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      Fungal Diversity Is Not Determined by Mineral and Chemical Differences in Serpentine Substrates

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          Abstract

          The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter.

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          Most cited references27

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          MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment.

          S. KUMAR (2004)
          With its theoretical basis firmly established in molecular evolutionary and population genetics, the comparative DNA and protein sequence analysis plays a central role in reconstructing the evolutionary histories of species and multigene families, estimating rates of molecular evolution, and inferring the nature and extent of selective forces shaping the evolution of genes and genomes. The scope of these investigations has now expanded greatly owing to the development of high-throughput sequencing techniques and novel statistical and computational methods. These methods require easy-to-use computer programs. One such effort has been to produce Molecular Evolutionary Genetics Analysis (MEGA) software, with its focus on facilitating the exploration and analysis of the DNA and protein sequence variation from an evolutionary perspective. Currently in its third major release, MEGA3 contains facilities for automatic and manual sequence alignment, web-based mining of databases, inference of the phylogenetic trees, estimation of evolutionary distances and testing evolutionary hypotheses. This paper provides an overview of the statistical methods, computational tools, and visual exploration modules for data input and the results obtainable in MEGA.
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            Microbial diversity and function in soil: from genes to ecosystems.

            Soils sustain an immense diversity of microbes, which, to a large extent, remains unexplored. A range of novel methods, most of which are based on rRNA and rDNA analyses, have uncovered part of the soil microbial diversity. The next step in the era of microbial ecology is to extract genomic, evolutionary and functional information from bacterial artificial chromosome libraries of the soil community genomes (the metagenome). Sophisticated analyses that apply molecular phylogenetics, DNA microarrays, functional genomics and in situ activity measurements will provide huge amounts of new data, potentially increasing our understanding of the structure and function of soil microbial ecosystems, and the interactions that occur within them. This review summarizes the recent progress in studies of soil microbial communities with focus on novel methods and approaches that provide new insight into the relationship between phylogenetic and functional diversity.
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              ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases

              Background During the last 15 years the internal transcribed spacer (ITS) of nuclear DNA has been used as a target for analyzing fungal diversity in environmental samples, and has recently been selected as the standard marker for fungal DNA barcoding. In this study we explored the potential amplification biases that various commonly utilized ITS primers might introduce during amplification of different parts of the ITS region in samples containing mixed templates ('environmental barcoding'). We performed in silico PCR analyses with commonly used primer combinations using various ITS datasets obtained from public databases as templates. Results Some of the ITS primers, such as ITS1-F, were hampered with a high proportion of mismatches relative to the target sequences, and most of them appeared to introduce taxonomic biases during PCR. Some primers, e.g. ITS1-F, ITS1 and ITS5, were biased towards amplification of basidiomycetes, whereas others, e.g. ITS2, ITS3 and ITS4, were biased towards ascomycetes. The assumed basidiomycete-specific primer ITS4-B only amplified a minor proportion of basidiomycete ITS sequences, even under relaxed PCR conditions. Due to systematic length differences in the ITS2 region as well as the entire ITS, we found that ascomycetes will more easily amplify than basidiomycetes using these regions as targets. This bias can be avoided by using primers amplifying ITS1 only, but this would imply preferential amplification of 'non-dikarya' fungi. Conclusions We conclude that ITS primers have to be selected carefully, especially when used for high-throughput sequencing of environmental samples. We suggest that different primer combinations or different parts of the ITS region should be analyzed in parallel, or that alternative ITS primers should be searched for.
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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, USA )
                1932-6203
                2012
                20 September 2012
                : 7
                : 9
                : e44233
                Affiliations
                [1 ]Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy
                [2 ]Interdepartmental Centre “G. Scansetti” for Studies on Asbestos and Other Toxic Particulates, University of Torino, Torino, Italy
                [3 ]Unité Mixte de Recherche de l'Institut National de la Recherche Agronomique - Lorraine Université ‘Interactions Arbres/Microorganismes’, Centre de Nancy - Champenoux, France
                Dowling College, United States of America
                Author notes

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

                Conceived and designed the experiments: SD CM. Performed the experiments: SD ES. Analyzed the data: SD CM ES MG. Contributed reagents/materials/analysis tools: SP. Wrote the paper: SD CM MG SP.

                Article
                PONE-D-12-15636
                10.1371/journal.pone.0044233
                3447857
                23028507
                1e04587d-a9cf-47e9-8806-e3e2ac9fd91a
                Copyright @ 2012

                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
                : 28 May 2012
                : 30 July 2012
                Page count
                Pages: 13
                Funding
                Dr. Daghino was supported by a fellowship from Regione Piemonte. Local funding by the University of Torino and Fondazione San Paolo is also gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Ecology
                Biodiversity
                Microbial Ecology
                Soil Ecology
                Genomics
                Metagenomics
                Microbiology
                Mycology
                Fungal Classification
                Fungi
                Microbial Ecology
                Earth Sciences
                Mineralogy
                Minerals

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                Uncategorized

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