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Plankton seascape genomics show different trends from large-scale weak differentiation to micro-scale structures. Prior studies underlined the influence of environment and seascape on a few single species differentiation and adaptation. However, these works generally focused on few single species, sparse molecular markers, or local scales. Here, we investigate the genomic differentiation of plankton at macro-scale in a holistic approach using Tara Oceans metagenomic data together with a reference-free computational method to reconstruct the FST-based genomic differentiation of 113 marine planktonic species using metavariant species (MVS). These MVSs, modelling the species only by their polymorphism, include a wide range of taxonomic groups comprising notably 46 Maxillopoda/Copepoda, 24 Bacteria, 5 Dinoflagellates, 4 Haptophytes, 3 Cnidarians, 3 Mamiellales, 2 Ciliates, 1 Collodaria, 1 Echinoidea, 1 Pelagomonadaceae, 1 Cryptophyta and 1 Virus. The analyses showed that differentiation between populations was significantly lower within basins and higher in bacteria and unicellular eukaryotes compared to zooplantkon. By partitioning the variance of pairwise-FSTmatrices, we found that the main drivers of genomic differentiation were Lagrangian travel time, salinity and temperature. Furthermore, we classified MVSs into parameter-driven groups and showed that taxonomy poorly determines which environmental factor drives genomic differentiation. This holistic approach of plankton genomic differentiation for large geographic scales, a wide range of taxa and different oceanic basins, offers a systematic framework to analyse population genomics of non-model and undocumented marine organisms.

Among marine phytoplankton, Mamiellales encompass several species from the genera Micromonas, Ostreococcus and Bathycoccus, which are important contributors to primary production. Previous studies based on single gene markers described their wide geographical distribution but led to discussion because of the uneven taxonomic resolution of the method. Here, we leverage genome sequences for six Mamiellales species, two from each genus Micromonas, Ostreococcus and Bathycoccus, to investigate their distribution across 133 stations sampled during the Tara Oceans expedition. Our study confirms the cosmopolitan distribution of Mamiellales and further suggests non-random distribution of species, with two triplets of co-occurring genomes associated with different temperatures: Ostreococcus lucimarinus, Bathycoccus prasinos and Micromonas pusilla were found in colder waters, whereas Ostreococcus spp. RCC809, Bathycoccus spp. TOSAG39-1 and Micromonas commoda were more abundant in warmer conditions. We also report the distribution of the two candidate mating-types of Ostreococcus for which the frequency of sexual reproduction was previously assumed to be very low. Indeed, both mating types were systematically detected together in agreement with either frequent sexual reproduction or the high prevalence of a diploid stage. Altogether, these analyses provide novel insights into Mamiellales&rsquo, biogeography and raise novel testable hypotheses about their life cycle and ecology.

Among marine phytoplankton, Mamiellales encompass several species from the genera Micromonas, Ostreococcus and Bathycoccus, which are important contributors to primary production. Previous studies based on single gene markers described their wide geographical distribution but led to discussion because of the uneven taxonomic resolution of the method. Here, we leverage genome sequences for six Mamiellales species, two from each genus Micromonas, Ostreococcus and Bathycoccus, to investigate their distribution across 133 stations sampled during the Tara Oceans expedition. Our study confirms the cosmopolitan distribution of Mamiellales and further suggests non-random distribution of species, with two triplets of co-occurring genomes associated with different temperatures: Ostreococcus lucimarinus, Bathycoccus prasinos and Micromonas pusilla were found in colder waters, whereas Ostreococcus spp. RCC809, Bathycoccus spp. TOSAG39-1 and Micromonas commoda were more abundant in warmer conditions. We also report the distribution of the two candidate mating-types of Ostreococcus for which the frequency of sexual reproduction was previously assumed to be very low. Indeed, both mating types were systematically detected together in agreement with either frequent sexual reproduction or the high prevalence of a diploid stage. Altogether, these analyses provide novel insights into Mamiellales’ biogeography and raise novel testable hypotheses about their life cycle and ecology.

Members of the class Mamiellophyceae comprise species that can dominate picophytoplankton diversity in polar waters. Yet polar species are often morphologically indistinguishable from temperate species, although clearly separated by molecular features. Here we examine four Mamiellophyceae strains from the Canadian Arctic. The 18S rRNA and Internal Transcribed Spacer 2 (ITS2) gene phylogeny place these strains within the family Mamiellaceae (Mamiellales, Mamiellophyceae) in two separate clades of the genus Mantoniella. ITS2 synapomorphies support their placement as two new species, Mantoniella beaufortii and Mantoniella baffinensis. Both species have round green cells with diameter between 3–5 μm, one long flagellum and a short reduced flagellum (~1 μm) and are covered by spiderweb-like scales, making both species similar to other Mantoniella species. Morphologically, M. beaufortii and M. baffinensis are most similar to the cosmopolitan M. squamata with only minor differences in scale structure distinguishing them. Screening of global marine metabarcoding datasets indicates M. beaufortii has only been recorded in seawater and sea ice samples from the Arctic while no environmental barcode matches M. baffinensis. Like other Mamiellophyceae genera that have distinct polar and temperate species, the polar distribution of these new species suggests they are cold or ice-adapted Mantoniella species.

Planktonic photosynthetic organisms of the class Mamiellophyceae include the smallest eukaryotes (less than 2 µm), are globally distributed and form the basis of coastal marine ecosystems. Eight complete fully annotated 13–22 Mb genomes from three genera, Ostreococcus , Bathycoccus and Micromonas , are available from previously isolated clonal cultured strains and provide an ideal resource to explore the scope and challenges of analysing single cell amplified genomes (SAGs) isolated from a natural environment. We assembled data from 12 SAGs sampled during the Tara Oceans expedition to gain biological insights about their in situ ecology, which might be lost by isolation and strain culture. Although the assembled nuclear genomes were incomplete, they were large enough to infer the mating types of four Ostreococcus SAGs. The systematic occurrence of sequences from the mitochondria and chloroplast, representing less than 3% of the total cell's DNA, intimates that SAGs provide suitable substrates for detection of non-target sequences, such as those of virions. Analysis of the non-Mamiellophyceae assemblies, following filtering out cross-contaminations during the sequencing process, revealed two novel 1.6 and 1.8 kb circular DNA viruses, and the presence of specific Bacterial and Oomycete sequences suggests that these organisms might co-occur with the Mamiellales. This article is part of a discussion meeting issue ‘Single cell ecology’.

Prasinophytes (Chlorophyta) are a diverse, paraphyletic group of planktonic microalgae for which benthic species are largely unknown. Here, we report a sand-dwelling, marine prasinophyte with several novel features observed in clonal cultures established from numerous locations around Australia. The new genus and species, which we name Microrhizoidea pickettheapsiorum (Mamiellophyceae), alternates between a benthic palmelloid colony, where cell division occurs, and a planktonic flagellate. Flagellates are short lived, settle and quickly resorb their flagella, the basal bodies then nucleate novel tubular appendages, termed "microrhizoids", that lack an axoneme and function to anchor benthic cells to the substratum. To our knowledge, microrhizoids have not been observed in any other green alga or protist, are slightly smaller in diameter than flagella, generally contain nine microtubules, are long (3-5 times the length of flagella) and are not encased in scales. Following settlement, cell divisions result in a loose, palmelloid colony, each cell connected to the substratum by two microrhizoids. Flagellates are round to bean-shaped with two long, slightly uneven flagella. Both benthic cells and flagellates, along with their flagella, are encased in thin scales. Phylogenies based on the complete chloroplast genome of Microrhizoidea show that it is clearly a member of the Mamiellophyceae, most closely related to Dolichomastix tenuilepsis. More taxon-rich phylogenetic analyses of the 18S rRNA gene, including metabarcodes from the Tara Oceans and Ocean Sampling Day projects, confidently show the distinctive nature of Microrhizoidea, and that the described biodiversity of the Mamiellophyceae is a fraction of its real biodiversity. The discovery of a largely benthic prasinophyte changes our perspective on this group of algae and, along with the observation of other potential benthic lineages in environmental sequences, illustrates that benthic habitats can be a rich ground for algal biodiscovery.

Prasinophytes are a paraphyletic assemblage of nine heterogeneous lineages in the Chlorophyta clade of Archaeplastida. Until now, seven complete mitochondrial genomes have been sequenced from four prasinophyte lineages. Here, we report the mitochondrial genome of Pyramimonas parkeae, the first representative of the prasinophyte clade I. The circular-mapping molecule is 43,294 bp long, AT rich (68.8%), very compact and it comprises two 6,671 bp long inverted repeat regions. The gene content is slightly smaller than the gene-richest prasinophyte mitochondrial genomes. The single identified intron is located in the cytochrome c oxidase subunit 1 gene (cox1). Interestingly, two exons of cox1 are encoded on the same strand of DNA in the reverse order and the mature mRNA is formed by trans-splicing. The phylogenetic analysis using the data set of 6,037 positions assembled from 34 mtDNA-encoded proteins of 48 green algae and plants is not in compliance with the branching order of prasinophyte clades revealed on the basis of 18S rRNA genes and cpDNA-encoded proteins. However, the phylogenetic analyses based on all three genomic elements support the sister position of prasinophyte clades Pyramimonadales and Mamiellales.

The molecular diversity of freshwater microeukaryotes, particularly phytoplankton, in the Arctic Svalbard, has been relatively unexplored. Freshwater algae are considered biological indicators of environmental change and can be useful in assessing the impact of global climate change and increased environmental pollution. In this study, freshwater microeukaryotes in an Arctic reservoir at Ny-Ǻlesund (Svalbard, Norway) were studied using the hypervariable V1–V3 small subunit rRNA and 454 pyrosequencing. On the basis of 8,956 reads, we revealed high genetic diversity in eukaryotes, representing all known eukaryotic supergroups, except Excavata. “Chromalveolata” (previously supergroup Chromalveolata) and Archaeplastida were the most and least abundant supergroups, respectively. After data mining, 57 phylotypes were detected from 7,398 pyrosequences. They were dominated by stramenopiles (84 %) and Dinoflagellata (13 %), with minor contributions from Cryptophyta, Chlorophyta, and Telonemida. The detection of algae belonging to the orders Mamiellales and Monomastigales provides a window into a fraction of the ‘rare biosphere’ that had previously been undetected in such environments. Interestingly, no haptophytes were recorded. Stramenopiles and Dinoflagellata mainly comprised taxa belonging to the families Chrysophyceae, Synurophyceae, and Dinophyceae. On the basis of the proportion of operational taxonomic units, the dominant phylotypes were found to include Ochromonas spp., Mallomonas spp., and Uroglena americana. These results demonstrate the significance of a chrysophyte-dominated microeukaryotic community, which is of great potential for future studies in terms of reconstruction of past climate trends and monitoring of environmental change in the Arctic.

Summary The Atacama Desert is one of the oldest and driest deserts in the world, and its hyper-arid core is described as ‘the most barren region imaginable’. We used a combination of high-throughput sequencing and microscopy methods to characterize the endolithic microbial assemblages of halite pinnacles (salt rocks) collected in several hyper-arid areas of the desert. We found communities dominated by archaea that relied on a single phylotype of Halothece cyanobacteria for primary production. A few other phylotypes of salt-adapted bacteria and archaea, including Salinibacter, Halorhabdus, and Halococcus were major components of the halite communities, indicating specific adaptations to the unique halite environments. Multivariate statistical analyses of diversity metrics clearly separated the halite communities from that of the surrounding soil in the Yungay area. These analyses also revealed distribution patterns of halite communities correlated with atmospheric moisture. Microbial endolithic communities from halites exposed to coastal fogs and high relative humidity were more diverse; their archaeal and bacterial assemblages were accompanied by a novel algae related to oceanic picoplankton of the Mamiellales. In contrast, we did not find any algae in the Yungay pinnacles, suggesting that the environmental conditions in this habitat might be too extreme for eukaryotic photosynthetic life.

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of world's oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae., Author Summary We propose that chromosome 19 of O. tauri is specialized in defence against viral attack, a constant threat for all planktonic life, and that the most likely cause of resistance is the over-expression of numerous predicted glycosyltransferase genes. O. tauri thus provides an amenable model for molecular analysis of genome evolution under environmental stress and for investigating glycan-mediated host-virus interactions, such as those seen in herpes, influenza, HIV, PBCV and mimivirus.

Flow cytometric sorting, based on cellular optical properties and macromolecule content, has been successfully employed to taxonomically affiliate bacterioplankton. However, this approach has not been much used for eukaryotic plankton. To redress this imbalance, we identified a conspicuous group of red autofluorescent picoplankton in surface waters of the South Atlantic Ocean. Using catalysed reporter deposition fluorescence in situ hybridization, virtually, all cells sorted from that group were affiliated with the Mamiellales clade II (84 ± 4%, division Chlorophyta) with a size of 1.6 ± 0.03 μm. Based on electron microscopy, the Mamiellales clade II-sorted cells have a simple morphology with apparently no scales, flagella or surface features. Their latitudinal distribution resembled the distribution of Synechococcus with very low concentrations in the surface waters of the Southern subtropical gyre (0.6-1.6 × 10(3) cells mL(-1)) and increased concentrations in the Southern temperate waters 8.3 × 10(3) cells mL(-1). Identification of the flow cytometric group as Mamiellales clade II allowed us to characterize the morphology of these enigmatic uncultured picoplanktonic cells by electron microscopy and to determine their apparent preference for temperate rather than subtropical oceanic photic waters.

Prasinoviruses infecting unicellular green algae in the order Mamiellales (class Mamiellophyceae) are commonly found in coastal marine waters where their host species frequently abound. We tested 40 Ostreococcus tauri viruses on 13 independently isolated wild-type O. tauri strains, 4 wild-type O. lucimarinus strains, 1 Ostreococcus sp. (“ Ostreococcus mediterraneus ”) clade D strain, and 1 representative species of each of two other related species of Mamiellales, Bathycoccus prasinos and Micromonas pusilla . Thirty-four out of 40 viruses infected only O. tauri , 5 could infect one other species of the Ostreococcus genus, and 1 infected two other Ostreococcus spp., but none of them infected the other genera. We observed that the overall susceptibility pattern of Ostreococcus strains to viruses was related to the size of two host chromosomes known to show intraspecific size variations, that genetically related viruses tended to infect the same host strains, and that viruses carrying inteins were strictly strain specific. Comparison of two complete O. tauri virus proteomes revealed at least three predicted proteins to be candidate viral specificity determinants.

The prasinophyte order Mamiellales contains several widespread marine picophytoplankton (≤2 μm diameter) taxa, including Micromonas and Ostreococcus. Complete genome sequences are available for two Micromonas isolates, CCMP1545 and RCC299. We performed in silico analyses of nitrogen transporters and related assimilation genes in CCMP1545 and RCC299 and compared these with other green lineage organisms as well as Chromalveolata, fungi, bacteria, and archaea. Phylogenetic reconstructions of ammonium transporter (AMT) genes revealed divergent types contained within each Mamiellales genome. Some were affiliated with plant and green algal AMT1 genes and others with bacterial AMT2 genes. Land plant AMT2 genes were phylogenetically closer to archaeal transporters than to Mamiellales AMT2 genes. The Mamiellales represent the first green algal genomes to harbor AMT2 genes, which are not found in Chlorella and Chlamydomonas or the chromalveolate algae analyzed but are present in oomycetes. Fewer nitrate transporter (NRT) than AMT genes were identified in the Mamiellales. NRT1 was found in all but CCMP1545 and showed highest similarity to Mamiellales and proteobacterial NRTs. NRT2 genes formed a bootstrap-supported clade basal to other green lineage organisms. Several nitrogen-related genes were colocated, forming a nitrogen gene cluster. Overall, RCC299 showed the most divergent suite of nitrogen transporters within the various Mamiellales genomes, and we developed TaqMan quantitative polymerase chain reaction primer-probes targeting a subset of these, as well as housekeeping genes, in RCC299. All those investigated showed expression either under standard growth conditions or under nitrogen depletion. Like other recent publications, our findings show a higher degree of "mixed lineage gene affiliations" among eukaryotes than anticipated, and even the most phylogenetically anomalous versions appear to be functional. Nitrogen is often considered a regulating factor for phytoplankton populations. This study provides a springboard for exploring the use and functional diversification of inorganic nitrogen transporters and related genes in eukaryotic phytoplankton. © 2010 The Author.

Summary Viruses play an important role in the regulation of phytoplankton populations. In the Mediterranean Sea, prasinophyte green algae are abundant and wide- spread, and within this group the genera Bathycoc- cus, Micromonas and Ostreococcus (Mamiellales) are the most common. Although these organisms consti- tute a significant part of the marine ecosystem, little is known about the viruses infecting them. We showed that double-stranded DNA viruses, likely members of the Phycodnaviridae family, can infect and grow in different host laboratory prasinophyte strains. Differ- ent pairs of degenerate primers were designed to PCR amplify a region of the conserved viral polymerase gene in order to characterize these viral strains. Twenty-seven new viral strains from five different host strains were thus analysed. We established phy- logenetic trees for the hosts (18S) and their associ- ated viruses (partial polymerase gene) and discuss the taxonomic significance of Phycodnaviridae. Within eukaryotic double-stranded DNA viruses, we showed that viruses from Bathycoccus, Micromonas and Ostreococcus form a monophyletic group that we refer to as 'Prasinovirus'.

Because they represent the earliest divergences of the Chlorophyta and include the smallest known eukaryotes (e.g., the coccoid Ostreococcus), the morphologically diverse unicellular green algae making up the Prasinophyceae are central to our understanding of the evolutionary patterns that accompanied the radiation of chlorophytes and the reduction of cell size in some lineages. Seven prasinophyte lineages, four of which exhibit a coccoid cell organization (no flagella nor scales), were uncovered from analysis of nuclear-encoded 18S rDNA data; however, their order of divergence remains unknown. In this study, the chloroplast genome sequences of the scaly quadriflagellate Pyramimonas parkeae (clade I), the coccoid Pycnococcus provasolii (clade V), and the scaly uniflagellate Monomastix (unknown affiliation) were determined, annotated, and compared with those previously reported for green algae/land plants, including two prasinophytes (Nephroselmis olivacea, clade III and Ostreococcus tauri, clade II). The chlorarachniophyte Bigelowiella natans and the euglenid Euglena gracilis, whose chloroplasts originate presumably from distinct green algal endosymbionts, were also included in our comparisons. The three newly sequenced prasinophyte genomes differ considerably from one another and from their homologs in overall structure, gene content, and gene order, with the 80,211-bp Pycnococcus and 114,528-bp Monomastix genomes (98 and 94 conserved genes, respectively) resembling the 71,666-bp Ostreococcus genome (88 genes) in featuring a significantly reduced gene content. The 101,605-bp Pyramimonas genome (110 genes) features two conserved genes (rpl22 and ycf65) and ancestral gene linkages previously unrecognized in chlorophytes as well as a DNA primase gene putatively acquired from a virus. The Pyramimonas and Euglena cpDNAs revealed uniquely shared derived gene clusters. Besides providing unequivocal evidence that the green algal ancestor of the euglenid chloroplasts belonged to the Pyramimonadales, phylogenetic analyses of concatenated chloroplast genes and proteins elucidated the position of Monomastix and showed that the Mamiellales, a clade comprising Ostreococcus and Monomastix, are sister to the Pyramimonadales + Euglena clade. Our results also revealed that major reduction in gene content and restructuring of the chloroplast genome occurred in conjunction with important changes in cell organization in at least two independent prasinophyte lineages, the Mamiellales and the Pycnococcaceae.

Summary The genetic diversity of picoplanktonic (i.e. cells that can pass through a 3 mm pore-size filter) green algae was investigated in the Mediterranean Sea in late summer by a culture-independent approach. Genetic libraries of the 18S rRNA gene were constructed using two different primer sets. The first set is com- monly used to amplify the majority of eukaryotic lin- eages, while the second was composed of a general eukaryotic forward primer and a reverse primer biased towards the phylum Chloroplastida. A total of 3980 partial environmental sequences were obtained: 1668 using the general eukaryotic primer set and 2312 using the Chloroplastida-biased primer set. Of these sequences, 65 (4%) and 594 (26%) belonged to the Chloroplastida respectively. A 99.5% sequence simi- larity cut-off value allowed classification of these 659 Chloroplastida sequences into 74 different opera- tional taxonomic units. A majority of the Chloroplas- tida sequences (99%) belonged to the prasinophytes. In addition to the seven independent prasinophyte lineages previously described, we discovered two new clades (clades VIII and IX), as well as a significant genetic diversity at the species and subspecies levels, notably among the genera Crustomastix, Doli- chomastix and Mamiella (Mamiellales), but also within Pyramimonas and Halosphaera (Pyramimonadales). Such diversity within prasinophytes has not previ- ously been observed by cloning approaches, illustrat- ing the power of using targeted primers for clone library construction. Prasinophyte assemblages dif- fered especially in relation to nutrient levels. Micro- monas and Ostreococcus were mainly recovered from mesotrophic areas, whereas Mamiella, Crusto- mastix and Dolichomastix were mostly detected in oligotrophic surface waters. Within genera such as Ostreococcus or Crustomastix for which several clades were observed, depth seemed to be the main factor controlling differential distribution of genotypes.

Eukaryotic ultraphytoplankton (

The extraordinary molecular phylogenetic diversity of picoeukaryotes and their contributions to marine processes have been highlighted recently. Herein, picoeukaryotic diversity in the coastal Pacific Ocean is characterized for the first time. Close to full length small subunit ribosomal RNA (ssu rRNA) gene amplicons were cloned and sequenced from size fractionated samples (< 2 μm) taken between September 2000 and October 2001. Sequences belonging to the order Mamiellales were abundant, with Micromonas pusilla-like sequences in all 5 libraries (4 operational taxonomic units, OTUs, at ≥99 sequence similarity), Ostreococcus in 4 (1 OTU) and Bathycoccus in 2 (1 OTU). Phylogenetic reconstruction showed distinct Micromonas clades at this site (although not for other Mamiellales), supporting the idea that the designation M. pusilla harbors cryptic species. In combination with picophytoplankton dynamics previously reported for the same period, the data indicates that picoplanktonic primary production at this site is likely to be dominated by prasinophytes. Furthermore, a Micromonas-clade with no cultured representatives was identified bearing highest identity to Sargasso Sea shotgun clone sequences. Comparison of the Pacific sequences with the shotgun clones showed Ostreococcus and Micromonas at 1 Sargasso site with elevated chlorophyll (chl) levels, but not at other Sargasso sites. Other Pacific clones were primarily Novel Alveolate Group II, which were highly diverse based on OTU analyses. Novel Alveolate Group I, Ciliophora, Cercozoa-like and stramenopile sequences were also retrieved. Although picoeukaryotic diversity has been characterized in only 1 other Pacific Ocean sample (equatorial Pacific), most stramenopile and alveolate sequences corresponded to previously identified phylogenetic clades from studies conducted in other oceans and for which no cultured representatives exist. © Inter-Research 2006.

The basal position of the Mamiellales (Prasinophyceae) within the green lineage makes these unicellular organisms key to elucidating early stages in the evolution of chlorophyll a/b-binding light-harvesting complexes (LHCs). Here, we unveil the complete and unexpected diversity of Lhc proteins in Ostreococcus tauri, a member of the Mamiellales order, based on results from complete genome sequencing. Like Mantoniella squamata, O. tauri possesses a number of genes encoding an unusual prasinophyte-specific Lhc protein type herein designated "Lhcp". Biochemical characterization of the complexes revealed that these polypeptides, which bind chlorophylls a, b, and a chlorophyll c-like pigment (Mg-2,4-divinyl-phaeoporphyrin a 5 monomethyl ester) as well as a number of unusual carotenoids, are likely predominant. They are retrieved to some extent in both reaction center I (RCI)- and RCII-enriched fractions, suggesting a possible association to both photosystems. However, in sharp contrast to previous reports on LHCs of M. squamata, O. tauri also possesses other LHC subpopulations, including LHCI proteins (encoded by five distinct Lhca genes) and the minor LHCII polypeptides, CP26 and CP29. Using an antibody against plant Lhca2, we unambiguously show that LHCI proteins are present not only in O. tauri, in which they are likely associated to RCI, but also in other Mamiellales, including M. squamata. With the exception of Lhcp genes, all the identified Lhc genes are present in single copy only. Overall, the discovery of LHCI proteins in these prasinophytes, combined with the lack of the major LHCII polypeptides found in higher plants or other green algae, supports the hypothesis that the latter proteins appeared subsequent to LHCI proteins. The major LHC of prasinophytes might have arisen prior to the LHCII of other chlorophyll a/b-containing organisms, possibly by divergence of a LHCI gene precursor. However, the discovery in O. tauri of CP26-like proteins, phylogenetically placed at the base of the major LHCII protein clades, yields new insight to the origin of these antenna proteins, which have evolved separately in higher plants and green algae. Its diverse but numerically limited suite of Lhc genes renders O. tauri an exceptional model system for future research on the evolution and function of LHC components. © The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

7 pages, 3 tables, Pigment analysis performed on 30 Prasinophyceae strains revealed two main groups: the prasinoxanthin-containing and prasinoxanthin-less Prasinophyceae. Prasinoxanthin-containing Prasinophyceae comprised the orders Mamiellales, Pseudoscourfieldiales (Pycnococcaceae), and Prasinococcales. For this group, classification with pigment composition showed a good agreement with molecular phylogeny. Mamiellales, except Crustomastix stigmatica, accumulated uriolide, micromonal, dihydrolutein, and the pigment Unidentified M1 as characteristic pigments. Prasinococcales and Pseudoscourfieldiales (Pycnococcaceae) lacked micromonal and Unidentified M1. In addition, Pseudoscourfieldiales (Pycnococcaceae) lacked uriolide. A chl c3-like pigment was present in prasinoxanthin-containing strains isolated from the deep sea. Common green algae pigments, a loroxanthin derivative, and siphonaxanthin plus derivatives were found in the prasinoxanthin-less Prasinophyceae, which included strains from Pyramimonadales, Pseudoscourfieldiales (Nephroselmidiaceae), Chlorodendrales, and a new order. Although some associations could be observed, the correspondence between pigments and molecular taxonomy was less clear for this group, This work has been carried out in the framework of the EU project PICODIV (EVK3-CT-1999-00021) with additional support from the Spanish Ministry of Science and Technology (REN2000-2913-E). L. G. was supported by the EU Marie Curie Grant (EVK3-CT-1999-50004)

9 pages, 4 figures, 4 tables, 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, This work was funded by the following programs: PICODIV, supported by the European Union (EVK3-CT-1999-00021); PICMANCH, supported by the Région Bretagne; and BIOSOPE, supported by PROOF (CNRS). F.N. was supported by a doctoral fellowship from the French Research Ministry

In surveys of marine phytoplankton diversity, prasinophyte algae are commonly found and are often a major component, especially in coastal areas. They form a paraphyletic assemblage of at least nine clades, with the Mamiellophyceae (prasinophyte clade II) being a major group. In the order Mamiellales, the three genera Bathycoccus, Micromonas and Ostreococcus are the dominant picoeukaryotic organisms in many different oceanic areas, as witnessed by their abundance in analyses of environmental DNA samples. Each of the six complete genomes analyzed from cultured representatives of this group has revealed densely packed coding sequences, with strong evolutionary divergence from its nearest phylogenetically defined neighbors. These species lie at the base of the green lineage, but various metabolic processes reflect their marine lifestyles and distinguish them from land plants, including a high proportion of selenoprotein enzymes and C4 photosynthesis. They all possess two unusual chromosomes with lower GC content and atypical gene content, whose function so far remains enigmatic.

A new marine microalga from the Mediterranean Sea, Crustomastix stigmatica Zingone, is investigated by means of LM, SEM, TEM, and pigment and molecular analyses (nuclear-encoded small subunit [SSU] rDNA and plastid-encoded rbcL). Pigment and molecular information is also provided for the related species Dolichomastix tenuilepis Throndsen et Zin-gone. Crustomastix stigmatica has a bean-shaped cell body 3-5 μm long and 1.5-2.8 μm wide, with two flagella four to five times the body length. The single chloroplast is pale yellow-green, cup-shaped, and lacks a pyrenoid. A small bright yellow stigma is located in the mid-dorsal part of the cell under the chloroplast membrane. An additional accumulation of osmiophilic globules is at times seen in a chloroplast lobe. Cells lack flat scales, whereas three different types of hair-like scales are present on the flagella. The main pigments of C. stigmatica are those typical of Mamiellales, though siphonein/siphonax-anthin replaces prasinoxanthin and uriolide is absent. The pigment pool of D. tenuilepis is more similar to that of Micromonas pusilla (Butcher) Manton et Parke and of other Mamiellales. The nuclear SSU rDNA phylogeny shows that the inclusion of C. stigmatica and D. tenuilepis in the Mamiellales retains monophyly for the order. The two species form a distinct clade, which is sister to a clade including all the other Mamiellales. Results of rbcL analyses failed to provide phylogenetic information at both the order and species level. No unique morphological or pigment characteristics circumscribe the mamiellalean clade as a whole nor its two daughter clades.

Phylogenetic analyses of 18S rDNA sequences from 25 prasinophytes, including 10 coccoid isolates, reveals that coccoid organisms are found in at least three prasinophyte lineages. The coccoid Ostreococcus tauri is included in the Mamiellales lineage and P ycnococcus provasolii is allied with the flagellate P seudoscourfieldia marina. A previously undescribed prasinophyte lineage is comprised of the coccoid Prasinococcus cf. capsulatus (CCMP 1407) and other isolates tentatively identified as Prasinococcus sp. (CCMP 1202, CCMP 1614, and CCMP 1194), as well as three unnamed coccoids (CCMP 1193, CCMP 1413, and CCMP 1220). No flagellate organisms are known from this lineage. Organisms of this new lineage share some characteristics of both the Pycnococcaceae and the Mamiellales, although relationships among these separate lineages were not supported by bootstrap analyses. An additional unnamed coccoid isolate (CCMP 1205) is separate from all major prasinophyte lineages. The analyses did not resolve the relationships among the major prasinophyte lineages, although they support previous conclusions that the Prasinophyceae are not monophyletic.

A new prasinophycean flagellate, Crustomastix didyma Nakayama, Kawachi & Inouye gen. & sp. nov., is described from the northwestern Pacific. This alga is elongate and bean-shaped and has two ventrally inserted, subequal flagella, with three types of hair scales. The entire cell and flagella are covered by a thin coat, which is probably derived from the Golgi apparatus and released through the duct system. The yellowish chloroplast is divided into two lobes connected by a narrow bridge. There is no pyrenoid or eyespot. The flagellar apparatus comprises the distal and proximal fibres, the Id and Is microtubular roots, and a duct fibre. A multilayered structure (MLS) is associated with the Id root. Some features, such as the swimming behaviour, the transitional region, and the absence of microtubular roots associated with the number 2 basal body, suggest a close relationship to the Mamiellales, but this alga differs from other members of the Mamiellales in having a MLS and a duct system, which are f...

18S rDNA sequences were obtained for the prasinophytes Pseudoscourfieldia marina (Throndsen) Manton and Pycnococcus provasolii Guillard. These sequences, along with those of additional prasinophytes and other green algae and land plants, were used for phylogenetic analyses by the neighbor-joining, maximum parsimony, and quartet puzzling methods. Results indicate that Ps. marina and Py. provasolii are closely allied and that both should be included in the Pycnococcaceae. In addition, results of these sequence analyses and additional pigment analysis indicate that the organism previously identified as Ps. marina, isolate CCMP 717, is, in fact, a Nephroselmis sp. or is closely related to that genus.

A new species of the genus Dolichomastix Manton has been brought into culture and described from the oligotrophic waters off the Island of Capri (Naples, Mediterranean Sea). This is the first speci...

Coastal marine waters in many regions worldwide support abundant populations of extremely small (1-3 mu m diameter) unicellular eukaryotic green algae, dominant taxa including several species in the class Mamiellophyceae. Their diminutive size conceals surprising levels of genetic diversity and defies classical species' descriptions. We present a detailed analysis within the genus Ostreococcus and show that morphological characteristics cannot be used to describe diversity within this group. Karyotypic analyses of the best-characterized species O. tauri show it to carry two chromosomes that vary in size between individual clonal lines, probably an evolutionarily ancient feature that emerged before species' divergences within the Mamiellales. By using a culturing technique specifically adapted to members of the genus Ostreococcus, we purified >30 clonal lines of a new species, Ostreococcus mediterraneus sp. nov., previously known as Ostreococcus clade D, that has been overlooked in several studies based on PCR-amplification of genetic markers from environment-extracted DNA. Phylogenetic analyses of the S-adenosylmethionine synthetase gene, and of the complete small subunit ribosomal RNA gene, including detailed comparisons of predicted ITS2 (internal transcribed spacer 2) secondary structures, clearly support that this is a separate species. In addition, karyotypic analyses reveal that the chromosomal location of its ribosomal RNA gene cluster differs from other Ostreococcus clades. (c) 2013 Elsevier GmbH. All rights reserved.

A light-harvesting complex was isolated from thylakoid membranes of the marine green flagellate Pseudoscourfieldia marina by sucrose density gradient centrifugation in the presence of the detergent n-dodecyl β- d -maltoside, followed by DEAE-cellulose ion-exchange chromatography. Absorption and fluorescence spectroscopy indicated that the complex was intact and capable of transferring energy from accessory pigments to chlorophyll a. The light-harvesting complex contained one major and two minor polypeptides with apparent molecular masses of 24 kDa, 26 kDa and 23 kDa, and the pigments chlorophyll a, chlorophyll b, prasinoxanthin, neoxanthin, magnesium 2,4-divinyl phaeoporphyrin a5 monomethyl ester and one unknown carotenoid. Protein and pigment quantification indicated that one polypeptide is complexed with about 2.7 chlorophyll b, 1.8 chlorophyll a, 1.6 prasinoxanthin, 0.7 neoxanthin, 0.5 magnesium 2,4-divinyl phaeoporphyrin a5 monomethyl ester and 0.2 unknown carotenoid. Immunoblot analysis demonstrated that the polypeptides of the light-harvesting complex of P. marina are structurally similar to those of Mantoniella gilva and other members of the Mamiellales, all of which possess the pigments prasinoxanthin and magnesium 2,4-divinyl phaeoporphyrin a5 monomethyl ester.

A new genus, Prasinococcus, and a new species, P. capsulatus, are described on the basis of specimen that appeared in enriched cultures inoculated from water samples in the western Pacific Ocean. Cells of this unicellular alga are spherical and surrounded with a large amount of gelatinous matrix. The pigment composition, which includes chlorophylls a and b, prasinoxanthin, Mg 2, 4-diviriylphaeoporphyrin a5 monomethyl ester (Mg 2, 4-D) and 5, 6-epoxy-3, 3′-dihydroxy-5, 6, 7′, 8′-tetrahydro-β-e-caroten-11′, 19-olide (uriolide), is close to that observed in the Mamiellales (Prasinophyceae). The cell is non-flagellate and has a firm cell wall, but no scales on the cell body. The pyrenoid has a characteristic structure in which a mitochondrial lobe and chloroplast outer membranes protrude into the pyrenoid matrix. The cell wall has a projecting appendage like a circle collar surrounded by distinctive holes penetrating the cell wall. Since the cell contains no 3-deoxy-manno-octulosonic acid (KDO), the wall is considered to have a different origin from the cell coverings which consist of fused scales in Tetraselmis species. It has a characteristic way of asexual reproduction in which, after cell division, one cell remains within the parent cell wall while the other is extruded. This suggests that this alga be placed tentatively in the Pycnococcaceae (Order Mamiellales) on the basis of the pigment composition and morphological characteristics, but the taxonomic position of coccoid prasinophytes may need to be reconsidered.

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.

International audience; Background: Photosynthetic picoeukaryotes (PPE) with a cell size less than 3 mm 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 Findings: In 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/Significance: Our 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.

The new genus Pycnococcus Guillard is based on several clones from the western North Atlantic and Gulf of Mexico. The type and only described species, Pycnococcus provasolii Guillard, sp. nov., is typified by clone Ω48-23 from the North Atlantic. Cells of Pycnococcus provasolii are solitary, spherical, 1.5–4.0 μm in diameter, have a resistant cell wall lacking sporopollenin, and have the ultrastructural characteristics of green algae. With the light microscope they are scarcely distinguishable from cells of other coccoid planktonic organisms. In pigmentation P. provasolii resembles Micromonas pusilla, Mantoniella squamata, and Mamiella gilva in having chl a, much chl b, Mg 2,4-divinylphaeoporphyrin a5 monomethyl ester (presumably), and prasinoxanthin as a major xanthophyll. The pyrenoid of P. provasolii has a cytoplasmic channel, which is unique among species closely related to it. Flagellates, occurring rarely in culture, are similar to but distinguishable from known Pedinomonas species by size and shape. Pycnococcus provasolii is referred to the new family Pycnococcaceae Guillard, in the order Mamiellales of the class Micromonadophyceae (Chlorophyta). Clones of Pycnococcus provasolii are oceanic in nutritional characteristics, require only vitamin B12 in culture, and are well adapted to growth under blue or blue-violet light of low intensity.

The major light-harvesting pigment-protein complex of Mamiella gilva (Parke et Rayns) Moestrup (Micromonadophyceae, Chlorophyta) was isolated by lithium dodecyl sulphate polyacrylamide gel electrophoresis at 4°C. This complex is similar to that of Mantoniella squamata (Manton et Parke) Desikachary and Micromonas pusilla (Butcher) Manton et Parke with regard to molecular weight, absorption spectrum, and immunochemical cross-reactivity. On the other hand, the light-harvesting complex of Chlamydomonas reinhardtii P. Dangeard is quite different. The distribution of this unusual light-harvesting complex, which has to date been reported only in Mamiella, Mantoniella, and Micromonas, suggests a line of evolution in the Micromonadophyceae separate from all other green algae. In addition, the inclusion of the naked Micromonas in this group suggests that Micromonas should be included with Mamiella and Mantoniella in the Mamiellales, an order which is presently defined by scale type.

Mantoniella squamata (Prasinophyceae, Chlorophyta) is presently considered to be one of the most primitive green plants. The structure of the spider-web-like scales that cover the flagellar and body surfaces of this organism is described. By contrast to current theory, the scales may be divided into several different morphological types, one form being resricted to the flagella and two to the cell body. The cells contain trichocyst-like organelles similar to those found in the related species Mamiella gilva. In the order Mamiellales, which contains four genera and seven described species, the simplest and thus perhaps the most primitive species is the recently described Bathycoccus prasinos Eikrem et Throndsen, the cells of which lack flagella and are covered with only one type of plate scale.

A non-motile, picoplanktonic species, Bathycoccus prasinos gen. et sp. nov. (Mamiellaceae, Mamiellales, Prasinophyceae) is described from the Gulf of Naples, Italy. Resembling a comma, the cells ar...

11 pages, 5 figures, 1 table, The aim of our study was to describe the diversity and dynamics of size-fractionated phytoplankton at a fixed station (P2, Ría de Pontevedra, NW Spain) from late spring to early winter. Phytoplankton successions were assessed weekly from May to November 2004 using pigment analyses, light microscopy and fluorescence in situ hybridization coupled with tyramide signal amplification (FISH-TSA). Periodic upwelling events triggered the development of diatom populations followed by periods of relaxation/downwelling conditions where remineralization processes (maxima of NH4 +), raised temperatures and/or low salinity waters from rainfall favored the increase of picophytoplankton. Marked differences were observed between diversity and dynamics of the micronano (>3 μm) and pico (, F.R. was supported by a postdoctoral fellowship from Caixanova Foundation

15 páginas, 6 figuras, 3 tablas, Size-fractionated phytoplankton was studied using pigments and carbon (C) biomass derived from light microscopy at a fixed location on the NW Iberian shelf between May 2001 and June 2002. The annual contributions of pico-, nano- and microphytoplankton to the total autotrophic C biomass were 9.7, 63 and 27.3%, respectively, with unidentified nanoflagellates (63%) and diatoms (22%) being the most important components. The CHEMTAX program was used to determine the chlorophyll a (chl a) contribution of 9 pigment groups in nano-microplankton and 8 in picoplankton. ‘Diatoms II’ (species containing chl c3), ‘haptophytes II’ (species of the genus Chrysochromulina containing chl c2-MGDG [monogalactosyl diacylglyceride] [14:0/14:0] ) and ‘chlorophytes’, represented 34, 17 and 16%, respectively, of the total chl a in the nano-microplankton fraction. The major pigment groups in the picoplankton were ‘chlorophytes’, ‘Synechococcus’ and ‘prasinophytes II’ (Order Mamiellales), which accounted for 34, 22 and 10% of the total chl a in this size class. ‘Prochlorococcus’ was traced using divinyl (DV) chl a and was present seasonally, especially during downwelling and stratified poleward conditions (October to November). The lowest chl a and autotrophic C values throughout the study (152 ± 45 ng chl a l–1 and 31 ± 13 μg C l–1) corresponded to the homogenous poleward flow or Iberian Poleward Current (IPC) from December to February. During this period the phytoplankton composition changed abruptly to a diverse pico-nanoplanktonic community that represented ~95% of the total autotrophic C biomass. ‘Chlorophytes’, ‘cryptophytes’ and ‘haptophytes II’ in the nanoplankton, and ‘chlorophytes’ and ‘Synechococcus’ in the picoplankton were the main contributers to chl a in IPC waters, as calculated by CHEMTAX., Financial support for this work came from the Spanish DYBAGA project (MAR99-1039-C02- 01) and from the EU HABILE project (EVK3-CT-2001-00063). B.G.C. was funded by a pre-doctoral I3P fellowship of CSICESF.

14 pages, 3 figures, 5 tables, A quantitative PCR (QPCR) assay based on the use of SYBR Green I was developed to assess the abundance of specific groups of picoeukaryotes in marine waters. Six primer sets were designed targeting four different taxonomic levels: domain (Eukaryota), division (Chlorophyta), order (Mamiellales) and genus (Bathycoccus, Micromonas, and Ostreococcus). Reaction conditions were optimized for each primer set which was validated in silico, on agarose gels, and by QPCR against a variety of target and non-target cultures. The approach was tested by estimating gene copy numbers for Micromonas, Bathycoccus, and Ostreococcus in seawater samples to which cultured cells were added in various concentrations. QPCR was then used to determine that rRNA gene (rDNA) copy number varied from one to more than 12,000 in 18 strains of phytoplankton. Finally, QPCR was applied to environmental samples from a Mediterranean Sea coastal site and the results were compared to those obtained by Fluorescent in situ hybridization (FISH). The data obtained demonstrate that Chlorophyta and more specifically Mamiellales were important in these waters, especially during the winter picoplankton bloom. The timing of major abundance peaks of the targeted species was similar by QPCR and FISH. When used in conjunction with other techniques such as FISH or gene clone libraries, QPCR appears as very promising to quickly obtain data on the ecological distribution of important phytoplankton groups. Data interpretation must take into account primer specificity and the varying rRNA gene copy number among eukaryotes. © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved., The work was supported by the following programs: PICODIV (EVK3-CT-1999-00021), BIOSOPE (CNRS INSUE), PICMANCH (Région Bretagne), GenoMer (Région Bretagne et Departement du Finistère). FZ and FN benefited from fellowships from the French Ministry of Education and Research

22 page, 6 figures, 3 tables, Picoplanktonic prasinophytes are well represented in culture collections and marine samples. In order to better characterize this ecologically important group, we compared the phylogenetic diversity of picoplanktonic prasinophyte strains available at the Roscoff Culture Collection (RCC) and that of nuclear SSU rDNA sequences from environmental clone libraries obtained from oceanic and coastal ecosystems. Among the 570 strains avalaible, 91 belonged to prasinophytes, 65 were partially sequenced, and we obtained the entire SSU rDNA sequence for a selection of 14 strains. Within the 18 available environmental clone libraries, the prasinophytes accounted for 12% of the total number of clones retrieved (142 partial sequences in total), and we selected 9 clones to obtain entire SSU rDNA sequence. Using this approach, we obtained a subsequent genetic database that revealed the presence of seven independent lineages among prasinophytes, including a novel clade (clade VII). This new clade groups the genus Picocystis, two unidentified coccoid strains, and 4 environmental sequences. For each of these seven lineages, at least one representative is available in culture. The three picoplanktonic genera Ostreococcus, Micromonas, and Bathycoccus (order Mamiellales), were the best represented prasinophytes both in cultures and genetic libraries. SSU rDNA phylogenetic analyses suggest that the genus Bathycoccus forms a very homogeneous group. In contrast, the genera Micromonas and Ostreococcus turned out to be quite complex, consisting of three and four independent lineages, respectively. This report of the overall diversity of picoeukaryotic prasinophytes reveals a group of ecologically important and diverse marine microorganims that are well represented by isolated cultures, This work was supported by a Marie Curie fellowship (EVKE-CT-1999-50004) to LG, the EU project PICODIV (EVK3-CT-1999-00021), and the following French programs: PicManche funded by the Région Bretagne, the CNRS Aventis initiative, the “Souchothèque de Bretagne” funded in the frame of the Plan Etat-Région by the Région Bretagne and the Département du Finistère, the Centre de Ressources Biologiques, and the CNRS PROOF programs PROSOPE and BIOSOPE

Picoeukaryotes (cells of