Nathalie Simon

A Single Species, Micromonas pusilla (Prasinophyceae), Dominates the Eukaryotic Picoplankton in the Western English Channel

ABSTRACT 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 (>103 cells ml−1) during the sampling period, with maxima in summer (up to 2 × 104 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.

Patterns of Rare and Abundant Marine Microbial Eukaryotes

BACKGROUND Biological communities are normally composed of a few abundant and many rare species. This pattern is particularly prominent in microbial communities, in which most constituent taxa are usually extremely rare. Although abundant and rare subcommunities may present intrinsic characteristics that could be crucial for understanding community dynamics and ecosystem functioning, microbiologists normally do not differentiate between them. Here, we investigate abundant and rare subcommunities of marine microbial eukaryotes, a crucial group of organisms that remains among the least-explored biodiversity components of the biosphere. We surveyed surface waters of six separate coastal locations in Europe, independently considering the picoplankton, nanoplankton, and microplankton/mesoplankton organismal size fractions. RESULTS Deep Illumina sequencing of the 18S rRNA indicated that the abundant regional community was mostly structured by organismal size fraction, whereas the rare regional community was mainly structured by geographic origin. However, some abundant and rare taxa presented similar biogeography, pointing to spatiotemporal structure in the rare microeukaryote biosphere. Abundant and rare subcommunities presented regular proportions across samples, indicating similar species-abundance distributions despite taxonomic compositional variation. Several taxa were abundant in one location and rare in other locations, suggesting large oscillations in abundance. The substantial amount of metabolically active lineages found in the rare biosphere suggests that this subcommunity constitutes a diversity reservoir that can respond rapidly to environmental change. CONCLUSIONS 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.

Oligonucleotide probes for the identification of three algal groups by dot blot and fluorescent whole-cell hybridization.

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.

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing.

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.

Quantitative Assessment of Picoeukaryotes in the Natural Environment by Using Taxon-Specific Oligonucleotide Probes in Association with Tyramide Signal Amplification-Fluorescence In Situ Hybridization and Flow Cytometry

ABSTRACT Picoeukaryotes (cells of <3 μm 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-μm-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.

CHARACTERIZATION OF OCEANIC PHOTOSYNTHETIC PICOEUKARYOTES BY FLOW CYTOMETRY1

To interpret flow cytometric data that are routinely obtained on natural oceanic communities, 23 strains of photosynthetic picoeukaryotes belonging to four classes (Prasinophyceae, Chlorophyceae, Pelagophyceae, and Prymnesiophyceae) and six pigment types were investigated for their light scattering in the forward and right‐angle directions, chlorophyll fluorescence, and DNA content as measured by flow cytometry. Cell she was assessed by Coulter counter, and pigment composition was measured by reverse‐phase high‐performance liquid chromatography. The size and GC% of the nuclear genome of cultured picoeukaryotes was measured from the fluorescence of DNA‐specific dyes. Using these two parameters, we could discriminate species within pigment groups. DNA staining of preserved natural samples may also prove useful in discriminating cooccurring populations in situ as long as the communities are not too complex. Using the relationships that we established between size and light‐scattering properties of the cells, we estimated equivalent diameters of picoeukaryotes in natural populations to be between 1.3 and 2 μm. Chlorophyll a content was between 6 and 16 fg·cel−1 as calculated from relationships that we established between chlorophyll a content and red fluorescence of the cultured strains. With respect to size, chlorophyll a content, and pigment composition, Pelagomonas sp. strains (Pelagophyceae) appeared to be the most representative of the natural communities in subtropical ocean waters. In contrast, green coccoid strains, which often outcompete other strains in culture, might only be minor contributors to these communities.

IDENTIFICATION OF BACTERIA ASSOCIATED WITH DINOFLAGELLATES (DINOPHYCEAE) ALEXANDRIUM SPP. USING TYRAMIDE SIGNAL AMPLIFICATION–FLUORESCENT IN SITU HYBRIDIZATION AND CONFOCAL MICROSCOPY1

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.

Ecological niche partitioning in the picoplanktonic green alga Micromonas pusilla: evidence from environmental surveys using phylogenetic probes

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.

Diversity and evolution of marine phytoplankton

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.

Diversity and Ecology of Eukaryotic Marine Phytoplankton

Abstract Marine phytoplankton, the photosynthetic microorganisms drifting in the illuminated waters of our planet, are extremely diverse, being distributed across major eukaryotic lineages. About 5000 eukaryotic species have been described with traditional morphological methods, but recent environmental molecular surveys are unveiling an ever-increasing diversity, including entirely new lineages with no described representatives. Eukaryotic marine phytoplankton are significant contributors to major global processes (such as oxygen production, carbon fixation and CO2 sequestration, nutrient recycling), thereby sustaining the life of most other aquatic organisms. In modern oceans, the most diverse and ecologically significant eukaryotic phytoplankton taxa are the diatoms, the dinoflagellates, the haptophytes and the small prasinophytes, some of which periodically form massive blooms visible in satellite images. Evidence is now accumulating that many phytoplankton taxa are actually mixotrophs, exhibiting alternate feeding strategies depending on environmental conditions (e.g. grazing on prey or containing symbiotic organisms), thus blurring the boundary between autotrophs and heterotrophs in the ocean.