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      From the Tunnels into the Treetops: New Lineages of Black Yeasts from Biofilm in the Stockholm Metro System and Their Relatives among Ant-Associated Fungi in the Chaetothyriales

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          Abstract

          Rock-inhabiting fungi harbour species-rich, poorly differentiated, extremophilic taxa of polyphyletic origin. Their closest relatives are often well-known species from various biotopes with significant pathogenic potential. Speleothems represent a unique rock-dwelling habitat, whose mycobiota are largely unexplored. Isolation of fungi from speleothem biofilm covering bare granite walls in the Kungsträdgården metro station in Stockholm yielded axenic cultures of two distinct black yeast morphotypes. Phylogenetic analyses of DNA sequences from six nuclear loci, ITS, nuc18S and nuc28S rDNA, rpb1, rpb2 and β-tubulin, support their placement in the Chaetothyriales (Ascomycota). They are described as a new genus Bacillicladium with the type species B. lobatum, and a new species Bradymyces graniticola. Bacillicladium is distantly related to the known five chaetothyrialean families and is unique in the Chaetothyriales by variable morphology showing hyphal, meristematic and yeast-like growth in vitro. The nearest relatives of Bacillicladium are recruited among fungi isolated from cardboard-like construction material produced by arboricolous non-attine ants. Their sister relationship is weakly supported by the Maximum likelihood analysis, but strongly supported by Bayesian inference. The genus Bradymyces is placed amidst members of the Trichomeriaceae and is ecologically undefined; it includes an opportunistic animal pathogen while two other species inhabit rock surfaces. ITS rDNA sequences of three species accepted in Bradymyces and other undescribed species and environmental samples were subjected to phylogenetic analysis and in-depth comparative analysis of ITS1 and ITS2 secondary structures in order to study their intraspecific variability. Compensatory base change criterion in the ITS2 secondary structure supported delimitation of species in Bradymyces, which manifest a limited number of phenotypic features useful for species recognition. The role of fungi in the speleothem biofilm and relationships of Bacillicladium and Bradymyces with other members of the Chaetothyriales are discussed.

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          MRBAYES: Bayesian inference of phylogenetic trees.

          The program MRBAYES performs Bayesian inference of phylogeny using a variant of Markov chain Monte Carlo. MRBAYES, including the source code, documentation, sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.
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            The non-Watson-Crick base pairs and their associated isostericity matrices.

            RNA molecules exhibit complex structures in which a large fraction of the bases engage in non-Watson-Crick base pairing, forming motifs that mediate long-range RNA-RNA interactions and create binding sites for proteins and small molecule ligands. The rapidly growing number of three-dimensional RNA structures at atomic resolution requires that databases contain the annotation of such base pairs. An unambiguous and descriptive nomenclature was proposed recently in which RNA base pairs were classified by the base edges participating in the interaction (Watson-Crick, Hoogsteen/CH or sugar edge) and the orientation of the glycosidic bonds relative to the hydrogen bonds (cis or trans). Twelve basic geometric families were identified and all 12 have been observed in crystal structures. For each base pairing family, we present here the 4 x 4 'isostericity matrices' summarizing the geometric relationships between the 16 pairwise combinations of the four standard bases, A, C, G and U. Whenever available, a representative example of each observed base pair from X-ray crystal structures (3.0 A resolution or better) is provided or, otherwise, theoretically plausible models. This format makes apparent the recurrent geometric patterns that are observed and helps identify isosteric pairs that co-vary or interchange in sequences of homologous molecules while maintaining conserved three-dimensional motifs.
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              Oily yeasts as oleaginous cell factories.

              Oily yeasts have been described to be able to accumulate lipids up to 20% of their cellular dry weight. These yeasts represent a minor proportion of the total yeast population, and only 5% of them have been reported as able to accumulate more than 25% of lipids. The oily yeast genera include Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon, and Lipomyces. More specifically, examples of oleaginous yeasts include the species: Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, and Yarrowia lipolytica. Yeast do exhibit advantages for lipid production over other microbial sources, namely, their duplication times are usually lower than 1 h, are much less affected than plants by season or climate conditions, and their cultures are more easily scaled up than those of microalgae. Additionally, some oily yeasts have been reported to accumulate oil up to 80% of their dry weight and can indeed generate different lipids from different carbon sources or from lipids present in the culture media. Thus, they can vary their lipid composition by replacing the fatty acids present in their triglycerides. Due to the diversity of microorganisms and growth conditions, oily yeasts can be useful for the production of triglycerides, surfactants, or polyunsaturated fatty acids.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                12 October 2016
                2016
                : 11
                : 10
                : e0163396
                Affiliations
                [1 ]Department of Taxonomy, Institute of Botany of the Czech Academy of Sciences, 252 43, Průhonice, Czech Republic
                [2 ]Department of Botany, Faculty of Science, Charles University in Prague, 128 01, Prague, 2, Czech Republic
                [3 ]Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology of the Czech Academy of Sciences, 142 20, Prague, 4, Czech Republic
                [4 ]Department of Ecology, Environment and Plant Sciences, Stockholm University, 106 91, Stockholm, Sweden
                [5 ]Department of Botany, Swedish Museum of Natural History, 104 05, Stockholm, Sweden
                [6 ]Department of Biology, University of Southern Denmark, 5230, Odense, Denmark
                [7 ]Department of Palaeobiology, Swedish Museum of Natural History, 104 05, Stockholm, Sweden
                University of California Riverside, UNITED STATES
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceived and designed the experiments: MI MW JL VH OT MR.

                • Performed the experiments: VH MI JL OT TS.

                • Analyzed the data: MR VH MW OT TS.

                • Contributed reagents/materials/analysis tools: MI VH MW.

                • Wrote the paper: MR VH MI MW JL OT.

                Author information
                http://orcid.org/0000-0001-5229-1709
                Article
                PONE-D-16-19064
                10.1371/journal.pone.0163396
                5061356
                27732675
                68594376-6214-4cc2-bb4c-212e66efb594
                © 2016 Réblová et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 May 2016
                : 14 August 2016
                Page count
                Figures: 14, Tables: 0, Pages: 36
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Award ID: Contract No. 2012-4364
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001859, Swedish National Space Board;
                Award ID: Contract No. 83/10
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100004359, Vetenskapsrådet;
                Award ID: VR 621-2012-3990
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001824, Grantová Agentura České Republiky;
                Award ID: GA CR 506/12/0038
                Award Recipient :
                Funded by: BIOCEV
                Award ID: CZ.1.05/1.1.00/02.0109
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001732, Danmarks Grundforskningsfond;
                Award ID: DNRF53
                Award Recipient :
                The research was funded by grants from the Swedish Research Council (Contract No. 2012-4364 and grant VR 621-2012-3990; www.vr.se), the Swedish National Space Board (Contract No. 83/10; www.snsb.se), Danish National Research Foundation (DNRF53; dg.dk), Czech Science Foundation (GA ČR project 506/12/0038; www.gacr.cz), by the Ministry of Education, Youth and Sports of the Czech Republic (SVV project; www.msmt.cz), and by the project “BIOCEV—Biotechnology and Biomedicine Centre of the Czech Academy of Sciences and Charles University” (CZ.1.05/1.1.00/02.0109; www.biocev.eu) from the European Regional Development Fund. Additional support was provided by a long-term research development project of the Institute of Botany of the Czech Academy of Sciences (No. RVO 67985939) to MR, and a long-term research development project of the Institute of Microbiology of the Czech Academy of Sciences (No. RVO 61388971) to VH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Organisms
                Fungi
                Biology and Life Sciences
                Microbiology
                Biofilms
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Phylogenetic Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Phylogenetic Analysis
                Biology and Life Sciences
                Mycology
                Fungal Structure
                Research and Analysis Methods
                Computational Techniques
                Split-Decomposition Method
                Multiple Alignment Calculation
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Custom metadata
                All relevant data are within the paper and its Supporting Information file. The DNA sequences determined for this study are deposited in EMBL (accession numbers LT558702- LT558709) and GenBank (accession numbers KX179909- KX179914).

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