Microbiome in Cladonia squamosa Is Vertically Stratified According to Microclimatic Conditions.

Lichens are miniature ecosystems that contain fungi, microalgae, and bacteria. It is generally accepted that symbiosis between mycobiont and photobiont and microbial contribution to the ecosystem support the wide distribution of lichens in terrestrial ecosystems, including polar areas. The composition of symbiotic components can be affected by subtle microenvironmental differences within a thallus, as well as large-scale climate differences. In this study, we investigated fine-scale profiles of algal, fungal, and bacterial compositions through horizontal and vertical positions of the Antarctic lichen Cladonia squamosa colonies by next-generation sequencing of the nuclear large subunit rRNA gene (nucLSU) of eukaryotes and the 16S rRNA gene of bacteria. Apical parts of thalli were exposed to strong light, low moisture, and high variability of temperature compared with basal parts. Microbial diversity increased from apical parts to basal parts of thalli. Asterochloris erici was the major photobiont in apical positions of thalli, but other microalgal operational taxonomic units (OTUs) of Trebouxiophyceae and Ulvophyceae were major microalgal components in basal positions. Photochemical responses of algal components from apical and basal parts of thalli were quite different under variable temperature and humidity conditions. Several fungal OTUs that belonged to Arthoniomycetes and Lecanoromycetes, and diverse bacterial OTUs that belonged to Alphaproteobacteria, Acidobacteria_Gp1, and candidate division WPS-2 showed a clear distribution pattern according to their vertical positions within thalli. The overall lichen microbiome was significantly differentiated by the vertical position within a thallus. These results imply that different microclimate are formed at different lichen thallus parts, which can affect microbial compositions and physiological responses according to positions within the thalli.

Algal and fungal diversity in Antarctic lichens.

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7-100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen-associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen-associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen-associated fungi was not evident.

Development of SCAR marker for discrimination of Artemisia princeps and A. argyi from other Artemisia herbs.

Some Artemisia herbs are used for medicinal purposes. In particular, A. princeps and A. argyi are classified as 'Aeyup' and are used as important medicinal material in traditional Korean medicine. On the other hand, A. capillaris and A. iwayomogi, which are classified as 'Injinho' and 'Haninjin', respectively, are used for other purposes distinct from those of 'Aeyup'. However, sometimes 'Aeyup' is not clearly discriminated from 'Injinho' and/or 'Haninjin'. Furthermore, Artemisia capillaris and/or A. iwayomogi have been used in place of A. princeps and A. argyi. In this study, we developed an efficient method to discriminate A. argyi and A. princeps from other Artemisia plants. The RAPD (random amplified polymorphic DNA) method efficiently discriminated various Artemisia herbs. In particular, non-specific primer 329 (5'-GCG AAC CTC C-3'), which shows polymorphism among Artemisia herbs, amplified 838 bp products, which are specific to A. princeps and A. argyi only. Based on nucleotide sequence of the primer 329 product, we designed a Fb (5'-CAT CAA CCA TGG CTT ATC CT-3') and R7 (5'-GCG AAC CTC CCC ATT CCA-3') primer-set to amplify a 254 bp sized SCAR (sequence characterized amplified regions) marker, through which A. princeps and A. argyi can be efficiently discriminated from other Artemisia herbs, particularly, A. capillaris and A. iwayomogi.