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      Microbial Diversity in Hummock and Hollow Soils of Three Wetlands on the Qinghai-Tibetan Plateau Revealed by 16S rRNA Pyrosequencing

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          Abstract

          The wetlands of the Qinghai-Tibetan Plateau are believed to play an important role in global nutrient cycling, but the composition and diversity of microorganisms in this ecosystem are poorly characterized. An understanding of the effects of geography and microtopography on microbial populations will provide clues to the underlying mechanisms that structure microbial communities. In this study, we used pyrosequencing-based analysis of 16S rRNA gene sequences to assess and compare the composition of soil microbial communities present in hummock and hollow soils from three wetlands (Dangxiong, Hongyuan and Maduo) on the Qinghai-Tibetan Plateau, the world’s highest plateau. A total of 36 bacterial phyla were detected. Proteobacteria (34.5% average relative abundance), Actinobacteria (17.3%) and Bacteroidetes (11%) had the highest relative abundances across all sites. Chloroflexi, Acidobacteria, Verrucomicrobia, Firmicutes, and Planctomycetes were also relatively abundant (1–10%). In addition, archaeal sequences belonging to Euryarchaea, Crenarchaea and Thaumarchaea were detected. Alphaproteobacteria sequences, especially of the order Rhodospirillales, were significantly more abundant in Maduo than Hongyuan and Dangxiong wetlands. Compared with Hongyuan soils, Dangxiong and Maduo had significantly higher relative abundances of Gammaproteobacteria sequences (mainly order Xanthomonadales). Hongyuan wetland had a relatively high abundance of methanogens (mainly genera Methanobacterium, Methanosarcina and Methanosaeta) and methanotrophs (mainly Methylocystis) compared with the other two wetlands. Principal coordinate analysis (PCoA) indicated that the microbial community structure differed between locations and microtopographies and canonical correspondence analysis indicated an association between microbial community structure and soil properties or geography. These insights into the microbial community structure and the main controlling factors in wetlands of the Qinghai-Tibetan Plateau provide a valuable background for further studies on biogeochemical processes in this distinct ecosystem.

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          Fast UniFrac: Facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data

          Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis (PCoA) results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We demonstrate the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, revealing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way towards a broad range of applications and demonstrate some of the new features of Fast UniFrac.
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            Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction.

            Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.
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              Anaerolinea thermolimosa sp. nov., Levilinea saccharolytica gen. nov., sp. nov. and Leptolinea tardivitalis gen. nov., sp. nov., novel filamentous anaerobes, and description of the new classes Anaerolineae classis nov. and Caldilineae classis nov. in the bacterial phylum Chloroflexi.

              One thermophilic (strain IMO-1(T)) and two mesophilic (strains KIBI-1(T) and YMTK-2(T)) non-spore-forming, non-motile, Gram-negative, multicellular filamentous micro-organisms, which were previously isolated as members of the tentatively named class 'Anaerolineae' of the phylum Chloroflexi, were characterized. All isolates were strictly anaerobic micro-organisms. The length of the three filamentous isolates was greater than 100 microm and the width was 0.3-0.4 microm for strain IMO-1(T), 0.4-0.5 microm for strain KIBI-1(T) and thinner than 0.2 microm for strain YMTK-2(T). Strain IMO-1(T) could grow at pH 6.0-7.5 (optimum growth at pH 7.0). The optimal temperature for growth of strain IMO-1(T) was around 50 degrees C (growth occurred between 42 and 55 degrees C). Growth of the mesophilic strains KIBI-1(T) and YMTK-2(T) occurred at pH 6.0-7.2 with optimal growth at pH 7.0. Both of the mesophilic strains were able to grow in a temperature range of 25-50 degrees C with optimal growth at around 37 degrees C. Yeast extract was required for growth of all three strains. All the strains could grow with a number of carbohydrates in the presence of yeast extract. The G + C contents of the DNA of strains IMO-1(T), KIBI-1(T) and YMTK-2(T) were respectively 53.3, 59.5 and 48.2 mol%. Major fatty acids for thermophilic strain IMO-1(T) were anteiso-C(17 : 0), iso-C(15 : 0), C(16 : 0) and anteiso-C(15 : 0), whereas those for mesophilic strains KIBI-1(T) and YMTK-2(T) were branched C(14 : 0), iso-C(15 : 0), C(16 : 0) and branched C(17 : 0), and branched C(17 : 0), C(16 : 0), C(14 : 0) and C(17 : 0), respectively. Detailed phylogenetic analyses based on their 16S rRNA gene sequences indicated that the isolates belong to the class-level taxon 'Anaerolineae' of the bacterial phylum Chloroflexi, which for a long time had been considered as a typical uncultured clone cluster. Their morphological, physiological, chemotaxonomic and genetic traits strongly support the conclusion that these strains should be described as three novel independent taxa in the phylum Chloroflexi. Here, Anaerolinea thermolimosa sp. nov. (type strain IMO-1(T) = CM 12577(T) = DSM 16554(T)), Levilinea saccharolytica gen. nov., sp. nov. (type strain KIBI-1(T) = JCM 12578(T) = DSM 16555(T)) and Leptolinea tardivitalis gen. nov., sp. nov. (type strain YMTK-2(T) = JCM 12579(T) = DSM 16556(T)) are proposed. In addition, we formally propose to subdivide the tentative class-level taxon 'Anaerolineae' into Anaerolineae classis nov. and Caldilineae classis nov. We also propose the subordinate taxa Anaerolineales ord. nov., Caldilineales ord. nov., Anaerolineaceae fam. nov. and Caldilineaceae fam. nov.
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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
                2014
                31 July 2014
                : 9
                : 7
                : e103115
                Affiliations
                [1 ]Key Laboratory of Virtual Geographic Environment of the Ministry of Education, Nanjing Normal University, Nanjing, China
                [2 ]University of the Chinese Academy of Sciences, Beijing, China
                [3 ]Department of Biogeochemistry, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
                Argonne National Laboratory, United States of America
                Author notes

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

                Conceived and designed the experiments: YD XC MGD. Performed the experiments: YD. Analyzed the data: YD. Contributed reagents/materials/analysis tools: YD XC MH MGD. Wrote the paper: YD MGD.

                Article
                PONE-D-13-52198
                10.1371/journal.pone.0103115
                4117511
                25078273
                1f5c6c54-2321-4330-8da8-8737c2a37088
                Copyright @ 2014

                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
                : 12 December 2013
                : 27 June 2014
                Page count
                Pages: 10
                Funding
                YD was supported by a CAS/MPG doctoral promotion program fellowship and MH by a research fellowship of the Alexander von Humboldt Foundation. Support was also obtained from the National Natural Science Foundation of China (grant No. 11079053). YD acknowledges further support from the National Basic Research Program (2013CB956000), Strategic Priority Research Program (B) of the Chinese Academy of Sciences (XDB15010201), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. 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
                Biogeography
                Ecology
                Community Ecology
                Community Structure
                Biodiversity
                Ecosystems
                Microbial Ecology
                Microbiology
                Bacteriology
                Earth Sciences
                Geochemistry
                Biogeochemistry
                Ecology and Environmental Sciences
                Soil Science
                Soil Ecology

                Uncategorized
                Uncategorized

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