Use of  flow cytometric sorting to better assess the diversity of small photosynthetic eukaryotes in the English Channel

Abstract

Small photosynthetic eukaryotes are key primary producers in marine waters. In recent years, their diversity has been studied by the analysis of 18S rRNA gene sequences directly amplified and cloned from filtered natural samples. However, these clone libraries are often dominated by nonphotosynthetic organisms and few sequences from autotrophs are recovered. In the present paper, we developed a new approach based on flow cytometry. Photosynthetic pico-, nano- and phycoerythrin-containing (PE-) eukaryotes from the coastal English Channel were sorted based on their size and pigment fluorescence. 18S rRNA gene libraries were constructed from the DNA of sorted cells. We addressed methodological issues linked to the relatively low concentration of these cells. This novel approach confirmed that, in the English Channel, pico-eukaryotes are dominated by three genera Micromonas, Ostreococcus and Bathycoccus, while PE-eukaryotes are mainly cryptophytes from clade 4. It also revealed that nano-eukaryotes are dominated by haptophytes with important contributions from small diatoms and Prasinophyceae. It should be emphasized that haptophytes were nearly absent from clone libraries constructed from filtered samples, which explains why they have been overlooked in previous studies. The new strategy should be very useful to conduct similar studies on other specific populations that can be discriminated by flow cytometry (e.g. red tide organisms or uncultivated protists).

Design and Evaluation of Illumina MiSeq-Compatible, 18S rRNA Gene-Specific Primers for Improved Characterization of Mixed Phototrophic Communities

“…Consistent with this effect, all mismatches occurred in GC-rich regions and were predominantly substitution errors. Significantly, haptophytes have been shown to be routinely underrepresented when in the presence of other eukaryotic DNA (76,77), and Marie et al found that haptophytes present in marine samples had higher GC contents along the 18S rRNA gene than the other groups present (e.g., 49.2% for Haptophyta compared to 45.9% for Chlorophyta) (77).…”

Phylogeny and Molecular Evolution of the Green Algae

“…Beside portals dedicated to the annotation of each of the different genomes, web sites focused on comparative analysis between the different organisms belonging to the green lineage, as PLAZA (bioinformatics.psb.ugent.be/plaza/) (Proost et al, 2009) and its derivative Pico-PLAZA (bioinformatics.psb.ugent.be/pico-plaza/) are now available. Such tools for comparative approaches, coupled to the development of new sequencing possibilities for both new genomes and metagenomes (Cheung et al, 2010;Marie et al, 2010) from various environments open the door to a better understanding of the adaptations of the organisms to their environment.…”

Taming the smallest predators of the oceans

“…The 18 S Smallest predators of the oceans J del Campo et al Smallest predators of the oceans J del Campo et al rDNA sequence of our isolate was highly similar to environmental sequences retrieved from the Mediterranean Sea (BL000921.31 and BL010625. 12; Massana et al, 2004), the Sargasso Sea (SSRPE06; Not et al, 2007), and the English Channel (RA070625T.047; Marie et al, 2010). Therefore, we decided to focus in the rhizarian isolate for the rest of our study, given its taxonomic novelty and its similarity with environmental marine sequences.…”