Summary Although protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico‐, nano‐ and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta‐diversity analyses split the protist community structure into three main clusters: picoplankton‐nanoplankton‐dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA‐based surveys provided similar relative abundances for most class‐level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)‐I and MALV‐II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date.

The class Prasinophyceae (Chlorophyta) contains several photosynthetic picoeukaryotic species described from cultured isolates. The ecology of these organisms and their contributions to the picoeukaryotic community in aquatic ecosystems have received little consideration. We have designed and tested eight new 18S ribosomal DNA oligonucleotide probes specific for different Prasinophyceae clades, genera, and species. Using fluorescent in situ hybridization associated with tyramide signal amplification, these probes, along with more general probes, have been applied to samples from a marine coastal site off Roscoff (France) collected every 2 weeks between July 2000 and September 2001. The abundance of eukaryotic picoplankton remained high (>10 3 cells ml ؊1 ) during the sampling period, with maxima in summer (up to 2 ؋ 10 4 cells ml ؊1 ), and a single green algal species, Micromonas pusilla (Prasinophyceae), dominated the community all year round. Members of the order Prasinococcales and the species Bathycoccus prasinos (Mamiellales) displayed sporadic occurrences, while the abundances of all other Prasinophyceae groups targeted remained negligible.Several studies have demonstrated the importance of eukaryotic picoplankton (cell size, 0.2-to 3-m) in terms of biomass and productivity in the euphotic zone of oceanic oligotrophic waters (15), as well as in coastal waters (10). To date, only ϳ40 species belonging to nine algal classes (Chlorophyceae, Prasinophyceae, Trebouxiophyceae, Prymnesiophyceae, Bolidophyceae, Eustigmatophyceae, Pinguiophyceae, Bacillariophyceae, and Pelagophyceae) of photosynthetic picoplanktonic eukaryotes have been formerly described (41). However, phylogenetic analyses of sequences retrieved from natural samples in different oceanic regions have demonstrated much higher diversity, since many of these sequences do not correspond to any described taxa (19). The contributions of the different taxonomic groups to the picoplanktonic biomass, diversity, and ecology are poorly known because simple and reliable methods to detect and quantify such organisms in natural samples are lacking. Pigment signatures, scanning electron microscopy, and serial dilution cultures suggest that the classes Prasinophyceae (division Chlorophyta), Pelagophyceae (division Heterokontophyta), and Prymnesiophyceae are major components of the picoplankton biomass in different marine systems (20,35).Among these, the class Prasinophyceae contains several photosynthetic picoeukaryote species. This class is considered to be the most primitive in the green lineage and to have given rise to all other green algal classes, as well as to the land plants (34). Members are known to be common in temperate and cold regions and can occur as prominent constituents of marine picoplankton (38). Within these organisms, genera such as Ostreococcus, Bathycoccus, and Micromonas have been described in coastal waters (4b, 6). Micromonas pusilla (the only described species in the genus Micromonas) has been identified as a major component of the pic...

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We investigated marine picoeukaryotic diversity (cells Ͻ3 m) in samples collected in late summer 2002 at the boundary between the Norwegian, Greenland, and Barents Seas. The two main Arctic and Atlantic water masses in this region are separated by the polar front. We combined total counts of picoeukaryotes assemblages by flow cytometry and epifluorescence microscopy with taxa detection by tyramide signal amplification-fluorescent in situ hybridization (TSA-FISH) and high performance liquid chromatography (HPLC) pigment analyses. The picoeukaryotic community was primarily composed of photoautotrophs (75% of the cells on average). Members of the division Chlorophyta, in particular the species Micromonas pusilla (Butcher) Manton and Parke, were the major components in truly Arctic waters (32% of the picoeukaryotes, maximum 3,200 cells ml Ϫ1 ). M. pusilla was also well represented in coastal waters and at the polar front (25% of the picoeukaryotes, maximum 9,100 cells ml Ϫ1). Haptophyta were prominent in more typical Atlantic waters (up to 35% of the picoeukaryotes, maximum 4,500 cells ml Ϫ1 ). Quantification of haptophyte biomass by HPLC pigment analyses and CHEMTAX, and haptophyte abundances by TSA-FISH were in good agreement. This confirms previous studies, which suggested that M. pusilla is a dominant contributor of picoeukaryotic communities in both coastal and nutrient rich environments, whereas haptophytes seem to be more important in open seawaters.

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Photosynthetic picoeukaryotes (phytoplankton cells with a diameter smaller than 2 to 3 µm) contribute significantly to both biomass and primary production in the oligotrophic open ocean and coastal waters, at certain times of the year. The identification of these organisms is difficult because of their small size and simple morphology, therefore hindering detailed ecological studies of their distribution and role. In this paper, we demonstrate the use of oligonucleotide probes specific to algal classes or to lower order taxa in combination with fluorescent in situ hybridization and tyramide signal amplification (FISH-TSA) to determine eukaryotic picophytoplankton diversity. Target cells were detected and enumerated using epifluorescence microscopy. The sensitivity of the technique and the specificity of the probes were tested on pure and mixed picoplanktonic strains, as well as on natural samples from the English Channel. In these samples, the community was dominated by cells belonging to the division Chlorophyta. Haptophyta, Bolidophyceae and Pelagophyceae were also detected at low abundance. The FISH-TSA method is readily applicable to the study of picoplankton diversity in natural communities. KEY WORDS: Fluorescent in situ hybridization · Tyramide signal amplification · Picoplankton · Eukaryotes · Coastal waters · DiversityResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 28: 157-166, 2002 samples permit culture-independent assessments of diversity and indicate that there is a considerable number of yet uncultured species in eukaryotic picoplankton communities (Lopez-Garcia et al. 2001, Moon-van der Staay et al. 2001. The enumeration of specific taxonomic groups and the estimation of their contribution to eukaryotic picoplankton, however, are more difficult. Photosynthetic pigments have been widely used to estimate the contribution of algal classes to total chlorophyll a (chl a) biomass (Latasa & Bidigare 1998). However, this method is limited for several reasons. First, it cannot resolve diversity below the class level. Second, it is based on the assumption that the relative cellular content of diagnostic pigments is constant for a given algal group. This assumption is invalid because pigment content varies between species, as well as within species. For a given strain, it is also affected by environmental conditions (e.g. Stolte et al. 2000).Whole cell fluorescent in situ hybridization (FISH) with rRNA-targeted nucleic acid probes has been used more and more extensively to detect bacteria (Amann 1995). This method, which combines identification with quantitative determination of cell number, has been successfully applied to complex bacteria communities such as biofilms (Brümmer et al. 2000), marine sediments (Llobet-Brossa et al. 1998) and soils (Ravenschlag et al. 2000). Fluorescent rRNA probes have not been used as widely for eukaryotic phytoplankton (Simon et al. 1995, Scholin et al. 1996. Attempts to use mono-labeled oligonucleotide probes for...

Because of their tiny size (0.2 to 2 m), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico-and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.

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