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      Evaluation of lake sedimentary ancient DNA metabarcoding to assess fungal biodiversity in Arctic paleoecosystems

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          Abstract

          Fungi are crucial organisms in most ecosystems as they exert ecological key functions and are closely associated with land plants. Fungal community changes may, therefore, help reveal biodiversity changes in past ecosystems. Lake sediments contain the DNA of organisms in the catchment area, which allows reconstructing past biodiversity by using metabarcoding of ancient sedimentary DNA. We re‐evaluated various commonly used metabarcoding primers, and we developed a novel PCR primer combination for fungal metabarcoding to produce a short amplicon, thus accounting for amplification bias due to the degradation of ancient DNA. In silico PCRs showed higher diversity using this new primer combination, compared with previously established fungal metabarcoding primers. We analyzed data from sediment cores from four artic and one boreal lake in Siberia. These cores had been stored for 2–22 years after coring; we, therefore, examined the degradation effects of ancient DNA and storage time‐related bias affecting fungal communities. Amplicon lengths showed considerable variation within and between the major divisions of fungi, for example, amplicons of Basidiomycota were significantly longer than those of Mucoromycota; however, we observed no significant effect of sample age on amplicon length and GC content, suggesting the robustness of our results. We also found no indication of post‐coring fungal growth during storage regarding the proportions of common mold taxa, which would otherwise distort conclusions on past fungal communities. Terrestrial soil fungi, including mycorrhizal fungi and saprotrophs, were predominant in all lakes, whereas typical aquatic taxa were only represented to a negligible extent, which supports the use of lake sedimentary ancient DNA for reconstructing terrestrial communities.

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          ITS primers with enhanced specificity for basidiomycetes--application to the identification of mycorrhizae and rusts.

          We have designed two taxon-selective primers for the internal transcribed spacer (ITS) region in the nuclear ribosomal repeat unit. These primers, ITS1-F and ITS4-B, were intended to be specific to fungi and basidiomycetes, respectively. We have tested the specificity of these primers against 13 species of ascomycetes, 14 of basidiomycetes, and 15 of plants. Our results showed that ITS4-B, when paired with either a 'universal' primer ITS1 or the fungal-specific primer ITS1-F, efficiently amplified DNA from all basidiomycetes and discriminated against ascomycete DNAs. The results with plants were not as clearcut. The ITS1-F/ITS4-B primer pair produced a small amount of PCR product for certain plant species, but the quantity was in most cases less than that produced by the 'universal' ITS primers. However, under conditions where both plant and fungal DNAs were present, the fungal DNA was amplified to the apparent exclusion of plant DNA. ITS1-F/ITS4-B preferential amplification was shown to be particularly useful for detection and analysis of the basidiomycete component in ectomycorrhizae and in rust-infected tissues. These primers can be used to study the structure of ectomycorrhizal communities or the distribution of rusts on alternate hosts.
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            Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi.

            Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups.
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              The biomass distribution on Earth

              Significance The composition of the biosphere is a fundamental question in biology, yet a global quantitative account of the biomass of each taxon is still lacking. We assemble a census of the biomass of all kingdoms of life. This analysis provides a holistic view of the composition of the biosphere and allows us to observe broad patterns over taxonomic categories, geographic locations, and trophic modes.
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                Author and article information

                Contributors
                (View ORCID Profile)
                (View ORCID Profile)
                Journal
                Environmental DNA
                Environmental DNA
                Wiley
                2637-4943
                2637-4943
                September 2022
                May 29 2022
                September 2022
                : 4
                : 5
                : 1150-1163
                Affiliations
                [1 ] Department of Biology Limnological Institute, University of Konstanz Konstanz Germany
                [2 ] Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research Polar Terrestrial Environmental Systems Potsdam Germany
                [3 ] Section for Genetics and Evolutionary Biology, Department of Biosciences University of Oslo Oslo Norway
                [4 ] Experimental and Clinical Research Center Charité Universitätsmedizin Berlin and Max Delbrück Center for Molecular Medicine Berlin Germany
                [5 ] Max Delbrück Center for Molecular Medicine in the Helmholtz Association Berlin Germany
                [6 ] Institute of Environmental Science and Geography University of Potsdam Potsdam Germany
                [7 ] Institute of Biochemistry and Biology University of Potsdam Potsdam Germany
                Article
                10.1002/edn3.315
                be5750eb-744f-4c2a-b567-899a39ee70a5
                © 2022

                http://creativecommons.org/licenses/by-nc/4.0/

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