Environmental Barcoding Reveals Massive Dinoflagellate Diversity in Marine Environments

Rowena F. Stern is with University of British Columbia, Ales Horak is with University of British Columbia, Rose L. Andrew is with University of British Columbia, Mary-Alice Coffroth is with State University of New York at Buffalo, Robert A. Andersen is with the Bigelow Laboratory for Ocean Sciences, Frithjof C. Küpper is with the Scottish Marine Institute, Ian Jameson is with CSIRO Marine and Atmospheric Research, Mona Hoppenrath is with the German Center for Marine Biodiversity Research, Benoît Véron is with University of Caen Lower Normandy and the National Institute for Environmental Studies, Fumai Kasai is with the National Institute for Environmental Studies, Jerry Brand is with UT Austin, Erick R. James is with University of British Columbia, Patrick J. Keeling is with University of British Columbia.Background -- Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known “species”, as a reference to measure the natural diversity in three marine environments. Methodology/Principal Findings -- In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species. Conclusions/Significance -- COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.This project was funded by Genome Canada and the Canadian Barcode of Life Network. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

In situ identification and localization of bacteria associated with Gyrodinium instriatum (Gymnodiniales, Dinophyceae) by electron and confocal microscopy

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions

Seasonal dynamics of bacterioplankton community structure at a coastal station in the western English Channel

An annual study of the bacterioplankton community structure was carried out at Stn L4 (50° 15’ N, 04° 13’ W) in the western English Channel between August 2003 and July 2004. Bacterioplankton abundance and community structure were assessed using flow cytometry and fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes, respectively. The Eubacteria domain dominated over the Archaea domain (<15%) at the highest phylogenetic level. The Sphingobacteria-Flavobacteria group of the Bacteroidetes phylum (SFB) numerically dominated in spring and early summer. The ?-Proteobacteria dominated from late summer to winter. The SAR11 clade represented ~13% of the microbial community throughout the year and accounted for up to 69% of ?-Proteobacteria in late spring. Annually, ?-Proteobacteria were 2 or 3 times less abundant than the other groups and showed no obvious seasonal trend. The SAR86 cluster accounted for up to half of ?-Proteobacteria when it peaked in summer. Consequently, we found that community structure at higher taxonomic level did not change dramatically with season but lower level phylogenetic groups showed pronounced seasonal peaks

Nutrient control of N₂ fixation in the oligotrophic Mediterranean Sea and the impact of Saharan dust events

International audienceA better understanding of the factors controlling N2 fixation is a pre-requisite for improving our knowledge on the contribution of N2 fixation process in the nitrogen cycling. Trace-metal clean nutrient/dust addition bioassays (+P, +PFe, +dust) were performed at three stations located in the western, central and eastern Mediterranean Sea, in summer 2008 as part of the BOUM cruise. The main goals were (1) to investigate the nutrient factor(s) limiting N2 fixation (uptake of 15N2) and (2) to evaluate the potential impact of a Saharan dust event on this biological process during the stratification period. Initially, surface waters at the three stations were DIP-depleted (2 fixation (from 130 % to 430 %). The highest dust stimulation of N2 fixation was recorded at the station located in the eastern basin. The response of diazotrophic activity to nutrient additions was variable between the sampled stations suggesting a spatial variability of the factor controlling N2 fixation over the whole basin. At all stations, N2 fixation was not limited by Fe nor co-limited by P and Fe. At the western station, N2 fixation was DIP limited while at the eastern one, N2 fixation was first DIP limited, then was limited by one or several chemical element(s) released by dust. Our results demonstrated that a Saharan dust input was able to relieve these successive on going limitations. Very interestingly, at the station located in the central basin, N2 fixation was not limited by the availability of P yet it was strongly stimulated by dust addition (x3.1). A chemical element or a combination of several, released by the added dust may have been responsible for the observed stimulations of N2 fixation. These results indicated that Saharan dust pulses to the surface Mediterranean waters, in addition to P and Fe, could be a source of chemical(s) element(s) that are necessary for metabolic processes and therefore influence rates of N2 fixation

Project DIAPICNA -DIAzotrophic PIco-Cyanobacteria in the North Atlantic open ocean: their abundance and importance as a source of new nitrogen at the Azores Front/Current

International audienceNutrient concentrations indicate an oligotrophic area with nutrient bottom regeneration at the North of the Azores Current Front (Station 4, 35°N, Graph 2, Left). Nitrate isotopes indicate nitrate assimilation at Station 4, while depleted signatures South of the Front might result from N 2 fixation (Graph 2, Right)

Project DIAPICNA -DIAzotrophic PIco-Cyanobacteria in the North Atlantic open ocean: their abundance and importance as a source of new nitrogen at the Azores Front/Current

International audienceNutrient concentrations indicate an oligotrophic area with nutrient bottom regeneration at the North of the Azores Current Front (Station 4, 35°N, Graph 2, Left). Nitrate isotopes indicate nitrate assimilation at Station 4, while depleted signatures South of the Front might result from N 2 fixation (Graph 2, Right)

Project DIAPICNA -DIAzotrophic PIco-Cyanobacteria in the North Atlantic open ocean: their abundance and importance as a source of new nitrogen at the Azores Front/Current

International audienceNutrient concentrations indicate an oligotrophic area with nutrient bottom regeneration at the North of the Azores Current Front (Station 4, 35°N, Graph 2, Left). Nitrate isotopes indicate nitrate assimilation at Station 4, while depleted signatures South of the Front might result from N 2 fixation (Graph 2, Right)