Small eukaryotes, cells with a diameter of less than 5 m, are fundamental components of lacustrine planktonic systems. In this study, small-eukaryote diversity was determined by sequencing cloned 18S rRNA genes in three libraries from lakes of differing trophic status in the Massif Central, France: the oligotrophic Lake Godivelle, the oligomesotrophic Lake Pavin, and the eutrophic Lake Aydat. This analysis shows that the least diversified library was in the eutrophic lake (12 operational taxonomic units [OTUs]) and the most diversified was in the oligomesotrophic lake (26 OTUs). Certain groups were present in at least two ecosystems, while the others were specific to one lake on the sampling date. Cryptophyta, Chrysophyceae, and the strictly heterotrophic eukaryotes, Ciliophora and fungi, were identified in the three libraries. Among the small eukaryotes found only in two lakes, Choanoflagellida and environmental sequences (LKM11) were not detected in the eutrophic system whereas Cercozoa were confined to the oligomesotrophic and eutrophic lakes. Three OTUs, linked to the Perkinsozoa, were detected only in the Aydat library, where they represented 60% of the clones of the library. Chlorophyta and Haptophyta lineages were represented by a single clone and were present only in Godivelle and Pavin, respectively. Of the 127 clones studied, classical pigmented organisms (autotrophs and mixotrophs) represented only a low proportion regardless of the library's origin. This study shows that the small-eukaryote community composition may differ as a function of trophic status; certain lineages could be detected only in a single ecosystem.

The diversity of small eukaryotes (0.2 to 5 m) in a mesotrophic lake (Lake Bourget) was investigated using 18S rRNA gene library construction and fluorescent in situ hybridization coupled with tyramide signal amplification (TSA-FISH). Samples collected from the epilimnion on two dates were used to extend a data set previously obtained using similar approaches for lakes with a range of trophic types. A high level of diversity was recorded for this system with intermediate trophic status, and the main sequences from Lake Bourget were affiliated with ciliates (maximum, 19% of the operational taxonomic units [OTUs]), cryptophytes (33%), stramenopiles (13.2%), and cercozoa (9%). Although the comparison of TSA-FISH results and clone libraries suggested that the level of Chlorophyceae may have been underestimated using PCR with 18S rRNA primers, heterotrophic organisms dominated the small-eukaryote assemblage. We found that a large fraction of the sequences belonged to potential parasites of freshwater phytoplankton, including sequences affiliated with fungi and Perkinsozoa. On average, these sequences represented 30% of the OTUs (40% of the clones) obtained for each of two dates for Lake Bourget. Our results provide information on lacustrine small-eukaryote diversity and structure, adding to the phylogenetic data available for lakes with various trophic types.It has been recognized that the eukaryotic component of picoplankton plays a critical role in the functioning of aquatic ecosystems (29, 34). However, numerous members of this community possess very few morphological characteristics that can be used for identification by traditional microscopic methods. Thus, molecular techniques offer a very useful alternative for the elucidation and description of picoeukaryotic plankton diversity in aquatic environments. Numerous studies have revealed new lineages and unexpected diversity of picoeukaryotes in the open ocean (7, 29, 37), coastal areas (35, 46), anaerobic sediments (5), acid rivers (2), and deep-sea vents (11,30). However, the diversity, distribution, and natural abundance of small-eukaryote taxa in freshwater systems are still poorly known. Nevertheless, a few recent studies have reported high diversity of 18S rRNA sequences in this community (Ͻ5 m) in lakes and general dominance of heterotrophic cells in the small-eukaryote assemblage (25,27,45). A salient feature of the first findings is the presence of sequences affiliated with parasitic groups in lakes with different trophic types (25). This suggests that the ecological impact of parasitism may have been underestimated in marine systems (31) and also in freshwater systems. However, the data on lacustrine small-eukaryote diversity are still largely incomplete, and additional investigation of the composition and organization of freshwater small-eukaryote assemblages is essential; in particular, no data are available for mesotrophic lakes.To draw up an inventory of lacustrine picoplankton (Ͻ5 m) over a full trophic status range (from oligotrophic to hypereutro...

The structure and dynamics of small eukaryotes (cells with a diameter less than 5 m) were studied over two consecutive years in an oligomesotrophic lake (Lake Pavin in France). Water samples were collected at 5 and 30 m below the surface; when the lake was stratified, these depths corresponded to the epilimnion and hypolimnion. Changes in small-eukaryote structure were analyzed using terminal restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the 18S rRNA genes. Terminal restriction fragments from clones were used to reveal the dominant taxa in T-RFLP profiles of the environmental samples. Spumellalike cells (Chrysophyceae) did not dominate the small eukaryote community identified by molecular techniques in lacustrine ecosystems. Small eukaryotes appeared to be dominated by heterotrophic cells, particularly Cercozoa, which represented nearly half of the identified phylotypes, followed by the Fungi-LKM11 group (25%), choanoflagellates (10.3%) and Chrysophyceae (8.9%). Bicosoecida, Cryptophyta, and ciliates represented less than 9% of the community studied. No seasonal reproducibility in temporal evolution of the small-eukaryote community was observed from 1 year to the next. The T-RFLP patterns were related to bottom-up (resources) and top-down (grazing) variables using canonical correspondence analysis. The results showed a strong top-down regulation of small eukaryotes by zooplankton, more exactly, by cladocerans at 5 m and copepods at 30 m. Among bottom-up factors, temperature had a significant effect at both depths. The concentrations of nitrogenous nutrients and total phosphorus also had an effect on small-eukaryote dynamics at 5 m, whereas bacterial abundance and dissolved oxygen played a more important structuring role in the deeper zone.Small phototrophic and heterotrophic eukaryotes (Ͻ5 m) are found throughout the world's oceans and lakes at concentrations between 10 2 and 10 4 cells ml Ϫ1 in the photic zone (11). Small eukaryotes are known to be essential components in marine trophic food webs (20). The small-eukaryote assemblage is formed by picoalgae, which participate in primary production (55), by colorless heterotrophic cells, mostly flagellates, which are considered to be important grazers of prokaryotic and eukaryotic cells (11) and also play a significant role in the mineralization of organic matter, and finally by some small eukaryotes which can be mixotrophs. Despite the ecological importance of small eukaryotes and the general lack of distinct morphological features of these small cells, they have only recently been studied from a molecular perspective (20, 37). Thanks to these techniques, recent studies, conducted in various environments, have revealed a surprisingly high diversity of small eukaryotes and the existence of novel lineages (39). For example, the genetic diversity of small eukaryotes from coastal waters showed the dominance of novel alveolates (from 36% to 62% of total sequences obtained in their libraries) and the importance of novel stramenopiles, ...

Photosynthetic picoeukaryotes (PPEs), comprising organisms < 3 mum in size, are important primary producers in marine food webs and include representatives from all known algal lineages. Little is known, however, regarding the composition and distribution of PPE communities, particularly at large spatial scales, or in relation to the underlying biotic and abiotic factors that influence this structure. Here, we analysed PPE community structure along a transect in the South East Pacific Ocean (BIOSOPE cruise) that encompassed a large trophic gradient, including hyper-oligotrophic waters in the South Pacific Gyre (SPG), considered to be some of the 'clearest' natural waters on Earth. Using dot blot hybridizations with 16S rRNA oligonucleotide probes, we established that the PPE community was dominated by members of the classes Prymnesiophyceae and Chrysophyceae throughout the transect. Moreover, clone library construction followed by phylogenetic analysis of sequenced clones revealed several novel 16S rRNA gene lineages, including new clades of prymnesiophytes (designated Prym 16S-III) and prasinophytes (Pras 16S-VIII). Pras 16S-VIII was found at all five stations at which clone libraries were constructed, representing a range of trophic conditions, including the South Pacific Gyre, suggesting members of this clade have a broad distribution in this part of the South East Pacific at least. In contrast, Prym 16S-III sequences were largely restricted to oligotrophic stations of the SPG. Subsequent multivariate statistical analyses showed that, within the measured factors, chemical and biological factors seem to influence PPE community structure more than physical parameters. However, more than 50% of the variation in distribution of PPE classes remained unexplained.

Although they are widespread, diverse and involved in biogeochemical cycles, microbial eukaryotes attract less attention than their prokaryotic counterparts in environmental microbiology. In this study, we used publicly available 18S barcoding data to define biases that may limit such analyses and to gain an overview of the planktonic microbial eukaryotic diversity in freshwater ecosystems. The richness of the microbial eukaryotes was estimated to 100 798 operational taxonomic units (OTUs) delineating 1267 clusters or phylogenetic units (PUs, i.e. monophyletic groups of OTUs that are phylogenetically close). By summing the richness found in aquatic environments, we can predict the microbial eukaryotic richness to be around 200 000-250 000 species. The molecular diversity of protists in freshwater environments is generally higher than that of the morphospecies and cultivated species catalogued in public databases. Amoebozoa, Viridiplantae, Ichthyosporea, and Cryptophyta are the most phylogenetically diverse taxa, and characterisation of these groups is still needed. A network analysis showed that Fungi, Stramenopiles and Viridiplantae play central role in lake ecosystems. Finally, this work provides guidance for compiling metabarcoding data and identifies missing data that should be obtained to increase our knowledge on microbial eukaryote diversity.

Heterotrophic bacterioplankton, cyanobacteria and phototrophic picoeukaryotes (< 5 μm in size) numerically dominate planktonic oceanic communities. While feeding on bacterioplankton is often attributed to aplastidic protists, recent evidence suggests that phototrophic picoeukaryotes could be important bacterivores. Here, we present direct visual evidence from the surface mixed layer of the Atlantic Ocean that bacterioplankton are internalized by phototrophic picoeukaryotes. In situ interactions of phototrophic picoeukaryotes and bacterioplankton (specifically Prochlorococcus cyanobacteria and the SAR11 clade) were investigated using a combination of flow cytometric cell sorting and dual tyramide signal amplification fluorescence in situ hybridization. Using this method, we observed plastidic Prymnesiophyceae and Chrysophyceae cells containing Prochlorococcus, and to a lesser extent SAR11 cells. These microscopic observations of in situ microbial trophic interactions demonstrate the frequency and likely selectivity of phototrophic picoeukaryote bacterivory in the surface mixed layer of both the North and South Atlantic subtropical gyres and adjacent equatorial region, broadening our views on the ecological role of the smallest oceanic plastidic protists.

The genetic diversity of photosynthetic picoeukaryotes was investigated in the South East Pacific Ocean. Genetic libraries of the plastid 16S rRNA gene were constructed on picoeukaryote populations sorted by flow cytometry, using two different primer sets, OXY107F/OXY1313R commonly used to amplify oxygenic organisms, and PLA491F/OXY1313R, biased towards plastids of marine algae. Surprisingly, the two sets revealed quite different photosynthetic picoeukaryote diversity patterns, which were moreover different from what we previously reported using the 18S rRNA nuclear gene as a marker. The first 16S primer set revealed many sequences related to Pelagophyceae and Dictyochophyceae, the second 16S primer set was heavily biased toward Prymnesiophyceae, while 18S sequences were dominated by Prasinophyceae, Chrysophyceae and Haptophyta. Primer mismatches with major algal lineages is probably one reason behind this discrepancy. However, other reasons, such as DNA accessibility or gene copy numbers, may be also critical. Based on plastid 16S rRNA gene sequences, the structure of photosynthetic picoeukaryotes varied along the BIOSOPE transect vertically and horizontally. In oligotrophic regions, Pelagophyceae, Chrysophyceae, and Prymnesiophyceae dominated. Pelagophyceae were prevalent at the DCM depth and Chrysophyceae at the surface. In mesotrophic regions Pelagophyceae were still important but Chlorophyta contribution increased. Phylogenetic analysis revealed a new clade of Prasinophyceae (clade 16S-IX), which seems to be restricted to hyper-oligotrophic stations. Our data suggest that a single gene marker, even as widely used as 18S rRNA, provides a biased view of eukaryotic communities and that the use of several markers is necessary to obtain a complete image.

A central goal in ecology is to understand the factors affecting the temporal dynamics and spatial distribution of microorganisms and the underlying processes causing differences in community structure and composition. However, little is known in this respect for photosynthetic picoeukaryotes (PPEs), algae that are now recognised as major players in marine CO 2 fixation. Here, we analysed dot blot hybridisation and cloning-sequencing data, using the plastid-encoded 16S rRNA gene, from seven research cruises that encompassed all four ocean biomes. We provide insights into global abundance, a-and b-diversity distribution and the environmental factors shaping PPE community structure and composition. At the class level, the most commonly encountered PPEs were Prymnesiophyceae and Chrysophyceae. These taxa displayed complementary distribution patterns, with peak abundances of Prymnesiophyceae and Chrysophyceae in waters of high (25:1) or low (12:1) nitrogen:phosphorus (N:P) ratio, respectively. Significant differences in phylogenetic composition of PPEs were demonstrated for higher taxonomic levels between ocean basins, using Unifrac analyses of clone library sequence data. Differences in composition were generally greater between basins (interbasins) than within a basin (intrabasin). These differences were primarily linked to taxonomic variation in the composition of Prymnesiophyceae and Prasinophyceae whereas Chrysophyceae were phylogenetically similar in all libraries. These data provide better knowledge of PPE community structure across the world ocean and are crucial in assessing their evolution and contribution to CO 2 fixation, especially in the context of global climate change.