Daniel Vaulot

Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity

Picoplankton—cells with a diameter of less than 3 µm—are the dominant contributors to both primary production and biomass in open oceanic regions. However, compared with the prokaryotes, the eukaryotic component of picoplankton is still poorly known. Recent discoveries of new eukaryotic algal taxa based on picoplankton cultures suggest the existence of many undiscovered taxa. Conventional approaches based on phenotypic criteria have limitations in depicting picoplankton composition due to their tiny size and lack of distinctive taxonomic characters. Here we analyse, using an approach that has been very successful for prokaryotes but has so far seldom been applied to eukaryotes, 35 full sequences of the small-subunit (18S) ribosomal RNA gene derived from a picoplanktonic assemblage collected at a depth of 75 m in the equatorial Pacific Ocean, and show that there is a high diversity of picoeukaryotes. Most of the sequences were previously unknown but could still be assigned to important marine phyla including prasinophytes, haptophytes, dinoflagellates, stramenopiles, choanoflagellates and acantharians. We also found a novel lineage, closely related to dinoflagellates and not previously described.

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing.

Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the worlds oceans.

Photosynthetic picoplankton community structure in the subtropical North Pacific Ocean near Hawaii (station ALOHA)

The structure of the picoplankton community in the subtropical Pacific was examined on four depth profiles, one from each season, sampled at the Hawaii Ocean Time-series station ALOHA (22°45′N, 158°W). Three cell populations were discriminated by flow cytometry: Prochlorococcus prochlorophytes, Synechococcus cyanobacteria, and picoeukaryotes. Prochlorococcus were the most abundant component (maximum ca 2 × 105 cells ml−1). Unlike previous reports, their concentration was almost constant down to roughly 100 m, with a slight maximum at the surface or near the chlorophyll maximum. Cellular chlorophyll fluorescence increased 50-fold between surface and deep populations. One distinguishing feature of the community off Hawaii was the co-occurrence near the chlorophyll maximum of at least two distinct Prochlorococcus populations with different chlorophyll and DNA contents. Throughout the year, Synechococcus abundance was two orders of magnitude lower and there was no seasonal alteration between Prochlorococcus and Synechococcus, as observed in the northern Sargasso Sea. Synechococcus populations did not extend below 120 m and were dominated by high phycourobilin cell types. Picoeukaryote abundance was quite similar to that of Synechococcus, but these cells extended deeper in the water column. Their chlorophyll fluorescence exhibited much less depth variation than Prochlorococcus or Synechococcus. Seasonal variability was small (<2- to 3-fold) for all three components of the picoplankton, not only for cell abundance but also for cellular parameters such as light scatter or pigment fluorescence. Synechococcus populations exhibited the largest seasonal changes (e.g. abundance maximum and chlorophyll fluorescence varied 3-fold). Picoplankton community structure in the Pacific Ocean appears to be distinct from previous reports for other areas. In comparing station ALOHA to the Atlantic Ocean (especially the Sargasso Sea) and the Mediterranean Sea, depth-integrated abundances of Prochlorococcus were higher, that of Synechococcus were lower, and that of picoeukaryotes were similar. We believe this structure, dominated by Prochlorococcus, may be typical for subtropical open-ocean regions.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR2, http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Growth of Prochlorococcus, a Photosynthetic Prokaryote, in the Equatorial Pacific Ocean

The cell cycle of Prochlorococcus, a prokaryote that accounts for a sizable fraction of the photosynthetic biomass in the eastern equatorial Pacific, progressed in phase with the daily light cycle. DNA replication occurred in the afternoon and cell division occurred at night. Growth rates were maximal (about one doubling per day) at 30 meters and decreased toward the surface and the bottom of the ocean. Estimated Prochlorococcus production varied between 174 and 498 milligrams of carbon per square meter per day and accounted for 5 to 19 percent of total gross primary production at the equator. Because Prochlorococcus multiplies close to its maximum possible rate, it is probably not severely nutrient-limited in this region of the oceans.

Clade-Specific 16S Ribosomal DNA Oligonucleotides Reveal the Predominance of a Single Marine Synechococcus Clade throughout a Stratified Water Column in the Red Sea

ABSTRACT Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.

Unicellular Cyanobacterium Symbiotic with a Single-Celled Eukaryotic Alga

Fixing on a Marine Partnership Nitrogen fixation by microorganisms determines the productivity of the biosphere. Although plants photosynthesize by virtue of the ancient incorporation of cyanobacteria to form chloroplasts, no equivalent endosymbiotic event has occurred for nitrogen fixation. Nevertheless, in terrestrial environments, nitrogen-fixing symbioses between bacteria and plants, for example, are common. Thompson et al. (p. 1546) noticed that the ubiquitous marine cyanobacterium UCYN-A has an unusually streamlined genome lacking components of the photosynthetic machinery and central carbon metabolism—all suggestive of being an obligate symbiont. By using gentle filtration methods for raw seawater, a tiny eukaryote partner for UCYN-A of less than 3-µm in diameter was discovered. The bacterium sits on the cell wall of this calcifying picoeukaryote, donating fixed nitrogen and receiving fixed carbon in return. A nitrogen-fixing cyanobacterium that lacks photosynthesis and the tricarboxylic acid cycle possesses a tiny phytoplankton symbiont. Symbioses between nitrogen (N)2–fixing prokaryotes and photosynthetic eukaryotes are important for nitrogen acquisition in N-limited environments. Recently, a widely distributed planktonic uncultured nitrogen-fixing cyanobacterium (UCYN-A) was found to have unprecedented genome reduction, including the lack of oxygen-evolving photosystem II and the tricarboxylic acid cycle, which suggested partnership in a symbiosis. We showed that UCYN-A has a symbiotic association with a unicellular prymnesiophyte, closely related to calcifying taxa present in the fossil record. The partnership is mutualistic, because the prymnesiophyte receives fixed N in exchange for transferring fixed carbon to UCYN-A. This unusual partnership between a cyanobacterium and a unicellular alga is a model for symbiosis and is analogous to plastid and organismal evolution, and if calcifying, may have important implications for past and present oceanic N2 fixation.

The diversity of small eukaryotic phytoplankton (≤3 μm) in marine ecosystems

Small cells dominate photosynthetic biomass and primary production in many marine ecosystems. Traditionally, picoplankton refers to cells < or =2 microm. Here we extend the size range of the organisms considered to 3 microm, a threshold often used operationally in field studies. While the prokaryotic component of picophytoplankton is dominated by two genera, Prochlorococcus and Synechococcus, the eukaryotic fraction is much more diverse. Since the discovery of the ubiquitous Micromonas pusilla in the early 1950s, just over 70 species that can be <3 microm have been described. In fact, most algal classes contain such species. Less than a decade ago, culture-independent approaches (in particular, cloning and sequencing, denaturing gradient gel electrophoresis, FISH) have demonstrated that the diversity of eukaryotic picoplankton is much more extensive than could be assumed from described taxa alone. These approaches revealed the importance of certain classes such as the Prasinophyceae but also unearthed novel divisions such as the recently described picobiliphytes. In the last couple of years, the first genomes of photosynthetic picoplankton have become available, providing key information on their physiological capabilities. In this paper, we discuss the range of methods that can be used to assess small phytoplankton diversity, present the species described to date, review the existing molecular data obtained on field populations, and end up by looking at the promises offered by genomics.

Annual variability of phytoplankton and bacteria in the subtropical North Pacific Ocean at Station ALOHA during the 1991–1994 ENSO event

Time-series data on community structure in the upper 200 m at Station ALOHA in the subtropical North Pacific were collected at approximately monthly intervals from December 1990 through to March 1994 during an extended El Niiio-Southern Oscillation (ENSO) event. Samples were analyzed by flow cytometry to enumerate Prochlorococcus, Synechococcus, picoeucaryotes, 3–20 μm algae, and heterotrophic bacteria, as well as to quantify cellular chlorophyll fluorescence for the autotrophic components. A significant seasonal cycle was evident in cellular chlorophyll fluorescence for each of the autotrophic components, with maxima occurring each winter as a consequence of photoacclimation. Abundance of each picophytoplankton component exhibited temporal variability on both seasonal and interannual scales. Although the magnitude of the seasonal cycles in the abundance was relatively small, the cycles appeared to be out of phase. Typically, abundance maxima of Synechococcus occurred in winter, of picoeucaryotes in spring, and of Prochlorococcus during summer/fall. The different timing in these cycles may explain why the presence of a seasonal pattern in total phytoplankton biomass has been difficult to establish. Abundance of the larger 3–20 μm algae varied over two orders of magnitude during the time series, with no obvious seasonal pattern. The 3–20 μm algae were a small percentage of the total estimated carbon biomass (∼8%). Heterotrophic bacteria were the most numerous of the picoplankton, and the seasonal pattern in their 200-m integrated abundance paralleled Prochlorococcus over the time series. Together, the procaryotes contributed 60–90% of the total estimated microbial carbon. Significant interannual variation in the total 200-m integrated microbial carbon estimates may be related to the effects of the extended ENSO event, which began in 1991.

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.