Phylogenetic diversity in freshwater-dwelling Isochrysidales haptophytes with implications for alkenone production.

Members of the order Isochrysidales are unique among haptophyte lineages in being the exclusive producers of alkenones, long-chain ketones that are commonly used for paleotemperature reconstructions. Alkenone-producing haptophytes are divided into three major groups based largely on molecular ecological data: Group I is found in freshwater lakes, Group II commonly occurs in brackish and coastal marine environments, and Group III consists of open ocean species. Each group has distinct alkenone distributions; however, only Groups II and III Isochrysidales currently have cultured representatives. The uncultured Group I Isochrysidales are distinguished geochemically by the presence of tri-unsaturated alkenone isomers (C37:3b Me, C38:3b Et, C38:3b Me, C39:3b Et) present in water column and sediment samples, yet their genetic diversity, morphology, and environmental controls are largely unknown. Using small-subunit (SSU) ribosomal RNA (rRNA) marker gene amplicon high-throughput sequencing of environmental water column and sediment samples, we show that Group I is monophyletic with high phylogenetic diversity and contains a well-supported clade separating the previously described "EV" clade from the "Greenland" clade. We infer the first partial large-subunit (LSU) rRNA gene Group I sequence phylogeny, which uncovered additional well-supported clades embedded within Group I. Relative to Group II, Group I revealed higher levels of genetic diversity despite conservation of alkenone signatures and a closer evolutionary relationship with Group III. In Group I, the presence of the tri-unsaturated alkenone isomers appears to be conserved, which is not the case for Group II. This suggests differing environmental influences on Group I and II and perhaps uncovers evolutionary constraints on alkenone biosynthesis.

Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis.

BACKGROUND: Human-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required. RESULTS: We characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%-99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples. CONCLUSIONS: Fungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

VAMPS: a website for visualization and analysis of microbial population structures.

BACKGROUND: The advent of next-generation DNA sequencing platforms has revolutionized molecular microbial ecology by making the detailed analysis of complex communities over time and space a tractable research pursuit for small research groups. However, the ability to generate 105-108 reads with relative ease brings with it many downstream complications. Beyond the computational resources and skills needed to process and analyze data, it is difficult to compare datasets in an intuitive and interactive manner that leads to hypothesis generation and testing. RESULTS: We developed the free web service VAMPS (Visualization and Analysis of Microbial Population Structures, http://vamps.mbl.edu) to address these challenges and to facilitate research by individuals or collaborating groups working on projects with large-scale sequencing data. Users can upload marker gene sequences and associated metadata; reads are quality filtered and assigned to both taxonomic structures and to taxonomy-independent clusters. A simple point-and-click interface allows users to select for analysis any combination of their own or their collaborators' private data and data from public projects, filter these by their choice of taxonomic and/or abundance criteria, and then explore these data using a wide range of analytic methods and visualizations. Each result is extensively hyperlinked to other analysis and visualization options, promoting data exploration and leading to a greater understanding of data relationships. CONCLUSIONS: VAMPS allows researchers using marker gene sequence data to analyze the diversity of microbial communities and the relationships between communities, to explore these analyses in an intuitive visual context, and to download data, results, and images for publication. VAMPS obviates the need for individual research groups to make the considerable investment in computational infrastructure and bioinformatic support otherwise necessary to process, analyze, and interpret massive amounts of next-generation sequence data. Any web-capable device can be used to upload, process, explore, and extract data and results from VAMPS. VAMPS encourages researchers to share sequence and metadata, and fosters collaboration between researchers of disparate biomes who recognize common patterns in shared data.