Diverse eukaryotic phytoplankton from around the Marquesas Islands documented by combined microscopy and molecular techniques.

Oceanic phytoplankton serve as a base for the food webs within the largest planetary ecosystem. Despite this, surprisingly little is known about species composition, function and ecology of phytoplankton communities, especially for vast areas of the open ocean. In this study we focus on the marine phytoplankton microflora from the vicinity of the Marquesas Islands in the Southern Pacific Ocean collected during the Tara Oceans expedition. Multiple samples from four sites and two depths were studied in detail using light microscopy, scanning electron microscopy, and automated confocal laser scanning microscopy. In total 289 taxa were identified, with Dinophyceae and Bacillariophyceae contributing 60% and 32% of taxa, respectively, to phytoplankton community composition. Notwithstanding, a large number of cells could not be assigned to any known species. Coccolithophores and other flagellates together contributed less than 8% to the species list. Observed cell densities were generally low, but at sites of high autotrophic biomass, diatoms reached the highest cell densities (1.26 × 104 cells L-1). Overall, 18S rRNA metabarcode-based community compositions matched microscopy-based estimates, particularly for the main diatom taxa, indicating consistency and complementarity between different methods, while the wide range of microscopy-based methods permitted several unknown and poorly studied taxa to be revealed and identified.

A robust approach to estimate relative phytoplankton cell abundances from metagenomes.

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.

Global Trends in Marine Plankton Diversity across Kingdoms of Life.

The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21st century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT.

The epibiotic life of the cosmopolitan diatom Fragilariopsis doliolus on heterotrophic ciliates in the open ocean.

Diatoms are a diverse and ecologically important group of phytoplankton. Although most species are considered free living, several are known to interact with other organisms within the plankton. Detailed imaging and molecular characterization of any such partnership is, however, limited, and an appraisal of the large-scale distribution and ecology of such consortia was never attempted. Here, observation of Tara Oceans samples from the Benguela Current led to the detection of an epibiotic association between a pennate diatom and a tintinnid ciliate. We identified the diatom as Fragilariopsis doliolus that possesses a unique feature to form barrel-shaped chains, associated with seven different genera of tintinnids including five previously undescribed associations. The organisms were commonly found together in the Atlantic and Pacific Ocean basins, and live observations of the interaction have been recorded for the first time. By combining confocal and scanning electron microscopy of individual consortia with the sequencing of high-resolution molecular markers, we analyzed their distribution in the global ocean, revealing morpho-genetically distinct tintinnid haplotypes and biogeographically structured diatom haplotypes. The diatom was among the most abundant in the global ocean. We show that the consortia were particularly prevalent in nutrient-replete conditions, rich in potential predators. These observations support the hypothesis of a mutualistic symbiosis, wherein diatoms acquire increased motility and tintinnids benefit from silicification through increased protection, and highlight that such associations may be more prevalent than currently appreciated.

Insights into global diatom distribution and diversity in the world's ocean.

Diatoms (Bacillariophyta) constitute one of the most diverse and ecologically important groups of phytoplankton. They are considered to be particularly important in nutrient-rich coastal ecosystems and at high latitudes, but considerably less so in the oligotrophic open ocean. The Tara Oceans circumnavigation collected samples from a wide range of oceanic regions using a standardized sampling procedure. Here, a total of ∼12 million diatom V9-18S ribosomal DNA (rDNA) ribotypes, derived from 293 size-fractionated plankton communities collected at 46 sampling sites across the global ocean euphotic zone, have been analyzed to explore diatom global diversity and community composition. We provide a new estimate of diversity of marine planktonic diatoms at 4,748 operational taxonomic units (OTUs). Based on the total assigned ribotypes, Chaetoceros was the most abundant and diverse genus, followed by Fragilariopsis, Thalassiosira, and Corethron We found only a few cosmopolitan ribotypes displaying an even distribution across stations and high abundance, many of which could not be assigned with confidence to any known genus. Three distinct communities from South Pacific, Mediterranean, and Southern Ocean waters were identified that share a substantial percentage of ribotypes within them. Sudden drops in diversity were observed at Cape Agulhas, which separates the Indian and Atlantic Oceans, and across the Drake Passage between the Atlantic and Southern Oceans, indicating the importance of these ocean circulation choke points in constraining diatom distribution and diversity. We also observed high diatom diversity in the open ocean, suggesting that diatoms may be more relevant in these oceanic systems than generally considered.

The sexual phase of the diatom Pseudo-nitzschia multistriata: cytological and time-lapse cinematography characterization.

Pseudo-nitzschia is a thoroughly studied pennate diatom genus for ecological and biological reasons. Many species in this genus, including Pseudo-nitzschia multistriata, can produce domoic acid, a toxin responsible for amnesic shellfish poisoning. Physiological, phylogenetic and biological features of P. multistriata were studied extensively in the past. Life cycle stages, including the sexual phase, fundamental in diatoms to restore the maximum cell size and avoid miniaturization to death, have been well described for this species. P. multistriata is heterothallic; sexual reproduction is induced when strains of opposite mating type are mixed, and proceeds with cells producing two functionally anisogamous gametes each; however, detailed cytological information for this process is missing. By means of confocal laser scanning microscopy and nuclear staining, we followed the nuclear fate during meiosis, and using time-lapse cinematography, we timed every step of the sexual reproduction process from mate pairing to initial cell hatching. The present paper depicts cytological aspects during gametogenesis in P. multistriata, shedding light on the chloroplast behaviour during sexual reproduction, finely describing the timing of the sexual phases and providing reference data for further studies on the molecular control of this fundamental process.

Ocean plankton. Environmental characteristics of Agulhas rings affect interocean plankton transport.

Agulhas rings provide the principal route for ocean waters to circulate from the Indo-Pacific to the Atlantic basin. Their influence on global ocean circulation is well known, but their role in plankton transport is largely unexplored. We show that, although the coarse taxonomic structure of plankton communities is continuous across the Agulhas choke point, South Atlantic plankton diversity is altered compared with Indian Ocean source populations. Modeling and in situ sampling of a young Agulhas ring indicate that strong vertical mixing drives complex nitrogen cycling, shaping community metabolism and biogeochemical signatures as the ring and associated plankton transit westward. The peculiar local environment inside Agulhas rings may provide a selective mechanism contributing to the limited dispersal of Indian Ocean plankton populations into the Atlantic.

Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean.

Marine plankton support global biological and geochemical processes. Surveys of their biodiversity have hitherto been geographically restricted and have not accounted for the full range of plankton size. We assessed eukaryotic diversity from 334 size-fractionated photic-zone plankton communities collected across tropical and temperate oceans during the circumglobal Tara Oceans expedition. We analyzed 18S ribosomal DNA sequences across the intermediate plankton-size spectrum from the smallest unicellular eukaryotes (protists, >0.8 micrometers) to small animals of a few millimeters. Eukaryotic ribosomal diversity saturated at ~150,000 operational taxonomic units, about one-third of which could not be assigned to known eukaryotic groups. Diversity emerged at all taxonomic levels, both within the groups comprising the ~11,200 cataloged morphospecies of eukaryotic plankton and among twice as many other deep-branching lineages of unappreciated importance in plankton ecology studies. Most eukaryotic plankton biodiversity belonged to heterotrophic protistan groups, particularly those known to be parasites or symbiotic hosts.

Ultrastructural features of the benthic dinoflagellate Ostreopsis cf. ovata (Dinophyceae).

The toxic benthic dinoflagellate Ostreopsis cf. ovata has considerably expanded its distribution range in the last decade, posing risks to human health. Several aspects of this species are still poorly known. We studied ultrastructural features of cultivated and natural populations of Ostreopsis cf. ovata from the Gulf of Naples (Mediterranean Sea) using confocal laser scanning, and scanning and transmission electron microscopy. New information on the morphology and location of several sulcal plates was gained and a new plate designation is suggested that better fits the one applied to other Gonyaulacales. The microtubular component of the cytoskeleton, revealed using an anti-ß-tubulin antibody, consisted of a cortical layer of microtubules arranged asymmetrically in the episome and in the hyposome, complemented by a complex inner microtubular system running from the sulcal area towards the internal part of the cell. The conspicuous canal was delimited by two thick, burin-shaped lobes ending in a tubular ventral opening. The canal was surrounded by mucocysts discharging their content into it. A similar structure has been reported in other benthic and planktonic dinoflagellates and may be interpreted as an example of convergent evolution in species producing large amounts of mucus.

The dynamics of sexual phase in the marine diatom Pseudo-nitzschia multistriata (Bacillariophyceae).

Sexual reproduction represents a fundamental phase in the life cycle of diatoms, linked to both the production of genotypic diversity and the formation of large-sized initial cells. Only cells below a certain size threshold can be sexualized, but various environmental factors can modulate the success of sexual reproduction. We investigated the role of cell density and physiological conditions of parental strains in affecting the success and timing of sexual reproduction in the marine heterothallic diatom Pseudo-nitzschia multistriata. We also studied the dynamics of the sexual phase in still conditions allowing cell sedimentation and in gently mixed conditions that keep cells in suspension. Our results showed that successful sexual reproduction can only be achieved when crossing parental strains in the exponential growth phase. Evidence was provided for the fact that sexual reproduction is a density-dependent event and requires a threshold cell concentration to start, although this might vary considerably amongst strains. Moreover, the onset of the sexual phase was coupled to a marked reduction in growth of the vegetative parental cells. The crosses carried out in physically mixed conditions produced a significantly reduced number of sexual stages as compared to crosses in still conditions, showing that mixing impairs sexualization. The results of our experiments suggest that the signaling that triggers the sexual phase is favored when cells can accumulate, reducing the distance between them and facilitating contacts and/or the perception of chemical cues. Information on the progression of the sexual phase in laboratory conditions help understanding the conditions at which sex occurs in the natural environment.

New palytoxin-like molecules in Mediterranean Ostreopsis cf. ovata (dinoflagellates) and in Palythoa tuberculosa detected by liquid chromatography-electrospray ionization time-of-flight mass spectrometry.

A rapid, high resolution liquid chromatography coupled with ElectroSpray Ionization Time-Of-Flight Mass Spectrometry (ESI/TOF/MS) method was developed for the determination of the toxin pattern in cultured cells of Ostreopsis cf. ovata from the Mediterranean Sea. The samples were separated on a Phenomenex Luna 3µ HILIC 200A (150 × 2.00 mm) and analyzed by LC/TOF/MS with electrospray ionization (ESI) interface in positive ion mode. The method developed here provides the capability for a fully automated analysis, which requires relatively easy sample preparation and gives clean and simple chromatograms. The method was successfully applied to the determination of ovatoxin-a, mascarenotoxin-a and four new palytoxins in O. cf. ovata. Another new palytoxin was detected in the standard material from Palythoa tuberculosa provided by Wako Chemicals.