We propose that marine planktonic microeukaryote assemblages incorporate dynamic and metabolically active abundant and rare subcommunities, with contrasting structuring patterns but fairly regular proportions, across space and time.

Very few studies have analysed the niches of pelagic protist in details. This is because for most protists, both an accurate species definition and methods for routine detection and quantification of cells are lacking. The morphospecies Micromonas pusilla, a marine unicellular green alga, is the most ubiquitous and cosmopolitan picoeukaryote described to date. This species comprises several independent genetic lineages or clades, which are not currently distinguishable based on comparison of their morphology or biogeographical distribution. Molecular probes were used to detect and quantify the genetic clades of M. pusilla in samples from temperate, polar and tropical environments in order to assess potential ecological niche partitioning. The three clades were detected in all biogeographical regions studied and were commonly found in sympatry. Cell abundances recorded for clades A and B were high, especially at coastal stations. Clade C, when detected, was always at low abundances and is suggested to be a low-light clade. Shifts in the contribution of clades to total M. pusilla abundance were observed along environmental gradients, both at local and basin-wide scales. This suggests that the phylogenetic clades occupy specific niches and confirms the existence of cryptic species within the morphospecies M. pusilla. Parameters which can precisely explain the distribution of these cryptic species remain to be elucidated.

Picoeukaryotes (cells of <3 m in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3-m-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.Picoeukaryotes (cells smaller than 3 m in diameter) (22) are widely distributed in aquatic environments. Their important biomass and their high productivity suggest that they play a major role in oceanic, coastal, and freshwater ecosystems (27,28,48). Because of their small size and their simple morphology, a detailed study of these organisms is difficult. It is only recently that their diversity has been revealed, in particular by phylogenetic studies based on 18S rDNA sequence analysis (31, 34). It is necessary to quantify the dominating phylogenetic groups of picoeukaryotes in the natural environment in order to understand their contribution to the microbial food web and biogeochemical cycles.So far, the abundance of marine picoeukaryotes has been estimated based on their natural fluorescence by flow cytometry (28, 54) or by analysis of their pigment composition by high-performance liquid chromatography (56). The taxonomic resolution of these techniques is limited (e.g., class level only for high-performance liquid chromatography) and they do not allow the quantification of heterotrophic organisms. Molecular techniques based on full or partial sequence analysis of 18S rDNA allow us to determine species composition very precisely but are labor-intensive, and only a few samples can be analyzed at once (31,34

ABSTRACT. Photosynthetic pico-and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group-specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole-cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group-or taxon-specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.

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Marine phytoplankton organisms account for more than 45% of the photosynthetic net primary production on Earth. They are distributed across many of the major clades of the tree of life and include prokaryotes, and eukaryotes that acquired photosynthesis through the process of endosymbiosis. If the number of extant described species is relatively low compared to the diversity of the terrestrial plants, recent insights into the genetic diversity of natural assemblages have revealed a large unsuspected diversity at different taxonomic levels. Wide infra-specific diversity is also being discovered in many widespread and well known morphological species. This review summarizes data obtained in the fields of ecology, evolutionary biology, physiology and genomics that have improved our understanding of the biodiversity and evolution of marine phytoplankton. To cite this article: N.

Abstract. Sequences affiliated to Syndiniales (Marine alveolate, MALV) regularly dominate 18S rDNA genetic libraries of nearly all marine ecosystems investigated so far. Among them, Amoebophryidae (MALV group II) is composed of numerous and genetically distant environmental sequences, where Amoebophrya is the only known and formally described genus. Amoebophrya species include virulent pathogens for a wide range of dinoflagellate species. Beside their regular occurrence in marine ecosystems, their quantitative distribution and the environmental factors triggering host infection have barely been studied in open oligotrophic waters. In order to understand the functional role of these parasites in natural environments, we studied the distribution and contribution to the eukaryotic community of the small free-living stage of Amoebophryidae (the dinospores) along a transect in the Mediterranean Sea, as well as their host diversity at three oligotrophic stations. Dinospores were more abundant at a coastal station (max. 1.5 × 10 3 cells ml −1 ) than in oligotrophic waters (max. 51 ± 16.3 cells ml −1 ), where they represented 10.3 to 34.9% of the total eukaryotic community at 40 and 30 m depth, respectively and 21.2% on average along the water column. Positive correlation was found between dinospore occurrence and higher concentration of NO 3 + NO 2 at the coastal station. At selected stations, out of 38 different dinoflagellates taxa identified, 15 were infected, among which a majority were not recognized as Amoebophryidae host so far. Prevalences (percentage of infected cells) generally varied between 1% and 10%, with a notable exception for Blepharocysta paulsenii for which 25% of cells were infected Correspondence to: L. Guillou (laure.guillou@sb-roscoff.fr) at the most oligotrophic station. The present study shows that dinospores are able to thrive and infect dinoflagellates both in coastal and ultra-oligotrophic open waters. Our results emphasize the role of parasitism in microbial food web dynamics and ultimately on biogeochemical cycles.

In the marine environment, phytoplankton and bacterioplankton can be physically associated. Such association has recently been hypothesized to be involved in the toxicity of the dinoflagellate genus Alexandrium. However, the methods, which have been used so far to identify, localize, and quantify bacteria associated with phytoplankton, are either destructive, time consuming, or lack precision. In the present study we combined tyramide signal amplification–fluorescent in situ hybridization (TSA‐FISH) with confocal microscopy to determine the physical association of dinoflagellate cells with bacteria. Dinoflagellate attached microflora was successfully identified with TSA‐FISH, whereas FISH using monolabeled probes failed to detect bacteria, because of the dinoflagellate autofluorescence. Bacteria attached to entire dinoflagellates were further localized and distinguished from those attached to empty theca, by using calcofluor and DAPI, two fluorochromes that stain dinoflagellate theca and DNA, respectively. The contribution of specific bacterial taxa of attached microflora was assessed by double hybridization. Endocytoplasmic and endonuclear bacteria were successfully identified in the nonthecate dinoflagellate Gyrodinium instriatum. In contrast, intracellular bacteria were not observed in either toxic or nontoxic strains of Alexandrium spp. Finally, the method was successfully tested on natural phytoplankton assemblages, suggesting that this combination of techniques could prove a useful tool for the simultaneous identification, localization, and quantification of bacteria physically associated with dinoflagellates and more generally with phytoplankton.