Carlos Pedrós-Alió

Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton

Phylogenetic information from ribosomal RNA genes directly amplified from the environment changed our view of the biosphere, revealing an extraordinary diversity of previously undetected prokaryotic lineages. Using ribosomal RNA genes from marine picoplankton, several new groups of bacteria and archaea have been identified, some of which are abundant. Little is known, however, about the diversity of the smallest planktonic eukaryotes, and available information in general concerns the phytoplankton of the euphotic region. Here we recover eukaryotes in the size fraction 0.2–5 µm from the aphotic zone (250–3,000 m deep) in the Antarctic polar front. The most diverse and relatively abundant were two new groups of alveolate sequences, related to dinoflagellates that are found at all studied depths. These may be important components of the microbial community in the deep ocean. Their phylogenetic position suggests a radiation early in the evolution of alveolates.

Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques

ABSTRACT We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.

Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing

ABSTRACT Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis

ABSTRACT The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the divisionProteobacteria. Sequences obtained from Lake Vilar samples were related to members of theCytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.

Bacterial Community Structure Associated with a Dimethylsulfoniopropionate-Producing North Atlantic Algal Bloom

ABSTRACT The bacteria associated with oceanic algal blooms are acknowledged to play important roles in carbon, nitrogen, and sulfur cycling, yet little information is available on their identities or phylogenetic affiliations. Three culture-independent methods were used to characterize bacteria from a dimethylsulfoniopropionate (DMSP)-producing algal bloom in the North Atlantic. Group-specific 16S rRNA-targeted oligonucleotides, 16S ribosomal DNA (rDNA) clone libraries, and terminal restriction fragment length polymorphism analysis all indicated that the marine Roseobacter lineage was numerically important in the heterotrophic bacterial community, averaging >20% of the 16S rDNA sampled. Two other groups of heterotrophic bacteria, the SAR86 and SAR11 clades, were also shown by the three 16S rRNA-based methods to be abundant in the bloom community. In surface waters, the Roseobacter, SAR86, and SAR11 lineages together accounted for over 50% of the bacterial rDNA and showed little spatial variability in abundance despite variations in the dominant algal species. Depth profiles indicated thatRoseobacter phylotype abundance decreased with depth and was positively correlated with chlorophyll a, DMSP, and total organic sulfur (dimethyl sulfide plus DMSP plus dimethyl sulfoxide) concentrations. Based on these data and previous physiological studies of cultured Roseobacter strains, we hypothesize that this lineage plays a role in cycling organic sulfur compounds produced within the bloom. Three other abundant bacterial phylotypes (representing a cyanobacterium and two members of the α Proteobacteria) were primarily associated with chlorophyll-rich surface waters of the bloom (0 to 50 m), while two others (representing Cytophagales and δProteobacteria) were primarily found in deeper waters (200 to 500 m).

A Few Cosmopolitan Phylotypes Dominate Planktonic Archaeal Assemblages in Widely Different Oceanic Provinces

ABSTRACT We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.

Phylogenetic and ecological analysis of novel marine stramenopiles

ABSTRACT Culture-independent molecular analyses of open-sea microorganisms have revealed the existence and apparent abundance of novel eukaryotic lineages, opening new avenues for phylogenetic, evolutionary, and ecological research. Novel marine stramenopiles, identified by 18S ribosomal DNA sequences within the basal part of the stramenopile radiation but unrelated to any previously known group, constituted one of the most important novel lineages in these open-sea samples. Here we carry out a comparative analysis of novel stramenopiles, including new sequences from coastal genetic libraries presented here and sequences from recent reports from the open ocean and marine anoxic sites. Novel stramenopiles were found in all major habitats, generally accounting for a significant proportion of clones in genetic libraries. Phylogenetic analyses indicated the existence of 12 independent clusters. Some of these were restricted to anoxic or deep-sea environments, but the majority were typical components of coastal and open-sea waters. We specifically identified four clusters that were well represented in most marine surface waters (together they accounted for 74% of the novel stramenopile clones) and are the obvious targets for future research. Many sequences were retrieved from geographically distant regions, indicating that some organisms were cosmopolitan. Our study expands our knowledge on the phylogenetic diversity and distribution of novel marine stramenopiles and confirms that they are fundamental members of the marine eukaryotic picoplankton.

Light stimulates growth of proteorhodopsin-containing marine Flavobacteria

Proteorhodopsins are bacterial light-dependent proton pumps. Their discovery within genomic material from uncultivated marine bacterioplankton caused considerable excitement because it indicated a potential phototrophic function within these organisms, which had previously been considered strictly chemotrophic. Subsequent studies established that sequences encoding proteorhodopsin are broadly distributed throughout the world’s oceans. Nevertheless, the role of proteorhodopsins in native marine bacteria is still unknown. Here we show, from an analysis of the complete genomes of three marine Flavobacteria, that cultivated bacteria in the phylum Bacteroidetes, one of the principal components of marine bacterioplankton, contain proteorhodopsin. Moreover, growth experiments in both natural and artificial seawater (low in labile organic matter, which is typical of the world’s oceans) establish that exposure to light results in a marked increase in the cell yield of one such bacterium (Dokdonia sp. strain MED134) when compared with cells grown in darkness. Thus, our results show that the phototrophy conferred by proteorhodopsin can provide critical amounts of energy, not only for respiration and maintenance but also for active growth of marine bacterioplankton in their natural environment.

Distribution, phylogeny, and growth of cold-adapted Picoprasinophytes in Arctic Seas

Our pigment analyses from a year‐long study in the coastal Beaufort Sea in the western Canadian Arctic showed the continuous prevalence of eukaryotic picoplankton in the green algal class Prasinophyceae. Microscopic analyses revealed that the most abundant photosynthetic cell types were Micromonas‐like picoprasinophytes that persisted throughout winter darkness and then maintained steady exponential growth from late winter to early summer. A Micromonas (CCMP2099) isolated from an Arctic polynya (North Water Polynya between Ellesmere Island and Greenland), an ice‐free section, grew optimally at 6°C–8°C, with light saturation at or below 10 μmol photons·m−2·s−1 at 0°C. The 18S rDNA analyses of this isolate and environmental DNA clone libraries from diverse sites across the Arctic Basin indicate that this single psychrophilic Micromonas ecotype has a pan‐Arctic distribution. The 18S rDNA from two other picoprasinophyte genera was also found in our pan‐Arctic clone libraries: Bathycoccus and Mantoniella. The Arctic Micromonas differed from genotypes elsewhere in the World Ocean, implying that the Arctic Basin is a marine microbial province containing endemic species, consistent with the biogeography of its macroorganisms. The prevalence of obligate low‐temperature, shade‐adapted species in the phytoplankton indicates that the lower food web of the Arctic Ocean is vulnerable to ongoing climate change in the region.

Diversity and Distribution of Marine Microbial Eukaryotes in the Arctic Ocean and Adjacent Seas

ABSTRACT We analyzed microbial eukaryote diversity in perennially cold arctic marine waters by using 18S rRNA gene clone libraries. Samples were collected during concurrent oceanographic missions to opposite sides of the Arctic Ocean Basin and encompassed five distinct water masses. Two deep water Arctic Ocean sites and the convergence of the Greenland, Norwegian, and Barents Seas were sampled from 28 August to 2 September 2002. An additional sample was obtained from the Beaufort Sea (Canada) in early October 2002. The ribotypes were diverse, with different communities among sites and between the upper mixed layer and just below the halocline. Eukaryotes from the remote Canada Basin contained new phylotypes belonging to the radiolarian orders Acantharea, Polycystinea, and Taxopodida. A novel group within the photosynthetic stramenopiles was also identified. One sample closest to the interior of the Canada Basin yielded only four major taxa, and all but two of the sequences recovered belonged to the polar diatom Fragilariopsis and a radiolarian. Overall, 42% of the sequences were <98% similar to any sequences in GenBank. Moreover, 15% of these were <95% similar to previously recovered sequences, which is indicative of endemic or undersampled taxa in the North Polar environment. The cold, stable Arctic Ocean is a threatened environment, and climate change could result in significant loss of global microbial biodiversity.