Picoeukaryote Community Research Articles

Phylogenetic composition and distribution of picoeukaryotes in the hypoxic northwestern coast of the Gulf of Mexico

Coastal marine hypoxic, or low-oxygen, episodes are an increasing worldwide phenomenon, but its effect on the microbial community is virtually unknown by far. In this study, the community structure and phylogeny of picoeukaryotes in the Gulf of Mexico, which are exposed to severe hypoxia in these areas was explored through a clone library approach. Both oxic surface waters and suboxic bottom waters were collected in August 2010 from three representative stations on the inner Louisiana shelf near the Atchafalaya and Mississippi River plumes. The bottom waters of the two more western stations were much more hypoxic in comparison to those of the station closest to the Mississippi River plume, which were only moderately hypoxic. A phylogenetic analysis of a total 175 sequences, generated from six 18S rDNA clone libraries, demonstrated a clear dominance of parasitic dinoflagellates from Marine alveolate clades I and II in all hypoxic waters as well as in the surface layer at the more western station closest to the Atchafalaya River plume. Species diversity was significantly higher at the most hypoxic sites, and many novel species were present among the dinoflagellate and stramenopile clades. We concluded that hypoxia in the Gulf of Mexico causes a significant shift in picoeukaryote communities, and that hypoxia may cause a shift in microbial food webs from grazing to parasitism.

The response of marine picoplankton to ocean acidification

Since industrialization global CO(2) emissions have increased, and as a consequence oceanic pH is predicted to drop by 0.3-0.4 units before the end of the century - a process coined 'ocean acidification'. Consequently, there is significant interest in how pH changes will affect the ocean's biota and integral processes. We investigated marine picoplankton (0.2-2 µm diameter) community response to predicted end of century CO(2) concentrations, via a 'high-CO(2) ' (∼ 750 ppm) large-volume (11 000 l) contained seawater mesocosm approach. We found little evidence of changes occurring in bacterial abundance or community composition due to elevated CO(2) under both phytoplankton pre-bloom/bloom and post-bloom conditions. In contrast, significant differences were observed between treatments for a number of key picoeukaryote community members. These data suggested a key outcome of ocean acidification is a more rapid exploitation of elevated CO(2) levels by photosynthetic picoeukaryotes. Thus, our study indicates the need for a more thorough understanding of picoeukaryote-mediated carbon flow within ocean acidification experiments, both in relation to picoplankton carbon sources, sinks and transfer to higher trophic levels.

Composition of the summer photosynthetic pico and nanoplankton communities in the Beaufort Sea assessed by T-RFLP and sequences of the 18S rRNA gene from flow cytometry sorted samples

The composition of photosynthetic pico and nanoeukaryotes was investigated in the North East Pacific and the Arctic Ocean with special emphasis on the Beaufort Sea during the MALINA cruise in summer 2009. Photosynthetic populations were sorted using flow cytometry based on their size and pigment fluorescence. Diversity of the sorted photosynthetic eukaryotes was determined using terminal-restriction fragment length polymorphism analysis and cloning/sequencing of the 18S ribosomal RNA gene. Picoplankton was dominated by Mamiellophyceae, a class of small green algae previously included in the prasinophytes: in the North East Pacific, the contribution of an Arctic Micromonas ecotype increased steadily northward becoming the only taxon occurring at most stations throughout the Beaufort Sea. In contrast, nanoplankton was more diverse: North Pacific stations were dominated by Pseudo-nitzschia sp. whereas those in the Beaufort Sea were dominated by two distinct Chaetoceros species as well as by Chrysophyceae, Pelagophyceae and Chrysochromulina spp.. This study confirms the importance of Arctic Micromonas within picoplankton throughout the Beaufort Sea and demonstrates that the photosynthetic picoeukaryote community in the Arctic is much less diverse than at lower latitudes. Moreover, in contrast to what occurs in warmer waters, most of the key pico- and nanoplankton species found in the Beaufort Sea could be successfully established in culture.

Structure and seasonal dynamics of the eukaryotic picophytoplankton community in a wind‐driven coastal upwelling ecosystem

We studied the structure and seasonal dynamics of the photosynthetic picoeukaryote community at a coastal site off central Chile that is strongly affected by seasonal wind‐driven upwelling. We determined the picoeukaryote phylogenetic diversity by cloning and sequencing the 18S ribosomal ribonucleic acid (rRNA) gene amplified with Chloroplastida‐biased primers. Chloroplastida made up 100% (autumn), 61% (spring), and 20% (summer) of the clone libraries. Most sequences belonged to Mamiellophyceae and clustered with Micromonas (clades A.BC.1, B.E.3, C.D.5), Ostreococcus (clade A), and Crustomastix (clade A). Fluorescent in situ hybridization coupled with tyramide signal amplification (FISH–TSA) was used to determine the time variability in abundance of the total picoeukaryotic community, the Chloroplastida, and the genera Micromonas, Ostreococcus, and Bathycoccus over a period of 2 yr with nearly monthly sampling. The abundance of photosynthetic picoeukaryotes was low during the upwelling season (spring and summer) and high during the nonupwelling season (late fall and winter). Chloroplastida made up, on average, 74% of the total picoeukaryotic community, and the sum of the three genera probed accounted for 100% of the Chloroplastida abundances. Ostreococcus dominated the picophytoplanktonic community numerically throughout the year and, thus, appears to be a key component of the upwelling picoplanktonic community in the eastern South Pacific.

Analysis of photosynthetic picoeukaryote community structure along an extended Ellett Line transect in the northern North Atlantic reveals a dominance of novel prymnesiophyte and prasinophyte phylotypes

Photosynthetic picoeukaryotes (PPEs) of a size <3 μm can contribute significantly to primary production. Here, PPE community structure was analysed along an extended Ellett Line transect, an area in the North Atlantic well studied by physical oceanographers but largely neglected in the field of microalgal ecology. Distribution patterns of specific PPE classes were determined using dot-blot hybridization analysis, while the taxonomic composition of specific PPE classes was revealed by phylogenetic analysis of plastid 16S rRNA gene sequences. In addition, we performed fluorescent in situ hybridization (FISH) analysis of seawater samples collected along the transect to provide a PCR-independent survey of class level PPE distribution patterns. We found the PPE community was dominated by members of the Prymnesiophyceae, Prasinophyceae and Mamiellophyceae. Interestingly, phylogenetic analysis revealed several novel Prymnesiophyceae and Prasinophyceae phylotypes (with only 85–96% identity to neighbouring sequences) within lineages for which cultured counterparts are unknown.

Distribution of eukaryotic plankton in the English Channel and the North Sea in summer

The distribution of eukaryotic plankton was investigated in the English Channel and the North Sea during the MICROVIR cruise in summer 2007. The size distribution of autotrophic, heterotrophic eukaryotes and species composition was analyzed with a focus on two major divisions, Haptophyta and Chlorophyta, targeted by 18S rRNA probes. Picoeukaryotes (< 2 μm) dominated over the larger eukaryotes at all stations. Eukaryotes larger than 5 μm were mainly composed of diatoms in the English Channel and of dinoflagellates in the North Sea. The contribution of Haptophyta was maximal in the 2 to 5 μm fraction and they appeared more abundant in the central region of the North Sea. Chlorophyta, especially Micromonas pusilla, generally dominated the picoplanktonic fraction in the English Channel. Micromonas contribution decreased between the South and the North-east of the North Sea and it was even absent at some stations. Although this species is dominant among the picoeukaryote community of the English Channel, other Chlorophyta species may also play an important ecological role in these temperate ecosystems.

Rapid shifts in picoeukaryote community structure in response to ocean acidification.

Rapid shifts in picoeukaryote community structure were observed during a CO(2) perturbation experiment in which we followed the development of phytoplankton blooms in nutrient-amended mesocosms under the present day or predicted future atmospheric pCO(2) (750 μatm, seawater pH 7.8). Analysis of rbcL clone libraries (encoding the large subunit of RubisCO) and specific quantitative PCR assays showed that two prasinophytes closely related to Micromonas pusilla and Bathycoccus prasinos were present, but responded very differently to high CO(2)/acidification. We found that the abundance of Micromonas-like phylotypes was significantly higher (>20-fold) under elevated CO(2)/low pH, whereas the Bathycoccus-like phylotypes were more evenly distributed between treatments and dominated the prasinophyte community under ambient conditions.

Genetic diversity of picoeukaryotes in eight lakes differing in trophic status

The genetic diversity of picoeukaryotes (0.2-5.0 µm) was investigated in 8 lakes differing in trophic status in Nanjing, China. Denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing of 18S rRNA genes were applied to analyze the picoeukaryotic communities. DGGE analysis showed that among the 8 lakes, the diversity of picoeukaryotes was greatest in the mesotrophic Lake Nan (24 bands) and least in the oligotrophic Lake Qian (12 bands). Cluster analysis of DGGE profiles revealed that the 8 lakes were grouped into 2 distinct clusters. Cluster 1 contained lakes Mochou, Zixia, Huashen, Nan, Pipa, and Qian, while cluster 2 contained lakes Xuanwu and Baijia. Clone libraries were constructed from the mesotrophic Lake Xuanwu and the oligotrophic Lake Zixia, and the 2 libraries were compared using the program LIBSHUFF. This analysis indicated that the picoeukaryotic community composition differed significantly between the 2 lakes (p = 0.001). A total of 25 operational taxonomic units were detected; 18 (62 clones) were related to known eukaryotic groups, while 7 (30 clones) were not affiliated with any known eukaryotic group. Alveolates and stramenopiles were the dominant groups in Lake Xuanwu, while alveolates and chlorophyta predominated in Lake Zixia. Multivariate statistical analysis indicated that the differences in the picoeukaryotic community composition of the 8 lakes might be related to trophic status and top-down regulation by metazooplankton.

Composition and seasonal variability of picoeukaryote communities at two subtropical coastal sites with contrasting trophic conditions

Phylogenetic diversity (18S rRNA gene) of picoeukaryotes in Hong Kong coastalwaters is dominated by heterotrophic forms and influenced by the seasonallydriven combination of freshwater discharge from the Pearl River, oceanic water ofthe South China Sea and coastal water from the China Coastal Current.KEYWORDS: picoeukaryote; subtropical coastal waters; 18S-rDNA; DGGE;seasonal variationPicoeukaryotes, small eukaryotic plankton with diam-eter ,3 mm, have ubiquitous distribution and areimportant contributors to global primary productionand carbon and mineral cycles (Li, 1994). They arephylogenetically, morphologically and metabolicallydiverse and are capable of phototrophy, phagotrophy,osmotrophy and mixotrophy (Moreira and Lo´pez-Garci´a, 2002).Traditionally, morphological features have been usedfor picoeukaryote identification and classification.However, those features alone are not sufficient to dis-tinguish clearly among species or groups of picoeukar-yotes due to their tiny sizes and cryptic morphologicalcharacteristics (Sims et al., 2002), leading to underesti-mation of the diversity at phylum level (Massana et al.,2002). In contrast, molecular approaches based on the18S rRNA gene provide new insights into picoeukaryotecommunity structure in various ecosystems. Over thepast several years, studies have demonstrated tremen-dous molecular phylogenetic diversity among theseorganisms in open marine systems, including the equa-torial Pacific Ocean (Moon-van der Staay et al., 2001),the Antarctic Polar Front (Lo´pez-Garci´a et al., 2001),the Mediterranean and Scotia Sea as well as the NorthAtlantic Ocean (Di´ez et al., 2001b). A large number ofnovel picoeukaryote phylogenetic groups, some without

Archaea, Bacteria, and Algal Plastids Associated with the Reef-Building Corals Siderastrea stellata and Mussismilia hispida from Búzios, South Atlantic Ocean, Brazil

Reef-building corals may be seen as holobiont organisms, presenting diverse associated microbial communities. Best known is the symbiotic relationship with zooxanthellae, but Archaea, Bacteria, fungi, viruses, and algal plastids are also abundant. Until now, there is little information concerning microbial communities associated with Brazilian corals. The present study aims to describe the diversity of Archaea, Bacteria, and eukaryotic algal plastid communities associated with two sympatric species, Siderastrea stellata and Mussismilia hispida, from Southeastern Brazil, using 16S rRNA gene libraries. Since corals present a high number of other associated invertebrates, coral barcoding (COI) was performed to confirm the exclusive occurrence of coral DNA in our samples. Our analysis yielded 354 distinct microbial OTUs, represented mainly by novel phylotypes. Richness (Chao1 and ACE) and diversity (H') estimations of the microbial communities associated with both species were high and comparable to other studies. Rarefaction analyses showed that microbial diversity of S. stellata is higher than that of M. hispida. Libshuff comparative analyses showed that the highest microbial community similarity between the two coral species occurred in the bacterial libraries, while archaeal and plastidial communities were significantly different. Crenarchaeota dominated archaeal communities, while Proteobacteria was the most abundant bacterial phylum, dominated by alpha-Proteobacteria. Plastids were also represented by novel phylotypes and did not match with any 16S rRNA sequences of Cyanobacteria and zooxanthellae from GenBank. Our data improves the pool of available information on Brazilian coral microbes and shows corals as sources of diverse prokaryotic and picoeukaryotic communities.

Photosynthetic picoeukaryote community structure in the South East Pacific Ocean encompassing the most oligotrophic waters on Earth

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.

Groups without Cultured Representatives Dominate Eukaryotic Picophytoplankton in the Oligotrophic South East Pacific Ocean

BackgroundPhotosynthetic picoeukaryotes (PPE) with a cell size less than 3 µm play a critical role in oceanic primary production. In recent years, the composition of marine picoeukaryote communities has been intensively investigated by molecular approaches, but their photosynthetic fraction remains poorly characterized. This is largely because the classical approach that relies on constructing 18S rRNA gene clone libraries from filtered seawater samples using universal eukaryotic primers is heavily biased toward heterotrophs, especially alveolates and stramenopiles, despite the fact that autotrophic cells in general outnumber heterotrophic ones in the euphotic zone.Methodology/Principal FindingsIn order to better assess the composition of the eukaryotic picophytoplankton in the South East Pacific Ocean, encompassing the most oligotrophic oceanic regions on earth, we used a novel approach based on flow cytometry sorting followed by construction of 18S rRNA gene clone libraries. This strategy dramatically increased the recovery of sequences from putative autotrophic groups. The composition of the PPE community appeared highly variable both vertically down the water column and horizontally across the South East Pacific Ocean. In the central gyre, uncultivated lineages dominated: a recently discovered clade of Prasinophyceae (IX), clades of marine Chrysophyceae and Haptophyta, the latter division containing a potentially new class besides Prymnesiophyceae and Pavlophyceae. In contrast, on the edge of the gyre and in the coastal Chilean upwelling, groups with cultivated representatives (Prasinophyceae clade VII and Mamiellales) dominated.Conclusions/SignificanceOur data demonstrate that a very large fraction of the eukaryotic picophytoplankton still escapes cultivation. The use of flow cytometry sorting should prove very useful to better characterize specific plankton populations by molecular approaches such as gene cloning or metagenomics, and also to obtain into culture strains representative of these novel groups.

Protistan assemblages across the Indian Ocean, with a specific emphasis on the picoeukaryotes

Protists, and among them the picoeukaryotes (cells<3 μm), have been described as significant contributors to both carbon biomass and production in oligotrophic regions of the oceans. However, protist assemblages remain largely undescribed in pelagic ecosystems and in particular in the Indian Ocean. In the present work, we investigated protists along an eastward transect across the sub-tropical gyre of the Indian Ocean (from South Africa to Australia), with a particular focus on picoeukaryotes. We combined inverted and epifluorescence microscopy, flow cytometry, pigment analysis, denaturing gel gradient electrophoresis (DGGE), 18S rDNA clone libraries, and fluorescent in situ hybridization (FISH). Overall the picophytoplankton fraction contributed 88% and 90% of total Chl a at the surface and DCM, respectively, with picoeukaryotes accounting for 38% and 50% of total Chl a at the surface and DCM. Considering only the Indian South Subtropical Gyre (ISSG) province, we observed greater shifts in the picoeukaryotic assemblage throughout the upper 200 m of the water column than along the ca. 10,000 km cruise track. In terms of taxonomic diversity and contribution of each taxon to the picoeukaryotic community, prasinophytes were well represented at more coastal stations with the genus Micromonas reaching densities up to 750 cell mL −1 in coastal waters and less than 100 cell mL −1 at open ocean stations. Haptophytes (56% and 45% of picoeukaryotic pigments at surface and DCM, respectively) and possibly pelagophytes (28% and 40% of picoeukaryotic pigments at surface and DCM, respectively) appeared to be dominant at open ocean stations. Other groups and in particular organisms affiliated to chrysophytes, and to a lesser extent to cryptophytes, appear as clear targets for future qualitative and quantitative studies. Moreover, the occurrence of many sequences related to radiolarians (5% and 27% at surface and DCM, respectively) will require further investigation.

Molecular analysis of photosynthetic picoeukaryote community structure along an Arabian Sea transect

We developed oligonucleotide probes, based on plastid 16S ribosomal DNA (rDNA) sequences, to target 10 different algal classes (Chlorarachniophyceae, Chrysophyceae, Cryptophyceae, Eustigmatophyceae, Pavlovophyceae, Pelagophyceae, Pinguiophyceae, Prasinophyceae [clade VI], Prymnesiophyceae, and Trebouxiophyceae), for use with dot blot hybridization technology. These class‐specific probes were then used to investigate the community structure of photosynthetic picoeukaryotes (≪3 µm) along a transect in the Arabian Sea during September 2001, using hybridization to plastid 16S rDNA polymerase chain reaction amplicons. The transect ranged from oligotrophic open‐ocean waters, just south of the Equator, through mesotrophic and eutrophic waters toward the north coast of Oman, crossing a region of monsoonal upwelling off the Omani northeast coast. Photosynthetic picoeukaryotes in the surface‐mixed layer (SML) and deep chlorophyll maximum ranged from 1.0 x 103 cells mL‐1 in southern oligotrophic waters to almost 30 x 103 cells mL‐1 at the region of upwelling. Chrysophytes were abundant along most of the transect throughout much of the euphotic zone. Prymnesiophytes were abundant in surface waters along much of the transect. By contrast, trebouxiophytes were confined to deeper waters, below the SML. Pelagophytes were found in surface waters that were more mesotrophic, whereas cryptophytes were only detected in the more nutrient‐rich waters at the northern end of the transect, between depths of 20‐30 m. Pinguiophytes were also detected, but only in the warm surface waters off the north coast of Oman. Chlorarachniophytes, eustigmatophytes, pavlovophytes, and clade VI prasinophytes were essentially below detection limits for the entire transect.

Unveiling fungal zooflagellates as members of freshwater picoeukaryotes: evidence from a molecular diversity study in a deep meromictic lake

This study presents an original 18S rRNA PCR survey of the freshwater picoeukaryote community, and was designed to detect unidentified heterotrophic picoflagellates (size range 0.6-5 microm) which are prevalent throughout the year within the heterotrophic flagellate assemblage in Lake Pavin. Four clone libraries were constructed from samples collected in two contrasting zones in the lake. Computerized statistic tools have suggested that sequence retrieval was representative of the in situ picoplankton diversity. The two sampling zones exhibited similar diversity patterns but shared only about 5% of the operational taxonomic units (OTUs). Phylogenetic analysis clustered our sequences into three taxonomic groups: Alveolates (30% of OTUs), Fungi (23%) and Cercozoa (19%). Fungi thus substantially contributed to the detected diversity, as was additionally supported by direct microscopic observations of fungal zoospores and sporangia. A large fraction of the sequences belonged to parasites, including Alveolate sequences affiliated to the genus Perkinsus known as zooparasites, and chytrids that include host-specific parasitic fungi of various freshwater phytoplankton species, primarily diatoms. Phylogenetic analysis revealed five novel clades that probably include typical freshwater environmental sequences. Overall, from the unsuspected fungal diversity unveiled, we think that fungal zooflagellates have been misidentified as phagotrophic nanoflagellates in previous studies. This is in agreement with a recent experimental demonstration that zoospore-producing fungi and parasitic activity may play an important role in aquatic food webs.

Late summer community composition and abundance of photosynthetic picoeukaryotes in Norwegian and Barents Seas

We investigated marine picoeukaryotic diversity (cells <3 µm) 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 (TSAFISH) 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 ml21). M. pusilla was also well represented in coastal waters and at the polar front (25% of the picoeukaryotes, maximum 9,100 cells ml21). Haptophyta were prominent in more typical Atlantic waters (up to 35% of the picoeukaryotes, maximum 4,500 cells ml21). 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.

Phytoplankton seasonal dynamics in a Mediterranean coastal lagoon: emphasis on the picoeukaryote community

The dynamics of the phytoplankton community were investigated in a marine coastal lagoon (Thau, NW Mediterranean) from February 1999 to January 2000. Dilution experiments, chlorophyll a (Chl a) size-fractionation and primary production measurements were conducted monthly. Maximum growth and microzooplankton grazing rates were estimated from Chl a biomass fractions to separate pico- from nano- and microphytoplankton and by flow cytometry to distinguish between picoeukaryotes and picocyanobacteria. In spring, the phytoplankton community was dominated by Chaetoceros sp. and Skeletonema costatum, which represented most of biomass (B) and primary production (P). Nano- and microphytoplankton growth was controlled by nutrient availability and exceeded losses due to microzooplankton grazing (g). Picoeukaryote and cyanobacteria growth was positively correlated with water temperature and/or irradiance, reaching maximum values in the summer (2.38 and 1.44 day(-1) for picoeukaryotes and cyanobacteria, respectively). Picophytoplankton accounted for 57% of the biomass-specific primary productivity (P/B). Picophytoplankton was strongly controlled by protist grazers (g = 0.09-1.66 day(-1) for picoeukaryotes, g = 0.25-1.17 day(-1) for cyanobacteria), and microzooplankton consumption removed 71% of the daily picoplanktonic growth. Picoeukaryotes, which numerically dominate the picoplankton community, are an important source of organic carbon for the protistan community and contribute to the carbon flow to higher trophic levels.

A single species, Micromonas pusilla (Prasinophyceae), dominates the eukaryotic picoplankton in the Western English Channel.

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 x 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.

Composition and temporal variability of picoeukaryote communities at a coastal site of the English Channel from 18S rDNA sequences

We analyzed picoeukaryote assemblages at a French coastal site of the English Channel by sequencing cloned eukaryotic 18S rRNA genes in eight genetic libraries constructed from environmental samples (seven coastal, one estuarine) collected at different periods of the year. Eight hundred clones were examined by amplified restriction fragment length polymorphism (RFLP) using the restriction enzyme HaeIII. The coverage value calculated from the relative distribution of RFLP types was low, indicating that the library diversity was not entirely recovered. A variable region of the rRNA gene was partially sequenced (550 bases) and analyzed for 397 clones. Thirty‐two clones were affiliated with metazoans. Of the remaining clones, 132 were affiliated to algal classes (especially Prasinophyceae, Cryptophyceae, Dinophyceae, and Prymnesiophyceae) and 107 to known heterotrophic groups (Cercozoa, choanoflagellates, stramenopiles, and ciliates). One hundred three sequences fell into uncultivated groups of stramenopiles (43 clones) and alveolates (60 clones). We also found two potentially novel eukaryotic lineages, represented by 9 and 14 clones, respectively, not belonging to any known eukaryotic group. The overall composition of the picoeukaryote community remained fairly stable at the class/division level except during the early summer diatom bloom, when groups such as the Cryptophyceae and the ciliates completely disappeared. However, at a finer taxonomic level (corresponding to 98% sequence identity), the majority of the operational taxonomic units (OTU) were only observed once.

Diversity and abundance of Bolidophyceae (Heterokonta) in two oceanic regions.

The diversity and abundance of the Bolidophyceae (Heterokonta), a newly described picoplanktonic algal class which is a sister group to the diatoms, was assessed in the equatorial Pacific Ocean and in the Mediterranean Sea by culture isolation, molecular biology techniques, and pigment analyses. Eight strains of Bolidophyceae were isolated in culture from different mesotrophic and oligotrophic areas. The corresponding small subunit (SSU) rRNA gene sequences allowed us to design two probes specific for the Bolidophyceae. These probes have been used in natural samples (i) to selectively amplify and detect Bolidophyceae sequences and (ii) to quantify the relative abundance of Bolidophyceae within the picoeukaryote community. Sequences available to date indicate that the class Bolidophyceae comprises at least three different clades, two corresponding to the previously described species Bolidomonas pacifica and Bolidomonas mediterranea and the third one corresponding to a subspecies of B. pacifica. Amplification of the SSU rRNA gene from natural samples with universal primers and hybridization using a Bolidomonas-specific probe followed by a eukaryote-specific probe allowed us to estimate the contribution of the Bolidophyceae to the eukaryotic DNA in both Pacific and Mediterranean waters to be lower than 1%. Similarly, high-performance liquid chromatography analyses of fucoxanthin, the major carotenoid present in Bolidophyceae, indicated that less than 4% of the total chlorophyll a in the picoplanktonic fraction in the equatorial Pacific was due to Bolidophyceae. Consequently, although strains of Bolidophyceae have been isolated from samples collected at several stations, this new class seems to have been a minor component of the natural picoeukaryotic populations in the ecosystems investigated, at least during the periods sampled.