Groups without cultured representatives dominate eukaryotic picophytoplankton in the oligotrophic South East Pacific Ocean

Background: Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone. Methodology/Principal Findings: In order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated. Conclusions/Significance: Our data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups

Evaluation of Alternative High-Throughput Sequencing Methodologies for the Monitoring of Marine Picoplanktonic Biodiversity Based on rRNA Gene Amplicons

16 pages, 6 figures, 2 tablesSequencing of rRNA gene polymerase chain reaction amplicons (rRNA tags) is the most common approach for investigating microbial diversity. The recent development of high-throughput sequencing (HTS) technologies has enabled the exploration of microbial biodiversity at an unprecedented scale, greatly expanding our knowledge on the microbiomes of marine ecosystems. These approaches provide accurate, fast, and cost efficient observations of the marine communities, and thus, may be suitable tools in biodiversity monitoring programs. To reach this goal, consistent and comparable methodologies must be used over time and within sites. Here, we have performed a cross-platform study of the two most common HTS methodologies, i.e., 454-pyrosequencing and Illumina tags to evaluate their usefulness in biodiversity monitoring and assessment of environmental status. Picoplankton biodiversity has been compared through both methodologies by sequencing the 16 and 18 S rRNA genes of a set of samples collected in the coast of Barcelona (NW Mediterranean). The results show that, despite differences observed in the rare OTUs retrieved, both platforms provide a comparable view of the marine picoplankton communities. On a taxonomic level, there was an accurate overlap in the detected phyla between the two methods and the overall estimates of alpha- and betadiversity were comparable. In addition, we explored the concept of “indicator species” and found that certain taxa (i.e., members of the Gammaproteobacteria among others) as well as the ratio between some phylogenetic groups (i.e., the ratio of Alphaproteobacteria/Gammaproteobacteria, Alteromonas/SAR11, and Alteromonas + Oceanospirillales/SAR11) have potential for being useful indicators of environmental status. The data show that implementing new protocols and identifying indicators of environmental status based on rRNA amplicon sequencing is feasible, and that is worth exploring whether the identified indices are universally applicableThis manuscript is a result of DEVOTES (DEVelopment Of innovative Tools for understanding marine biodiversity and assessing GEnS) project, funded by the European Union (grant agreement no. 308392), and a MINECO Grant GRADIENTS Fine-scale structure of cross-shore GRADIENTS along the Mediterranean coast (CTM2012-39476-C02)Peer Reviewe

SMASHing the LMC: A Tidally-induced Warp in the Outer LMC and a Large-scale Reddening Map

We present a study of the three-dimensional (3D) structure of the Large Magellanic Cloud (LMC) using ~2.2 million red clump (RC) stars selected from the Survey of the MAgellanic Stellar History. To correct for line-of-sight dust extinction, the intrinsic RC color and magnitude and their radial dependence are carefully measured by using internal nearly dust-free regions. These are then used to construct an accurate 2D reddening map (165 square degrees with ~10 arcmin resolution) of the LMC disk and the 3D spatial distribution of RC stars. An inclined disk model is fit to the 2D distance map yielding a best-fit inclination angle i = 25.86(+0.73,-1.39) degrees with random errors of +\-0.19 degrees and line-of-nodes position angle theta = 149.23(+6.43,-8.35) degrees with random errors of +/-0.49 degrees. These angles vary with galactic radius, indicating that the LMC disk is warped and twisted likely due to the repeated tidal interactions with the Small Magellanic Cloud (SMC). For the first time, our data reveal a significant warp in the southwestern part of the outer disk starting at rho ~ 7 degrees that departs from the defined LMC plane up to ~4 kpc toward the SMC, suggesting that it originated from a strong interaction with the SMC. In addition, the inner disk encompassing the off-centered bar appears to be tilted up to 5-15 degrees relative to the rest of the LMC disk. These findings on the outer warp and the tilted bar are consistent with the predictions from the Besla et al. simulation of a recent direct collision with the SMC.Comment: 25 pages, 15 figures, published in Ap

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Journal ArticleEnvironmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.Gordon and Betty Moore FoundationDeutsche ForschungsgemeinschaftMarie Curie International Outgoing FellowshipMarie Curie Intra-European FellowshipEMBO Long-Term fellowshi

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

International audienceThe interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists

State recognition for ‘contested languages’: a comparative study of Sardinian and Asturian, 1992–2010

While the idea of a named language as a separate and discrete identity is a political and social construct, in the cases of Sardinian and Asturian doubts over their respective ‘languageness’ have real material consequences, particularly in relation to language policy decisions at the state level. The Asturian example highlights how its lack of official status means that it is either ignored or subjected to repeated challenges to its status as a language variety deserving of recognition and support, reflecting how ‘official language’ in the Spanish context is often understood in practice as synonymous with the theoretically broader category of ‘language’. In contrast, the recent state recognition of Sardinian speakers as a linguistic minority in Italy (Law 482/1999) illustrates how legal recognition served to overcome existing obstacles to the implementation of regional language policy measures. At the same time, the limited subsequent effects of this Law, particularly in the sphere of education, are a reminder of the shortcomings of top-down policies which fail to engage with the local language practices and attitudes of the communities of speakers recognized. The contrastive focus of this article thus acknowledges the continued material consequences of top-down language classification, while highlighting its inadequacies as a language policy mechanism which reinforces artificial distinctions between speech varieties and speakers deserving of recognition

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.This work was supported by the projects CGL2012-39627-C03-01 and 02 of the Spanish Ministry of Economy and Competitiveness, which were also co-financed with FEDER support from the European Union. TG group research is funded in part by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2012-37161), a grant from the Qatar National Research Fund grant (NPRP 5-298-3-086) and a grant from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC (grant agreement no. ERC-2012-StG-310325)

In situ prey selection of mixotrophic and heterotrophic flagellates in Antarctic oligotrophic lakes: An analysis of the digestive vacuole content

We investigated the selective predation of mixotrophic and heterotrophic flagellates (MF and HF) on different heterotrophic prokaryote phylotypes (HPP; Bacteria + Archaea) living in natural assemblages from oligotrophic Antarctic lakes. In situ prey preference was analyzed for the first time on different mixotrophic taxa (Pseudopedinella sp., Ochromonas-like cells, Chrysophyceae >5 μm). The relative abundances of seven different HPP hybridized by CARD-FISH (catalyzed reporter deposition-fluorescent in situ hybridization) in natural community were compared with the proportions of hybridized cells inside digestive vacuoles. Our results showed some general trends to selectivity over some HPP. Alphaproteobacteria and Betaproteobacteria were the most abundant groups, and strikingly, a negative selection trend was detected in most samples by all bacterivorous protists. In contrast, for Actinobacteria a positive selection trend was observed in most samples, whereas Bacteroidetes seemed to be randomly preyed upon. Interestingly, similar prey preferences were observed in all bacterivorous flagellates. Our results suggest that phylogenetic affiliation determines part of the process of prey selection by protists in these lakes. Nevertheless, other features, such as cell size, morphology and the presence of the S-layer, might also significantly contribute to prey selectivity on the HPP. © 2012 The Author 2012. Published by Oxford University Press. All rights reserved.Fil:Queimaliños, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Schiaffino, M.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Izaguirre, I. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.Fil:Unrein, F. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina