Stimulating and toxic effect of chromium on growth and photosynthesis of a marine chlorophyte.

Marine phytoplankton can interchange trace metals in various biochemical functions, particularly under metal-limiting conditions. Here, we investigate the stimulating and toxicity effect of chromium (Cr) on a marine Chlorophyceae Osetreococcus tauri under Fe-replete and Fe-deficient conditions. We determined the growth, photosynthesis, and proteome expressions of Osetreococcus tauri cultured under different Cr and Fe concentrations. In Fe-replete conditions, the presence of Cr(VI) stimulated significantly the growth rate and the maximum yield of photochemistry of photosystem II (Fv /Fm ) of the phytoplankton, while the functional absorption cross-section of photosystem II (σPSII ) did not change. Minor additions of Cr(VI) partially rescued phytoplankton growth under Fe-limited conditions. Proteomic analysis of this alga grown in Fe-replete normal and Fe-replete with Cr addition media (10 µM Cr) showed that the presence of Cr significantly decreased the expression of phosphate-transporting proteins and photosynthetic proteins, while increasing the expression of proteins related to carbon assimilation. Cr can stimulate the growth and photosynthesis of O. tauri, but the effects are dependent on both the Cr(VI) concentration and the availability of Fe. The proteomic results further suggest that Cr(VI) addition might significantly increase starch production and carbon fixation.

Rapid diversification underlying the global dominance of a cosmopolitan phytoplankton.

Marine phytoplankton play important roles in the global ecosystem, with a limited number of cosmopolitan keystone species driving their biomass. Recent studies have revealed that many of these phytoplankton are complexes composed of sibling species, but little is known about the evolutionary processes underlying their formation. Gephyrocapsa huxleyi, a widely distributed and abundant unicellular marine planktonic algae, produces calcified scales (coccoliths), thereby significantly affects global biogeochemical cycles via sequestration of inorganic carbon. This species is composed of morphotypes defined by differing degrees of coccolith calcification, the evolutionary ecology of which remains unclear. Here, we report an integrated morphological, ecological and genomic survey across globally distributed G. huxleyi strains to reconstruct evolutionary relationships between morphotypes in relation to their habitats. While G. huxleyi has been considered a single cosmopolitan species, our analyses demonstrate that it has evolved to comprise at least three distinct species, which led us to formally revise the taxonomy of the G. huxleyi complex. Moreover, the first speciation event occurred before the onset of the last interglacial period (~140 ka), while the second followed during this interglacial. Then, further rapid diversifications occurred during the most recent ice-sheet expansion of the last glacial period and established morphotypes as dominant populations across environmental clines. These results suggest that glacial-cycle dynamics contributed to the isolation of ocean basins and the segregations of oceans fronts as extrinsic drivers of micro-evolutionary radiations in extant marine phytoplankton.

The mode of speciation during a recent radiation in open-ocean phytoplankton.

Despite the enormous ecological importance of marine phytoplankton, surprisingly little is known about how new phytoplankton species originate and evolve in the open ocean, in the absence of apparent geographic barriers that typically act as isolation mechanisms in speciation. To investigate the mechanism of open-ocean speciation, we combined fossil and climatic records from the late Quaternary with genome-wide evolutionary genetic analyses of speciation in the ubiquitous and abundant pelagic coccolithophore genus Gephyrocapsa (including G. huxleyi, formerly known as Emiliania huxleyi). Based on the analysis of 43 sequenced genomes, we report that the best-fitting scenario for all speciation events analyzed included an extended period of complete isolation followed by recent (Holocene) secondary contact, supporting the role of geographic or oceanographic barriers in population divergence and speciation. Consistent with this, fossil data reveal considerable diachroneity of species first occurrence. The timing of all speciation events coincided with glacial phases of glacial-interglacial cycles, suggesting that stronger isolation between the ocean basins and increased segregation of ecological niches during glaciations are important drivers of speciation in marine phytoplankton. The similarity across multiple speciation events implies the generality of this inferred speciation scenario for marine phytoplankton.

Repeated species radiations in the recent evolution of the key marine phytoplankton lineage Gephyrocapsa.

Phytoplankton account for nearly half of global primary productivity and strongly affect the global carbon cycle, yet little is known about the forces that drive the evolution of these keystone microscopic organisms. Here we combine morphometric data from the fossil record of the ubiquitous coccolithophore genus Gephyrocapsa with genomic analyses of extant species to assess the genetic processes underlying Pleistocene palaeontological patterns. We demonstrate that all modern diversity in Gephyrocapsa (including Emiliania huxleyi) originated in a rapid species radiation during the last 0.6 Ma, coincident with the latest of the three pulses of Gephyrocapsa diversification and extinction documented in the fossil record. Our evolutionary genetic analyses indicate that new species in this genus have formed in sympatry or parapatry, with occasional hybridisation between species. This sheds light on the mode of speciation during evolutionary radiation of marine phytoplankton and provides a model of how new plankton species form.

Recent Reticulate Evolution in the Ecologically Dominant Lineage of Coccolithophores.

The coccolithophore family Noëlaerhabdaceae contains a number of taxa that are very abundant in modern oceans, including the cosmopolitan bloom-forming Emiliania huxleyi. Introgressive hybridization has been suggested to account for incongruences between nuclear, mitochondrial and plastidial phylogenies of morphospecies within this lineage, but the number of species cultured to date remains rather limited. Here, we present the characterization of 5 new Noëlaerhabdaceae culture strains isolated from samples collected in the south-east Pacific Ocean. These were analyzed morphologically using scanning electron microscopy and phylogenetically by sequencing 5 marker genes (nuclear 18S and 28S rDNA, plastidial tufA, and mitochondrial cox1 and cox3 genes). Morphologically, one of these strains corresponded to Gephyrocapsa ericsonii and the four others to Reticulofenestra parvula. Ribosomal gene sequences were near identical between these new strains, but divergent from G. oceanica, G. muellerae, and E. huxleyi. In contrast to the clear distinction in ribosomal phylogenies, sequences from other genomic compartments clustered with those of E. huxleyi strains with which they share an ecological range (i.e., warm temperate to tropical waters). These data provide strong support for the hypothesis of past (and potentially ongoing) introgressive hybridization within this ecologically important lineage and for the transfer of R. parvula to Gephyrocapsa. These results have important implications for understanding the role of hybridization in speciation in vast ocean meta-populations of phytoplankton.

Morphological and Phylogenetic Characterization of New Gephyrocapsa Isolates Suggests Introgressive Hybridization in the Emiliania/Gephyrocapsa Complex (Haptophyta).

The coccolithophore genus Gephyrocapsa contains a cosmopolitan assemblage of pelagic species, including the bloom-forming Gephyrocapsa oceanica, and is closely related to the emblematic coccolithophore Emiliania huxleyi within the Noëlaerhabdaceae. These two species have been extensively studied and are well represented in culture collections, whereas cultures of other species of this family are lacking. We report on three new strains of Gephyrocapsa isolated into culture from samples from the Chilean coastal upwelling zone using a novel flow cytometric single-cell sorting technique. The strains were characterized by morphological analysis using scanning electron microscopy and phylogenetic analysis of 6 genes (nuclear 18S and 28S rDNA, plastidial 16S and tufA, and mitochondrial cox1 and cox3 genes). Morphometric features of the coccoliths indicate that these isolates are distinct from G. oceanica and best correspond to G. muellerae. Surprisingly, both plastidial and mitochondrial gene phylogenies placed these strains within the E. huxleyi clade and well separated from G. oceanica isolates, making Emiliania appear polyphyletic. The only nuclear sequence difference, 1bp in the 28S rDNA region, also grouped E. huxleyi with the new Gephyrocapsa isolates and apart from G. oceanica. Specifically, the G. muellerae morphotype strains clustered with the mitochondrial ß clade of E. huxleyi, which, like G. muellerae, has been associated with cold (temperate and sub-polar) waters. Among putative evolutionary scenarios that could explain these results we discuss the possibility that E. huxleyi is not a valid taxonomic unit, or, alternatively the possibility of past hybridization and introgression between each E. huxleyi clade and older Gephyrocapsa clades. In either case, the results support the transfer of Emiliania to Gephyrocapsa. These results have important implications for relating morphological species concepts to ecological and evolutionary units of diversity.

PhytoREF: a reference database of the plastidial 16S rRNA gene of photosynthetic eukaryotes with curated taxonomy.

Photosynthetic eukaryotes have a critical role as the main producers in most ecosystems of the biosphere. The ongoing environmental metabarcoding revolution opens the perspective for holistic ecosystems biological studies of these organisms, in particular the unicellular microalgae that often lack distinctive morphological characters and have complex life cycles. To interpret environmental sequences, metabarcoding necessarily relies on taxonomically curated databases containing reference sequences of the targeted gene (or barcode) from identified organisms. To date, no such reference framework exists for photosynthetic eukaryotes. In this study, we built the PhytoREF database that contains 6490 plastidial 16S rDNA reference sequences that originate from a large diversity of eukaryotes representing all known major photosynthetic lineages. We compiled 3333 amplicon sequences available from public databases and 879 sequences extracted from plastidial genomes, and generated 411 novel sequences from cultured marine microalgal strains belonging to different eukaryotic lineages. A total of 1867 environmental Sanger 16S rDNA sequences were also included in the database. Stringent quality filtering and a phylogeny-based taxonomic classification were applied for each 16S rDNA sequence. The database mainly focuses on marine microalgae, but sequences from land plants (representing half of the PhytoREF sequences) and freshwater taxa were also included to broaden the applicability of PhytoREF to different aquatic and terrestrial habitats. PhytoREF, accessible via a web interface (http://phytoref.fr), is a new resource in molecular ecology to foster the discovery, assessment and monitoring of the diversity of photosynthetic eukaryotes using high-throughput sequencing.

Life-cycle modification in open oceans accounts for genome variability in a cosmopolitan phytoplankton.

Emiliania huxleyi is the most abundant calcifying plankton in modern oceans with substantial intraspecific genome variability and a biphasic life cycle involving sexual alternation between calcified 2N and flagellated 1N cells. We show that high genome content variability in Emiliania relates to erosion of 1N-specific genes and loss of the ability to form flagellated cells. Analysis of 185 E. huxleyi strains isolated from world oceans suggests that loss of flagella occurred independently in lineages inhabiting oligotrophic open oceans over short evolutionary timescales. This environmentally linked physiogenomic change suggests life cycling is not advantageous in very large/diluted populations experiencing low biotic pressure and low ecological variability. Gene loss did not appear to reflect pressure for genome streamlining in oligotrophic oceans as previously observed in picoplankton. Life-cycle modifications might be common in plankton and cause major functional variability to be hidden from traditional taxonomic or molecular markers.

Genetic delineation between and within the widespread coccolithophore morpho-species Emiliania huxleyi and Gephyrocapsa oceanica (Haptophyta).

Emiliania huxleyi and Gephyrocapsa oceanica are abundant coccolithophore morpho-species that play key roles in ocean carbon cycling due to their importance as both primary producers and cal-cifiers. Global change processes such as ocean acidification impact these key calcifying species. The physiology of E. huxleyi, a developing model species, has been widely studied, but its genetic delineation from G. oceanica remains unclear due to a lack of resolution in classical genetic markers. Using nuclear (18S rDNA and 28S rDNA), mitochondrial (cox1, cox2, cox3, rpl16, and dam), and plastidial (16S rDNA, rbcL, tufA, and petA) DNA markers from 99 E. huxleyi and 44 G. oceanica strains, we conducted a multigene/multistrain survey to compare the suitability of different markers for resolving phylogenetic patterns within and between these two morpho-species. The nuclear genes tested did not provide sufficient resolution to discriminate between the two morpho-species that diverged only 291Kya. Typical patterns of incomplete lineage sorting were generated in phylogenetic analyses using plastidial genes. In contrast, full morpho-species delineation was achieved with mitochondrial markers and common intra-morpho-species phylogenetic patterns were observed despite differing rates of DNA substitution. Mitochondrial genes are thus promising barcodes for distinguishing these coccolithophore morpho-species, in particular in the context of environmental monitoring.

Integrative taxonomy of the Pavlovophyceae (Haptophyta): a reassessment.

The Pavlovophyceae (Haptophyta) contains four genera (Pavlova, Diacronema, Exanthemachrysis and Rebecca) and only thirteen characterised species, several of which are important in ecological and economic contexts. We have constructed molecular phylogenies inferred from sequencing of ribosomal gene markers with comprehensive coverage of the described diversity, using type strains when available, together with additional cultured strains. The morphology and ultrastructure of 12 of the described species was also re-examined and the pigment signatures of many culture strains were determined. The molecular analysis revealed that sequences of all described species differed, although those of Pavlova gyrans and P. pinguis were nearly identical, these potentially forming a single cryptic species complex. Four well-delineated genetic clades were identified, one of which included species of both Pavlova and Diacronema. Unique combinations of morphological/ultrastructural characters were identified for each of these clades. The ancestral pigment signature of the Pavlovophyceae consisted of a basic set of pigments plus MV chl cPAV, the latter being entirely absent in the Pavlova + Diacronema clade and supplemented by DV chl cPAV in part of the Exanthemachrysis clade. Based on this combination of characters, we propose a taxonomic revision of the class, with transfer of several Pavlova species to an emended Diacronema genus. The evolution of the class is discussed in the context of the phylogenetic reconstruction presented.

NEW EVIDENCE FOR MORPHOLOGICAL AND GENETIC VARIATION IN THE COSMOPOLITAN COCCOLITHOPHORE EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) FROM THE COX1b-ATP4 GENES(1).

Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler is a cosmopolitan coccolithophore occurring from tropical to subpolar waters and exhibiting variations in morphology of coccoliths possibly related to environmental conditions. We examined morphological characters of coccoliths and partial mitochondrial sequences of the cytochrome oxidase 1b (cox1b) through adenosine triphosphate synthase 4 (atp4) genes of 39 clonal E. huxleyi strains from the Atlantic and Pacific Oceans, Mediterranean Sea, and their adjacent seas. Based on the morphological study of culture strains by SEM, Type O, a new morphotype characterized by coccoliths with an open central area, was separated from existing morphotypes A, B, B/C, C, R, and var. corona, characterized by coccoliths with central area elements. Molecular phylogenetic studies revealed that E. huxleyi consists of at least two mitochondrial sequence groups with different temperature preferences/tolerances: a cool-water group occurring in subarctic North Atlantic and Pacific and a warm-water group occurring in the subtropical Atlantic and Pacific and in the Mediterranean Sea.