefore, these polysaccharides could be considered suitable candidates in tolerance and anti-inflammatory studies, as IL-10 is one of the major cytokines involved in tolerance and anti-inflammatory responses

Antimicrobial Activity of Marine Bacterial Symbionts Retrieved from Shallow Water Hydrothermal Vents

Marine sponges and other sessile macro-organisms were collected at a shallow water hydrothermal site in Eyjafjörður, Iceland. Bacteria were isolated from the organisms using selective media for actinomycetes, and the isolates were screened for antimicrobial activity. A total of 111 isolates revealed antimicrobial activity displaying different antimicrobial patterns which indicates production of various compounds. Known test strains were grown in the presence of ethyl acetate extracts from one selected isolate, and a clear growth inhibition of Staphylococcus aureus was observed down to 0.1 % extract concentration in the medium. Identification of isolates shows different species of Actinobacteria with Streptomyces sp. playing the largest role, but also members of Bacilli, Alphaproteobacteria and Gammaproteobacteria. Sponges have an excellent record regarding production of bioactive compounds, often involving microbial symbionts. At the hydrothermal vents, however, the majority of active isolates originated from other invertebrates such as sea anemones or algae. The results indicate that antimicrobial assays involving isolates in full growth can detect activity not visible by other methods. The macro-organisms inhabiting the Eyjafjörður hydrothermal vent area host diverse microbial species in the phylum Actinobacteria with antimicrobial activity, and the compounds responsible for the activity will be subject to further research

Immunomodulating polysaccharides from the lichen Thamnolia vermicularis var. subuliformis

To access publisher full text version of this article. Please click on the hyperlink in Additional Links fieldThree heteroglycans Ths-4, Ths-5 and thamnolan and a beta-glucan, Ths-2, isolated from the lichen Thamnolia vermicularis var. subuliformis were tested for in vitro immunomodulating activities and shown to have various influences on the immune system. All the polysaccharides except Ths-4 caused a stimulation of rat spleen cell proliferation. In contrast, Ths-4 caused cell death early in the culture, probably due to over-stimulation. Moreover, the galactofuranomannans, Ths-4, Ths-5 and the beta-glucan Ths-2, induced rat spleen cells to secrete IL-10 significantly above background levels. In addition, Ths-4 and Ths-5 stimulated significant TNF-alpha secretion by rat peritoneal macrophages. The galactofuranomannans Ths-4 and Ths-5 have similar structures apart from the molecular weight. Thus, it may be concluded that the molecular size might influence the potency but not the pattern of activity for Ths-4 and Ths-5. The galactofuranorhamnan thamnolan had less mitogenic effect than Ths-5 and Ths-2 and neither induced IL-10 secretion by rat spleen cells nor TNF-alpha secretion by peritoneal macrophages to significant levels. This shows that thamnolan with its unusual galactofuranorhamnan structure differs from the other Thamnolia polysaccharides in its immunomodulatory activity

Flustramine Q, a Novel Marine Origin Acetylcholinesterase Inhibitor from Flustra foliacea

The bryozoan Flustra foliacea produces a range of indole alkaloids, and some have shown weak antibiotic, muscle-relaxant and cytotoxic properties; however, most of them have not been tested for bioactivity. Many of these alkaloids possess a physostigmine scaffold, and physostigmine is a well-known acetylcholinesterase (AChE) inhibitor. AChE inhibitors are of interest as drug leads in neurodegenerative diseases and are currently used in symptomatic treatment of Alzheimer&rsquo;s disease (AD). In this study, the AChE inhibitory activity of Flustra alkaloids was studied in vitro using the colorimetric method of Ellman and AChE from Electrophorus electricus. Twenty-five compounds isolated from the Icelandic bryozoan F. foliacea were screened at a 100 &micro;M concentration. Two of them, flustramine E and flustramine I, showed inhibition of 48%, and flustramine Q showed 82% inhibition. For flustramine Q, the IC50 was 9.6 &micro;M. Molecular modelling and docking studies indicated that simple in silico designed derivatives of flustramine Q could have potential for increased potency. Marine natural products including brominated indole alkaloids from Flustra foliacea are an interesting new source of AChE inhibitors with potential towards central nervous system disorders, e.g., Alzheimer&rsquo;s disease

Aqueous extracts from Menyanthes trifoliate and Achillea millefolium affect maturation of human dendritic cells and their activation of allogeneic CD4+ T cells in vitro.

To access publisher full text version of this article. Please click on the hyperlink in Additional Links field.Ethnopharmacological relevance: Menyanthes trifoliate and Achillea millefolium have been used in traditional medicine to ameliorate chronic inflammatory conditions. The aim of this study was to identify the effects of ethanol and aqueous extracts of Menyanthes trifoliate and Achillea millefolium on maturation of dendritic cells (DCs) and their ability to activate allogeneic CD4(+) T cells. MATERIALS AND METHODS: Human monocyte-derived DCs were matured in the absence or presence of lyophilised aqoueous or ethanol extracts from Menyanthes trifoliate or Achillea millefolium and their expression of surface molecules analysed with flow cytometry and cytokine secretion measured by ELISA. DCs matured in the presence of aqueous extracts from Menyanthes trifoliate and Achillea millefolium were co-cultured with allogeneic CD4(+) T cells and the expression of surface molecules by T cells and their cytokine secretion and cell proliferation determined. RESULTS: Maturation of DCs in the presence of aqueous extracts from Menyanthes trifoliate or Achillea millefolium did not affect expression of the surface molecules examined but reduced the ratio of secreted IL-12p40/IL-10, compared with that by DCs matured in the absence of extracts. Allogeneic CD4(+) T cells co-cultured with DCs matured in the presence of aqueous extract from Menyanthes trifoliate secreted less IFN-γ, IL-10 and IL-17 than CD4(+) T cells co-cultured with DCs matured without an extract. Maturation of DCs in the presence of aqueous extract from Achillea millefolium decreased IL-17 secretion but did not affect IFN-γ and IL-10 secretion by allogeneic CD4(+) T cells. CONCLUSIONS: Aqueous extract from Menyanthes trifoliate induces a suppressive phenotype of DCs that has reduced capacity to induce Th1 and Th17 stimulation of allogeneic CD4(+) T cells, whereas aqueous extract from Achillea millefolium reduces the capacity of DCs to induce a Th17 response.Landspitali University Hospital Memorial Fund of Bergthora Magnusdottir & Jakob Bjarnason Icelandic Research Fund Icelandic Research Fund for Graduate Student