Diversity of photosynthetic picoeukaryotes in eutrophic shallow lakes as assessed by combining flow cytometry cell-sorting and high throughput sequencing.

Photosynthetic picoeukaryotes (PPE) are key components of primary production in marine and freshwater ecosystems. In contrast with those of marine environments, freshwater PPE groups have received little attention. In this work, we used flow cytometry cell sorting, microscopy and metabarcoding to investigate the composition of small photosynthetic eukaryote communities from six eutrophic shallow lakes in South America, Argentina. We compared the total molecular diversity obtained from PPE sorted populations as well as from filtered total plankton samples (FTP). Most reads obtained from sorted populations belonged to the classes: Trebouxiophyceae, Chlorophyceae and Bacillariophyceae. We retrieved sequences from non-photosynthetic groups, such as Chytridiomycetes and Ichthyosporea which contain a number of described parasites, indicating that these organisms were probably in association with the autotrophic cells sorted. Dominant groups among sorted PPEs were poorly represented in FTP and their richness was on average lower than in the sorted samples. A significant number of operational taxonomic units (OTUs) were exclusively found in sorting samples, emphasizing that sequences from FTP underestimate the diversity of PPE. Moreover, 22% of the OTUs found among the dominant groups had a low similarity (<95%) with reported sequences in public databases, demonstrating a high potential for novel diversity in these lakes.

Active microbiome structure and its association with environmental factors and viruses at different aquatic sites of a high-altitude wetland.

Salar de Huasco is a high-altitude wetland characterized by a highly diverse microbial life adapted to extreme climatic and environmental conditions. Our study aims to determine active microbial community structure changes within different aquatic sites and its relationship with environmental factors and viruses as potential drivers of diversification in different aquatic areas of this ecosystem. In this study, bacteria and archaea composition (16S rRNA subunit pyrolibraries) and picoplankton and viral abundance were determined at ponds, springs and lagoon sites of the wetland during wet and dry seasons (February and July 2012, respectively). In general, mixosaline waters (1,400-51,000 µS/cm) usually found in ponds and lagoon presented higher picoplanktonic abundances compared to freshwater (<800 µS/cm) spring sites, ranging from 1.07 × 105 to 1.83 × 107  cells/ml. Viral abundance and viral to picoplankton ratio (VPR) also presented greater values at ponds compared to spring sites, reaching up to 4.78 × 108 viruses-like particles and up to 351 for VPR. In general, ponds hold a higher microbial diversity and complexity associated also with the presence of microbial mats compared with water sources or lagoon (Shannon index H' 2.6-3.9 vs. <2.0). A greater richness of archaea was also detected in ponds characterized by functional groups such as known methanogens and ammonia oxidizers, and uncultured groups. In total, our results indicate that among the different aquatic sites of the wetland, ponds presented a great microbial community diversification associated to a higher top-down control by viruses which may influence nutrient and greenhouse gases cycling.

Rare unclassified 16S rRNA operational taxonomic units from the uncharted Engaño Bay (Argentinean Patagonia).

Rare microbes make up most of the diversity of marine microbiomes, and recent works have highlighted their importance for microbial community dynamics and in fragmented habitats. Rare taxa have been infrequently studied in comparison with abundant groups, and rare unclassified sequences are common in culture-independent studies. Here, we describe a detailed analysis of nonclassifiable sequences from the Chubut river estuary at the Argentinean Patagonia. Standard taxonomic assignments of environmental 16S rRNA sequences resulted in about 13% unclassified operational taxonomic units (OTUs). The potential affiliations of these OTUs could be narrowed by mapping the classification software assignments on a phylogeny obtained directly from our environmental sequence data. Customized BLAST analyses were remarkably consistent with these phylogenetic assignments, especially when the unclassified OTUs were blasted against sequences from cultured and type microorganisms. In addition, our BLAST analyses revealed significant similarities between several unclassified OTUs and a plethora of unclassified sequences from around the world. Further phylogenetic comparisons with 6194 carefully selected reference sequences showed that these unclassified sequences may correspond to 5 unnamed groups, possibly encompassing ranks from subclass to family inside the Alphaproteobacteria, and to an unknown Gracilibacteria lineage. Overall, these results demonstrate the value of straight phylogenetic analysis, customized BLAST searches, and comparisons with sequences from type material, for the systematic study of rare unclassified sequences.

First freshwater member ever reported for the family Bathycoccaceae (Chlorophyta; Archaeplastida) from Argentinean Patagonia revealed by environmental DNA survey.

We characterized molecularly the first freshwater member ever reported for the family Bathycoccaceae in Lake Musters (Argentinean Patagonia). Members of this family are extremely numerous and play a key ecological role in marine systems as primary producers. We cloned a fragment comprising the SSU rRNA gene+ITS region from environmental DNA using specific mamiellophyte primers. The unique SSU rRNA gene sequence obtained clustered robustly with Bathycoccus prasinos. Analysis of the two-dimensional structure of the ITS region showed the presence of a typical supplementary helix in the ITS-2 region, a synapomorphy of Bathycoccaceae, which confirmed further its phylogenetic placement. We finally discuss the possible causes for the presence of this organism in Lake Musters.

Pico and nanoplankton abundance and carbon stocks along the Brazilian Bight.

Pico and nanoplankton communities from the Southwest Atlantic Ocean along the Brazilian Bight are poorly described. The hydrography in this region is dominated by a complex system of layered water masses, which includes the warm and oligotrophic Tropical Water (TW), the cold and nutrient rich South Atlantic Central Water (SACW) and the Coastal Water (CW), which have highly variable properties. In order to assess how pico- and nanoplankton communities are distributed in these different water masses, we determined by flow cytometry the abundance of heterotrophic bacteria, Prochlorococcus, Synechococcus and autotrophic pico and nanoeukaryotes along three transects, extending from 23°S to 31°S and 39°W to 49°W. Heterotrophic bacteria (including archaea, maximum of 1.5 × 106 cells mL-1) were most abundant in Coastal and Tropical Water whereas Prochlorococcus was most abundant in open-ocean oligotrophic waters (maximum of 300 × 103 cells mL-1). Synechococcus(up to 81 × 103 cells mL-1), as well as autotrophic pico and nanoeukaryotes seemed to benefit from the influx of nutrient-rich waters near the continental slope. Autotrophic pico and nanoeukaryotes were also abundant in deep chlorophyll maximum (DCM) layers from offshore waters, and their highest abundances were 20 × 103 cells mL-1 and 5 × 103 cells mL-1, respectively. These data are consistent with previous observations in other marine areas where Synechococcus and autotrophic eukaryotes dominate mesotrophic waters, whereas Prochlorococcus dominate in more oligotrophic areas. Regardless of the microbial community structure near the surface, the carbon stock dominance by autotrophic picoeukaryotes near the DCM is possibly linked to vertical mixing of oligotrophic surface waters with the nutrient-rich SACW and their tolerance to lower light levels.

Microbial food web components, bulk metabolism, and single-cell physiology of piconeuston in surface microlayers of high-altitude lakes.

Sharp boundaries in the physical environment are usually associated with abrupt shifts in organism abundance, activity, and diversity. Aquatic surface microlayers (SML) form a steep gradient between two contrasted environments, the atmosphere and surface waters, where they regulate the gas exchange between both environments. They usually harbor an abundant and active microbial life: the neuston. Few ecosystems are subjected to such a high UVR regime as high altitude lakes during summer. Here, we measured bulk estimates of heterotrophic activity, community structure and single-cell physiological properties by flow cytometry in 19 high-altitude remote Pyrenean lakes and compared the biological processes in the SML with those in the underlying surface waters. Phototrophic picoplankton (PPP) populations, were generally present in high abundances and in those lakes containing PPP populations with phycoerythrin (PE), total PPP abundance was higher at the SML. Heterotrophic nanoflagellates (HNF) were also more abundant in the SML. Bacteria in the SML had lower leucine incorporation rates, lower percentages of "live" cells, and higher numbers of highly-respiring cells, likely resulting in a lower growth efficiency. No simple and direct linear relationships could be found between microbial abundances or activities and environmental variables, but factor analysis revealed that, despite their physical proximity, microbial life in SML and underlying waters was governed by different and independent processes. Overall, we demonstrate that piconeuston in high altitude lakes has specific features different from those of the picoplankton, and that they are highly affected by potential stressful environmental factors, such as high UVR radiation.

Biomass distribution of heterotrophic and autotrophic microorganisms of the photic layer in Cuban southern oceanic waters

We measured the vertical and seasonal distribution of picoplankton (0.2-2 µm) and nanoplankton (2-20 µm) in the photic layer of Cuban southern oceanic and coastal waters. The concentration of the different fractions was estimated by epifluorescence microscopy. Heterotrophic components from the different fractions showed higher vertical stratification in the oceanic station in comparison to the coastal one. The autotrophic components showed an irregular vertical distribution pattern, both in coastal and oceanic stations. In all the analyzed stations, the heterotrophic bacteria showed an inverse correlation with the autotrophic (r= -0.98), and the heterotrophic nanoplankton (r= -0.96). Auto and heterotrophic nanoplankton probably regulate bacteria abundance by predation, although autotrophic nanoplankton may represent a source of organic matter for microorganisms. Rev. Biol. Trop. 55 (2): 449-457. Epub 2007 June, 29.

Determinamos la distribución vertical y estacional del picoplancton (0.2-2 µm) y el nanoplancton (2-20 µm) en la capa fótica de las aguas oceánicas y costeras al sur de Cuba. La concentración de las diferentes fracciones fue estimada mediante microscopía de epifluorescencia. Los componentes heterotróficos de las diferentes fracciones mostraron una elevada estratificación vertical en la estación oceánica en comparación con la estación costera. Por otro lado, los componentes autotróficos presentaron un patrón de distribución vertical irregular tanto en la estación costera como en la oceánica. En los análisis realizados las bacterias heterótrofas mostraron una correlación inversa con el nanoplancton autótrofo (r= -0.98), y con el nanoplancton heterótrofo (r= -0.96). Estos resultados sugieren que el nanoplancton (autótrofo y heterótrofo) probablemente regula la abundancia de bacterias mediante la depredación, además que el nanoplancton autótrofo pudiera representar una fuente de materia orgánica para los microorganismos.