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Planktonic protists are an essential component of marine pelagic ecosystems where they mediate important trophic and biogeochemical functions. Although these functions are largely influenced by their taxonomic affiliation, the composition and spatial variability of planktonic protist communities remain poorly characterized in vast areas of the ocean. Here, we investigated the diversity of these communities in contrasting oceanographic conditions of the southwest Pacific (33–58 °S) using DNA metabarcoding of the 18S rRNA gene. Seawater samples collected during twelve cruises ( = 482, 0–3100 m) conducted east of New Zealand were used to characterize protist communities in Subtropical (STW) and Subantarctic (SAW) surface water masses and the Subtropical Front (STF) that separates them. Diversity decreased with increasing latitude and increasing temperature but tended to be lowest in the STF. Sample ordination resulting from the abundance of amplicon single variants (ASVs) corresponded to the different water masses. Overall, Dinoflagellata (Syndiniales, 27%; Dinophyceae, 24% of standardized number of reads) dominated the euphotic zone followed by Chlorophyta (20%), but their relative abundance and composition at class and lower taxonomic levels varied consistently between water masses. Among Chlorophyta, several picoplanktonic algae species of the Mamiellophyceae class including Ostreococcus lucimarinus dominated in STW, while the Chloropicophyceae species Chloroparvula pacifica was most abundant in SAW. Bacillariophyta (5%), Prymnesiophyceae (5%), and Pelagophyceae (2%) classes were less abundant but showed analogous water mass specificity at class and finer taxonomic levels. Protist community composition in the STF had mixed characteristics and showed regional differences with the southern STF (50 °S) having more resemblance with subantarctic communities than the STF over the Chatham Rise region (42–44 °S). Below the euphotic zone, Syndiniales sequences (40%) dominated the dataset followed by Radiolaria (31%), Dinophyceae (14%) and other heterotrophic groups like Marine Stramenopiles and ciliates (1–1.5%). Among Radiolaria, several unidentified ASVs assigned to Spumellaria were most abundant, but showed significantly different distributions between STW and SAW highlighting the need to further investigate the taxonomy and ecology of this group. The present study represents a significant step forward towards characterizing protistan communities composition in relation to major physical oceanographic features in the southwest Pacific providing new insights about the biogeography and ecological preferences of different planktonic protist taxa from class to species and genotypic level., SI

Year-round reports of phytoplankton dynamics in the West Antarctic Peninsula are rare and mainly limited to microscopy and/or pigment-based studies. We analyzed the phytoplankton community from coastal waters of Fildes Bay in the West Antarctic Peninsula between January 2014 and 2015 using metabarcoding of the nuclear and plastidial 18/16S rRNA gene from both size-fractionated and flow cytometry sorted samples. Overall 14 classes of photosynthetic eukaryotes were present in our samples with the following dominating: Bacillariophyta (diatoms), Pelagophyceae and Dictyochophyceae for division Ochrophyta, Mamiellophyceae and Pyramimonadophyceae for division Chlorophyta, Haptophyta and Cryptophyta. Each metabarcoding approach yielded a different image of the phytoplankton community with for example Prymnesiophyceae more prevalent in plastidial metabarcodes and Mamiellophyceae in nuclear ones. Diatoms were dominant in the larger size fractions and during summer, while Prymnesiophyceae and Cryptophyceae were dominant in colder seasons. Pelagophyceae were particularly abundant towards the end of autumn (May). In addition of Micromonas polaris and Micromonas sp. clade B3, both previously reported in Arctic waters, we detected a new Micromonas 18S rRNA sequence signature, close to, but clearly distinct from M. polaris, which potentially represents a new clade specific of the Antarctic. These results highlight the need for complementary strategies as well as the importance of year-round monitoring for a comprehensive description of phytoplankton communities in Antarctic coastal waters. Ministry of Education (MOE) Published version This work was funded by INACH RG_31-15, INACH RT_34-17 and FONDECYT No. 1190879 grants. Collaboration between Chile and France was funded through ECOS-CONICYT No. C16B02 and CNRS International Research Network “Diversity, Evolution and Biotechnology of Marine Algae” (GDRI No. 0803). Catherine Géri-kas Ribeiro was supported by the FONDECYT Project No. 3190827. The authors thank Dr. Ernesto Molina for sampling during autumn and spring, as well the logistic support at the scientific station Professor Julio Escudero, INACH. Adriana Lopes dos Santos was supported by the Singapore Ministry of Education, Academic Research Fund Tier 1 (RG26/19). We thank the Roscoff ABIMS platform of the FR2424 (CNRS, Sorbonne Université) for bioinformatics resources.

The pelagophyte Aureococcus anophagefferens causes harmful brown tide blooms in marine embayments on three continents. Aureococcus anophagefferens was the first harmful algal bloom species to have its genome sequenced, an advance that evidenced genes important for adaptation to environmental conditions that prevail during brown tides. To expand the genomic tools available for this species, genomes for four strains were assembled, including three newly sequenced strains and one assembled from publicly available data. These genomes ranged from 57.11 to 73.62 Mb, encoding 13,191-17,404 potential proteins. All strains shared ~90% of their encoded proteins as determined by homology searches and shared most functional orthologs as determined by KEGG, although each strain also possessed coding sequences with unique functions. Like the original reference genome, the genomes assembled in this study possessed genes hypothesized to be important in bloom proliferation, including genes involved in organic compound metabolism and growth at low light. Cross-strain informatics and culture experiments suggest that the utilization of purines is a potentially important source of organic nitrogen for brown tides. Analyses of metatranscriptomes from a brown tide event demonstrated that use of a single genome yielded a lower read mapping percentage (~30% of library reads) as compared to a database generated from all available genomes (~43%), suggesting novel information about bloom ecology can be gained from expanding genomic space. This work demonstrates the continued need to sequence ecologically relevant algae to understand the genomic potential and their ecology in the environment.

Nano- and pico-eukaryotes play important roles in the diversity and functions of marine ecosystems. Warm, saline, and nutrient-depleted water that originates in the Kuroshio Current seasonally intrudes into the northern South China Sea (NSCS) from autumn to spring. To clarify the mechanisms in shaping the community structure of nano- and pico-eukaryotes as well as impacts of the Kuroshio intrusion on the NSCS ecosystem, genomic DNA and RNA were co-extracted from samples collected at two depths from nine stations, and then the V9 region of 18S rDNA and rRNA was sequenced with high-throughput sequencing. Our results showed that Dinophyceae was the most diverse and abundant nanoeukaryotic group during the study period revealed by both DNA and RNA surveys. In contrast, the relative read abundance of MAST, Pelagophyceae, and Dinophyceae in the size fraction of picoeukaryotes might be largely underestimated by the DNA survey. The RNA survey was the more reliable method to investigate the eukaryotic community structure. Environmental filtering played an important role in shaping the community structure, and the sampling depth became the governing factor of the beta diversity under the environmental setting of stratification during the study period. The spatial variations in the diversity of nanoeukaryotes were subject to the dispersal limitation under the size rule. The effects of the Kuroshio intrusion on the nanoeukaryotic community structure might also be explained by the dispersal limitation. Overall, neutral processes are critical in shaping the community structure of nanoeukaryotes. The relative metabolic activities of nanoeukaryotes were relatively stable in accordance with the high similarity of community structure between sampling sites. The responses of the relative metabolic activities of picoeukaryotes to environmental factors displayed two distinct patterns: positive correlations with salinity and nutrients and negative with temperature for Dinophyceae, MAST, and Pelagophyceae, while reversed patterns for Mamiellophyceae and Radiolaria. Our findings improve the understanding of the nano- and pico-eukaryotic communities in the NSCS and the mechanisms of their assembly.

Aureococcus anophagefferens Hargraves & Sieburth (Pelagophyceae, Ochrophyta) is a marine harmful microalga responsible for brown tides causing significant economic and ecological damage in China, USA, and South Africa. Here, the complete mitochondrial genome of A. anophagefferens strain CCMP 1984 has been constructed and characterized as a 42,401 bp circular-mapping molecule with the A + T content of 65.3%. The mitogenome encompassed 37 protein-coding genes (PCGs), 23 tRNAs, three rRNAs, and six genomic-specific orfs. A group I intron was present in the cox1 gene and encoded an LAGLIDADG homing endonuclease (LHE) in A. anophagefferens. One of the most interesting findings was the presence of three accessory PCGs (dam1, dam2, and dcm) encoding two DNA adenine methyltransferases and a DNA cytosine methyltransferase in the A. anophagefferens mitogenome, which had the nature of microbial origin, indicating that the DNA fragment of virus and bacteria might be obtained via horizontal gene transfer (HGT). This is the first published Pelagophyceae mitogenome, which improves our understanding of its evolutionary history and provides essential genomic information for tracking its ecological dynamics in field investigation on brown tides.

Assessment of methylmercury (MeHg) accumulation by marine microalgae is critical to understand the dynamics of mercury (Hg) and MeHg in marine environments. We conducted incubation experiments with added MeHg to reveal its bioaccumulation by four marine microalgal lineages. Cyanophyceae had a higher cellular MeHg accumulation than Pelagophyceae, Prymnesiophyceae, and Bacillariophyceae (diatom). MeHg accumulation was higher in living (than dead) diatom cells. Moreover, diatom cells did not release cellular MeHg during cell division and the stationary phase. Our findings suggest that the community composition and metabolic activity of marine microalgae can be critical for MeHg biomagnification in marine food webs.

Study of the occurrence and formation of sedimentary organic matter in the Sichuan Basin, southern China, provides valuable insights into the biological sources and compositions of Neoproterozoic to Lower Cambrian shales. Rock samples from two sets of shales in the Sichuan Basin and adjacent areas, including the Ediacaran Doushantuo shale and the Lower Cambrian Qiongzhusi shale in the Gaoshiti-Moxi and Ziyang-Weiyuan areas were collected and geochemically analyzed in this study. The distribution of biomarkers, stable carbon isotope compositions and microfossil characteristics reveal that eukaryotic algae including chlorophytes, pelagophyceae, dinoflagellates, diatoms (or their ancestors) and sponges proliferated widely during the Late Neoproterozoic to Early Cambrian in the Sichuan Basin. Cyanobacteria, some α-Proteobacteria, rhizobiales and aerobic methanotrophs also made a contribution to organic matter in the studied shales. Aerobic methanotrophs that utilize 13C-depleted substrates such as CH4 as carbon sources may have been the cause of anomalous 13C-depleted kerogen in the study area.

Marine chromophytic phytoplankton are a diverse group of algae and contribute significantly to the total oceanic primary production. However, the spatial distribution of chromophytic phytoplankton is understudied in the West Pacific Ocean (WPO). In this study, we have investigated the community structure and spatial distribution of chromophytic phytoplankton using RuBisCO genes (Form ID rbcL). Our results showed that Haptophyceae, Pelagophyceae, Cyanophyceae, Xanthophyceae, and Bacillariophyceae were the dominant groups. Further, chromophytic phytoplankton can be distinguished between upwelling and non-upwelling zones of the WPO. Surface and 75 m depths of a non-upwelling area were dominated by Prochlorococcus strains, whereas chromophytic phytoplankton were homogenously distributed at the surface layer in the upwelling zone. Meanwhile, Pelagomonas-like sequences were dominant at DCM (75 m) and 150 m depths of the upwelling zone. Non-metric multidimensional scaling (NMDS) analysis did not differentiate between chromophytic phytoplankton in the upwelling and non-upwelling areas, however, it showed clear trends of them at different depths. Further, redundancy analysis (RDA) showed the influence of physicochemical parameters on the distribution of chromophytic phytoplankton. Along with phosphate (p < 0.01), temperature and other dissolved nutrients were important in driving community structure. The upwelling zone was impacted by a decrease in temperature, salinity, and re-supplement of nutrients, where Pelagomonas-like sequences outnumbered other chromophytic groups presented.

The Pelagophyceae are marine stramenopile algae that include Aureoumbra lagunensis and Aureococcus anophagefferens, two microbial species notorious for causing harmful algal blooms. Despite their ecological significance, relatively few genomic studies of pelagophytes have been carried out. To improve understanding of the biology and evolution of pelagophyte algae, we sequenced complete mitochondrial genomes for A. lagunensis (CCMP1510), Pelagomonas calceolata (CCMP1756), and five strains of Aureoc. anophagefferens (CCMP1707, CCMP1708, CCMP1850, CCMP1984, and CCMP3368) using Nanopore long-read sequencing. All pelagophyte mitochondrial genomes assembled into single, circular mapping contigs between 39,376 bp (P. calceolata) and 55,968 bp (A. lagunensis) in size. Mitochondrial genomes for the five Aureoc. anophagefferens strains varied slightly in length (42,401–42,621 bp) and were 99.4–100.0% identical. Gene content and order were highly conserved between the Aureoc. anophagefferens and P. calceolata genomes, with the only major difference being a unique region in Aureoc. anophagefferens containingDNA adenine and cytosine methyltransferase (dam/dcm) genes that appear to be the product of lateral gene transfer from a prokaryotic or viral donor. Although the A. lagunensis mitochondrial genome shares seven distinct syntenic blocks with the other pelagophyte genomes, it has a tandem repeat expansion comprising ∼40% of its length, and lacks identifiable rps19 and glycine tRNA genes. Laterally acquired self-splicing introns were also found in the 23S rRNA (rnl) gene of P. calceolata and the coxI gene of the five Aureoc. anophagefferens genomes. Overall, these data provide baseline knowledge about the genetic diversity of bloom-forming pelagophytes relative to nonbloom-forming species.

The relationships among the Aurearenophyceae, Phaeothamniophyceae, Phaeophyceae and Xanthophyceae lineages of the Heterokontophyta SI clade are not well known. By adding previously unexamined taxa related to these classes in a five gene phylogeny (SSU rRNA, atpB, psaA, psaB, rbcL), we recovered an assemblage of taxa previously unrecognized. We propose the class Phaeosacciophyceae class. nov., that includes Phaeosaccion collinsii, Phaeosaccion multiseriatum sp. nov., Phaeosaccion okellyi sp. nov., Antarctosaccion applanatum, Tetrasporopsis fuscescens, Tetrasporopsis moei sp. nov., and Psammochrysis cassiotisii gen. & sp. nov. We re-examine the literature for Chrysomeris, Nematochrysis, Chrysowaernella and the invalid name “Giraudyopsis” and conclude some taxa in previous studies are misidentified or misnamed, i.e. Chrysomeris and Chrysowaernella, respectively. We also show that Nematochrysis sessilis var. vectensis and Nematochrysis hieroglyphica may belong in the recently described class Chrysoparadoxophyceae. The phylogenetic relationships of Phaeobotrys solitaria and Pleurochloridella botrydiopsis are not clearly resolved, but they branch near the Xanthophyceae. Here we describe a new class Phaeosacciophyceae, a new order Phaeosacciales, a new family Tetrasporopsidaceae, a new genus Psammochrysis and four new species.

Brown tides caused by Aureococcus anophagefferens have occurred annually and severely impacted on the local ecology and economy in the coastal waters of Qinhuangdao, China since 2009. The uptake preference, pigment composition and growth responses of the Chinese strain of A. anophagefferens to different nitrogen substrates were studied in the laboratory to better understand its nitrogen (N) strategies and compare the findings with those from the other strains in the U.S.A. Our results indicated that A. anophagefferens had a high absolute uptake rate (ρmax) for NH4+ and low half-saturation constants (Ks) for NO3− and urea. It grew faster on the organic substrates (urea and glutamic acid) than that on the inorganic substrates (NO3− and NH4+). A. anophagefferens had higher contents of cellular pigments for both photosynthesis (chlorophyll a, chlorophyll c2, 19′-butanoyloxyfucoxanthin and fucoxanthin) and photoprotection (β-Carotene and diadinoxanthin) on urea than did on NO3− and NH4+, suggesting a stronger capability for photosynthesis on urea. A. anophagefferens had higher contents of cellular carbon, N and proteins on urea than did on NO3− and NH4+, suggesting a stronger capability for the acclimation and storage of C and N on urea. A. anophagefferens produced more β-carotene rather than chlorophyll a on glutamic acid and thus inhibited photosynthesis and carbon fixation. We found in this study that urea was the best N substrate for the growth of A. anophagefferens. A. anophagefferens might have a competitive advantage over other phytoplankton in waters enriched with DON, particularly with urea.

Systematists increasingly use molecular markers to identify species; however, most microalgae were described before gene sequencing and type specimens were often ink drawings. Cryptic speciation and biogeographic isolation are other potential problems when anchoring an old species name with a modern gene sequence. Therefore when biological type material is absent, the best approach is to recollect the alga from the type locality and sequence genes. Sarcinochrysis marina, described in 1930 by Geitler from the Canary Islands, Spain, is the oldest Pelagophyceae genus. Geitler used two cultures in his study, but these cultures no longer exist. We re-isolated S. marina from the type locality near Las Palmas, Gran Canaria. Furthermore, we included additional Pelagophyceae strains that were isolated from natural habitats for this study or were obtained from culture collections. We produced 85 sequences, representing the nuclear-encoded SSU rRNA and the plastid-encoded rbcL, psaA, psaB, psbA, and psbC genes. The sequences were used to infer maximum likelihood phylogenetic trees. We anchored the name Sarcinochrysis marina using the Las Palmas isolate, and we described four new genera (Arachnochrysis, Pelagospilus, Sargassococcus, Sungminbooa) and nine new species in the Sarcinochrysidales. We also described a new family, Chrysocystaceae, based upon molecular phylogenetic analyses.

Phytoplankton are the basis of primary production and play important roles in regulating energy export in marine ecosystems. Compared to other regions, chromophytic phytoplankton is considerably understudied in the Bay of Bengal (BOB). Here we investigated community structure and spatial distribution of chromophytic phytoplankton in the BOB by using RuBisCO genes (Form ID rbcL). High throughput sequencing of rbcL genes revealed that Diatoms, Cyanobacteria (Cyanophyceae), Pelagophyceae, Haptophyceae, Chrysophyceae, Eustigamatophyceae, Xanthophyceae, Cryptophyceae, Dictyochophyceae, and Pinguiophyceae were the most abundant groups recovered in the BOB. Abundances and distribution of Bacillariophyceae and Pelagophyceae were further verified using quantitative PCR analyses which showed the dominance of these groups near the Equator region (p < 0.01) where upwelling was likely the source of nutrients. Further, redundancy analysis demonstrated that temperature was an important environmental driver in structuring distributions of Cyanophyceae and dominant chromophytic phytoplankton. Morphological identification and quantification confirmed the dominance of Diatoms, and also detected other cyanobacteria and dinoflagellates that were missing in our molecular characterizations. Pearson correlations of these morphologically identified phytoplankton with environmental gradients also indicated that nutrients and temperature were key variables shaping community structure. Combination of molecular characterization and morphological identification provided a comprehensive overview of chromophytic phytoplankton. This is the first molecular study of chromophytic phytoplankton accomplished in the BOB, and our results highlight a combination of molecular analysis targeting rbcL genes and microscopic detection in examining phytoplankton composition and diversity.

Chrysophaeum taylorii Lewis & Bryan (Pelagophyceae) is a mucilage-producing benthic microalga that has recently begun to spread in the Mediterranean Sea, where a range expansion is occurring. This paper presents the results of three field experiments that aimed to increase the knowledge on mucilage provision mechanisms for this benthic microalga and to evaluate the importance of mucilage in its range expansion. By means of two correlative field experiments (several years of data were considered to encompass the variability of mucilage cover) we found that, on the sea bottom, mucilage cover does not depend on epilithic cell density and that both its cover and settling are affected by water flow. We also tested the hypothesis that cells embedded in floating mucilage fall on the underlying substratum, where their abundance depends on water flow. To this aim, in the field we manipulated the presence of floating mucilaginous aggregates in cages with different levels of exposure to winds. The abundance of C. taylorii cells on the substratum under cages with mucilage was compared with that of two control treatments: cages without mucilage and mucilage in still water, in the field and lab, respectively. The results suggested that mucilage can represent an excellent strategy for the species to disperse, as C. taylorii cells fall from the floating mucilage and, if the water flow is unimportant, settle on hard substrata just underneath the cage. This study enriches the portfolio of knowledge of the dispersal strategies of microalgae and contributes to the understanding of the spread of invasive species.

Picoeukaryotes play an important role in the biogenic element cycle and energy flow in oligotrophic ecosystems. However, their biodiversity and activity are poorly studied in open ocean systems, such as the northwestern Pacific Ocean, which is characterized by a complex hydrological setting. Here, we investigated the diversity and activity of picoeukaryotes in the northwestern Pacific Ocean using high-throughput sequencing targeting the V9 region of 18S rDNA and rRNA. Our results showed that the DNA picoeukaryotic communities were mainly represented by Mamiellophyceae, MAST, MALV-II, Spirotrichea, Prymnesiophyceae, and MALV-I (69.33% of the total DNA reads), and the RNA communities were dominated by Spirotrichea, Mamiellophyceae, MAST, Pelagophyceae, and MALV-II (67.46% of the total RNA reads). The number of operational taxonomic units (OTUs) was significantly affected by temperature and salinity, and was decreased with the increasing nutrient concentration both in the DNA and RNA surveys. Significant differences were observed in the community composition between DNA-based and RNA-based molecular approaches, and these differences were mainly attributed to Mamiellophyceae, Spirotrichea, and Pelagophyceae. The RNA: DNA ratio was used as a proxy for relative metabolic activity of the individual OTUs. We found that the relative metabolic activities of Mamiellophyceae, Spirotrichea, and Pelagophyceae species in the northwestern Pacific Ocean were highly affected by the nutrient concentration, i.e., the NO3 + NO2 and SiO3 concentration. Overall, our study shed light on picoeukaryotic diversity and distribution in the northwestern Pacific Ocean and revealed the correlation between the diversity, relative metabolic activities of marine picoeukaryotes, and the environmental factors.

This paper focuses on the interactive short and long-term effect of three different stressors on a macroalgal assemblage. Three stressors are considered: herbivory, nutrients and mucilage. The experiment was conducted in Tavolara Punta Coda Cavallo Marine Protected Area (Mediterranean Sea) during a bloom of the benthic mucilage-producing microalga Chrysophaeum taylorii (Pelagophyceae); this microalga is recently spreading in the Mediterranean Sea. On a rocky substratum, 36 plots 20 × 20 cm in size were prepared. Factorial combinations of three experimental treatments were applied in triplicate, including three grazing levels crossed with two nutrient enrichment and two mucilage removal treatments. Significant differences were observed among treatments 8 weeks later, at the end of summer. In particular, dark filamentous algae were more abundant in all enriched plots, especially where mucilage and macroalgae had been removed; a higher percent cover of crustose coralline algae was instead observed where nutrients had been increased and no grazing pressure acted. Furthermore, the abundance of Dictyota spp. and Laurencia spp. was significantly higher in enriched mucilage-free plots where the grazing pressure was null or low. However, the effects of the treatments on the overall assemblage of the macroalgal community were not long persistent (36 weeks later). These results illustrate the capacity of a shallow-water macroalgal community to quickly recover from the simultaneous impacts of herbivory, nutrient enrichment, and mucilage.

Xylosyltransferases (XylTs) play key roles in the biosynthesis of many different polysaccharides. These enzymes transfer D-xylose from UDP-xylose to substrate acceptors. In this study, we identified 30 XylTs from primary endosymbionts (green algae, red algae, and glaucophytes) and secondary or higher endosymbionts (brown algae, diatoms, Eustigmatophyceae, Pelagophyceae, and Cryptophyta). We performed comparative phylogenetic studies on key XylT subfamilies, and investigated the functional divergence of genes using RNA-Seq. Of the 30 XylTs, one β-1,4-XylT IRX14-related, one β-1,4 XylT IRX10L-related, and one xyloglucan 6-XylT 1-related gene were identified in the Charophyta, showing strong similarities to their land plant descendants. This implied the ancient occurrence of xylan and xyloglucan biosynthetic machineries in Charophyta. The other 27 XylTs were identified as UDP-D-xylose: L-fucose-α-1,3-D-XylT (FucXylT) type that specifically transferred D-xylose to fucose. We propose that FucXylTs originated from the last eukaryotic common ancestor, rather than being plant specific, because they are also distributed in Choanoflagellatea and Echinodermata. Considering the evidence from many aspects, we hypothesize that the FucXylTs likely participated in fucoidan biosynthesis in brown algae. We provide the first insights into the evolutionary history and functional divergence of FucXylT in algal biology.

To evaluate microbial eukaryotic diversity and distribution in mesoscale processes, we investigated 18S rDNA diversity in a river plume and cyclonic eddy-influenced ecosystem in the southwestern South China Sea (SCS). Restriction fragment length polymorphism analysis was carried out using multiple primer sets. Relative to a wide range of previous similar studies, we observed a significantly higher proportion of sequences of pigmented taxa. Among the photosynthetic groups, Haptophyta accounted for 27.7% of the sequenced clones, which belonged primarily to Prymnesiophyceae. Unexpectedly, five operational taxonomic units of Cryptophyta were closely related to freshwater species. The Chlorophyta mostly fell within the Prasinophyceae, which was comprised of six clades, including Clade III, which is detected in the SCS for the first time in this study. Among the photosynthetic stramenopiles, Chrysophyceae was the most diverse taxon, which included seven clades. The majority of 18S rDNA sequences affiliated with the Dictyochophyceae, Eustigmatophyceae, and Pelagophyceae were closely related to those of pure cultures. The results of redundancy analysis and the permutation Mantel test based on unweighted UniFrac distances, conducted for spatial analyses of the Haptophyta subclades suggested that the Mekong River plume and cyclonic eddy play important roles in regulating microbial eukaryotic diversity and distribution in the southwestern SCS.

Chrysophaeum taylorii Lewis and Bryan (Pelagophyceae) is an allochthonous benthic microalga recently recorded in the Mediterranean Sea where it is spreading quickly. Although this species secretes mucilage which is seriously affecting marine communities, it has been scarcely studied and no information on its ecologyisavailableyet.Wepresenttheresultsofa fiveyearstudyaimedtoincreaseknowledgeonsubstratum and depth preferences of this bloom-forming microalga with special emphasis on its colonisation efficiency. Three separate field experiments established that cell abundances of Chrysophaeum taylorii were significantly lower on macroalgae, mollusc shells and sand than on hard granitic substrata, where the microalga settled efficiently and proliferated immediately independently of the colonisation level of the substratum (bare vs colonised) and the substratum stability (mobility) (cobbles vs rocks). Furthermore, C. taylorii abundance (density) decreased with depth >2 m, showing it prefers shallow waters in which it is able to resist to high light intensities. These results offer some important insights into the ecology of this bloom-forming microalga that will contribute to the planning of future research and strategies for management of bloom events.

Marine picoplankton, unicellular organisms with cell sizes up to 3 μm in diameter, numerically dominate marine ecosystems, encompassing Archaea, Bacteria, Eukarya (protists and fungi) as well as viruses. Autotrophic and heterotrophic picoplankton abundance and community composition with a focus on picoeukaryotes (PEs) were investigated in the winter of 2016 at three stations along a coast-to-offshore transect in the southern Adriatic Sea. Abundances were estimated by flow cytometry, while community composition by Illumina High-Throughput Sequencing (HTS) of 16S and 18S rRNA genes. The photosynthetic picoplankton diversity was also investigated by High-Performance Liquid Chromatography (HPLC) of liposoluble pigments. Heterotrophic bacteria and cyanobacteria (Prochlorococcus and Synechococcus) accounted for up to 7 × 105; 2.3 × 104 and 2.5 × 104 cells mL−1, respectively, while photosynthetic picoeukaryotes peaked with 3 × 103 cells mL−1. Prokaryotes, as revealed by HTS were dominated by Alphaproteobacteria (mainly SAR11, 44.91% of total 16S sequence reads), followed by Gammaproteobacteria (Oceanospirillales and Pseudomonadales, 14.96%), Bacteroidetes (mainly Flavobacteriales, 13%), Cyanobacteria (Prochlorococcus and Synechococcus, 9.52%), Marinimicrobia (SAR406, 7.97%), Deltaproteobacteria (SAR324, 3.83%), Actinobacteria (2.24%) and Chloroflexi (SAR202, 1.90%). Photosynthetic pigment concentrations were very low (12.12 μgL−1 at the most) and taxonomic pigments could be attributed to Prochlorococcus, Synechococcus, Prymnesiophyceae, Bacillariophyceae, Chrysophyceae, and Prasinophyceae. HTS data revealed that PEs were dominated by heterotrophs, such as Syndiniophyceae, parasitic dinoflagellates (79.67% of total 18S sequence reads), Dinophyceae (8.7%) and the radiolarians Collodaria belonging to Sphaerozoidae (22.1%) and Spumellaria (5.0%). On the other hand, photoautotrophs, including Chlorophyta (Mamiellophyceae, Prasinophyceae, Trebouxiophyceae, and Ulvophyceae), Stramenopiles (Bacillariophyta, Chrysophyceae, Dictyochophyceae, Pelagophyceae), photoautotrophic Cryptophyta and some Haptophyta (Prymnesiophyceae), did not exceed 5% of total sequence reads. This study provides the first snapshot of the PEs diversity in oligotrophic euphotic waters of the southern Adriatic Sea, hence setting the stage for large-scale surveying and characterization of the eukaryotic diversity in the entire basin.

Chrysophaeum taylorii (Pelagophyceae) is an allochthonous benthic microalga recently recorded in the Mediterranean Sea. During summer, the occurrence of C. taylorii is usually visible to the naked eye due to the large amount of mucilage this species produces. Information on the spatio-temporal variability of this species and on the predictability of massive mucilage events is still scarce and requires to define ad hoc managing strategies of major bloom events. The aim of this work was to identify the relevant scales of variation in the abundance of C. taylorii abundance and to estimate the relative recurrence of its blooms, testing the hypothesis that mucilage was depend- ent on the cell density. The first approach was the identi- fication of the most appropriate sampling procedure to estimate benthic cell abundance of C. taylorii. The second one was the estimation of the magnitude of variation in C. taylorii cell abundance attributable to each of several spa- tial (areas, sites, zones and replicates) and temporal scales (fortnights and years) in the Marine Protected area of Tavolara Punta Coda Cavallo (Western Mediterranean Sea). The results indicate fortnight and year as the most relevant scales of variability in the cell abundance of C. taylorii and

Phytoplankton forms the basis of primary production in mangrove environments. The phylogeny and diversity based on the amplification and sequencing of rbcL, the large subunit encoding the key enzyme ribulose-1, 5-bisphosphate carboxylase/oxygenase was investigated for improved understanding of the community structure and temporal trends of chromophytic eukaryotic phytoplankton assemblages in Sundarbans, the world's largest continuous mangrove. Diatoms (Bacillariophyceae) were by far the most frequently detected group in clone libraries (485 out of 525 clones), consistent with their importance as a major bloom-forming group. Other major chromophytic algal groups including Cryptophyceae, Haptophyceae, Pelagophyceae, Eustigmatophyceae, and Raphidophyceae which are important component of the assemblages were detected for the first time from Sundarbans based on rbcL approach. Many of the sequences from Sundarbans rbcL clone libraries showed identity with key bloom forming diatom genera namely Thalassiosira, Skeletonema and Nitzschia. Similarly, several rbcL sequences which were diatom-like were also detected highlighting the need to explore diatom communities from the study area. Some of the rbcL sequences detected from Sundarbans were ubiquitous in distribution showing 100% identities with uncultured rbcL sequences targeted previously from the Gulf of Mexico and California upwelling system that are geographically separated from study area. Novel rbcL lineages were also detected highlighting the need to culture and sequence phytoplankton from the ecoregion. Principal component analysis revealed that nitrate is an important variable that is associated with observed variation in phytoplankton assemblages (operational taxonomic units). This study applied molecular tools to highlight the ecological significance of diatoms, in addition to other chromophytic algal groups in Sundarbans.

Most harmful algae require B vitamins. We investigated vitamin use by the B1 and B12 auxotroph, Aureococcus anophagefferens, a harmful alga that dominates plankton communities during dense “brown tides” in North America, Africa, and Asia. B12-depleted cultures of A. anophagefferens (clone CCMP1984) adapted to lower ambient B12 concentrations by reducing half-saturation constants (Ks) of B12 uptake and increasing maximum uptake rates (Vmax) compared to vitamin-replete cultures. In contrast, Vmax of vitamin B1 was higher in replete compared to the depleted cultures, whereas the Ks values were similar for both. Ks values for B12 (5.0–21 pmol L−1) were similar to or higher than concentrations measured during brown tides, suggesting that B12 may restrict the growth of this alga in the field. Over the course of a dense brown tide (> 106 cells mL−1) in Quantuck Bay, New York, vitamin B1 and B12 concentrations declined from > 100 pmol L−1 to < 8 pmol L−1, suggesting there was rapid uptake by A. anophagefferens and its associated microbial community. Experiments performed using radioisotope-labeled vitamins B1 and B12 and 14C-bicarbonate indicated that plankton in the size range of A. anophagefferens (1–5 µm) were responsible for the majority of primary production and the majority of vitamin B1 uptake but shared vitamin B12 uptake with smaller picoplankton (< 1 µm). Vitamin uptake rates during the brown tide were capable of turning over standing stocks of vitamin B12 in 15 h, whereas B1 depletion was slower with maximal turnover times of 2.8 d. As the brown tide intensified and vitamin B12 levels declined, the experimental enrichment of brown tide water with vitamin B12 significantly enhanced the growth rates of A. anophagefferens. Collectively, this study demonstrates that vitamin B12 can influence the intensity of harmful algal blooms caused by A. anophagefferens.

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC-MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3β-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3β-ol (dinosterol) was detected in addition to cholest-5-en-3β-ol (cholesterol), (22E)-ergosta-5,22-dien-3β-ol, (22E)-stigmasta-5,22-dien-3β-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3β-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3β-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3β-ol, stigmast-5-en-3β-ol and campest-5-en-3β-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3β-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3β-ol is not a good indicator for diatoms.

The benthic mucilage producing microalga Chrysophaeum taylorii Lewis and Bryan ( Pelagophyceae ) has recently received attention for its rapid spread in the Mediterranean Sea, where its blooms have remarkable detrimental effects. So far no information on C. taylorii response to multiple stressors, especially in terms of mucilage hyperproduction, is available in the literature yet, and a manipulative field experiment in this topic was designed in Tavolara Punta Coda Cavallo Marine Protected Area. The aim of the study was to test the effects of nutrient enrichment (addition of nutrients), mechanical disturbance (partial and total benthic organisms removal) and hydrodynamics (increased water turbulence) on C. taylorii cell density and mucilage abundance. To the purpose, the three above mentioned stressors were simulated and the three treatments were assigned to 20 × 20 cm plots following a full-factorial design (n = 3). Interactive effects of the three stressors affected significantly both benthic C. taylorii cell density and mucilage cover although differently. Mechanical disturbance and high hydrodynamics produced consistent effects on cell density and mucilage production (i.e. the former factor enhancing and the latter decreasing). Nutrient enrichment on the contrary led to contrasting effects, promoting cell abundance and inhibiting mucilage production. Therefore, important mucilage blooms are expected in oligotrophic sheltered coastal locations where barren areas are present.

Large-scale blooms suspected to be "brown tides" occurred in early summer for three consecutive years from 2009 to 2011 in the coastal waters of Qinhuangdao, China, and had significant negative impacts on the shellfish mariculture industry. To identify the causative species of the blooms, phytoplankton samples were collected from regions with and without bloom in the coastal waters of Qinhuangdao in 2011, and clone libraries were built using eukaryote-specific 18S ribosomal RNA gene (18S rDNA). Altogether 50 clones, including 17 clones from bloom area and 33 clones from nearby regions without bloom were amplified. Blasted in GenBank, 17 clones amplified from the bloom area were assigned to Pelagophyceae (8 clones), Mediophyceae (2 clones), Cryptophyta (2 clones), Dinophyceae (2 clones) and unidentified eukaryotic species (3 clones). Those from the non-bloom site were assigned to Cryptophyta, Eustigmatophyceae, Prasinophyceae, Coscinodiscophyceae, Mediophyceae, Raphidophyceae and Dinophyceae, but not Pelagophyceae. All 8 pelagophyte clones from the bloom area were 99.7-100% similar to a single species, Aureococcus anophagefferens Hargraves et Sieburth, the causative species of brown tides on the east coast of USA. For nearly the entire length of the 18S rDNA, there were 0-6 base pair differences between the 8 amplicons and those of A. anophagefferens from USA. Furthermore, all of the 8 clones were clustered into the same well-supported clade with A. anophagefferens (posterior probability = 0.99) in a phylogenetic tree established for pelagophytes and other related microalgae. In our previous studies, the causative species of the bloom was tentatively identified as a pelagophyte, haptophyte or silicoflagellate, based on the pigment profile of the size-fractioned phytoplankton samples. Based on this study, we conclude that blooms in the coastal waters of Qinhuangdao of the Bohai Sea were brown tides caused by A. anophagefferens. As far as we know, this is the first report of brown tide events caused by A. anophagefferens in China, which is the third country in the world reporting A. anophagefferens blooms in addition to USA and South Africa. (c) 2012 Elsevier B.V. All rights reserved.

Brown tide blooms, caused by excessive growth of the pelagophyte Aureococcus anophagefferens Hargraves et Sieburth (Ochrophyta, Pelagophyceae), have been observed to be spreading in new water bodies with substantial human influences. This study quantified A. anophagefferens growth potential as affected by common pollutants—heavy metals, herbicide metolachlor, and secondary-treated sewage effluent. Culture studies show that A. anophagefferens growth potential was not affected by environmentally relevant levels of metolachlor but was reduced at low salinity or addition of 10–15 % sewage effluent. When exposed to Cd, Cu, and Zn at high but environmentally relevant concentrations, A. anophagefferens growth potential was reduced, but Zn antagonized inhibitive effect from Cu and CdCu. Preliminary field incubation showed that adding Ni, Cu, Zn, or EDTA did not significantly alter A. anophagefferens survival in filtered Newark Bay seawaters (NJ, USA), suggesting tolerance of A. anophagefferens to anthropogenic toxicants. These results may aid on the understanding and modeling of A. anophagefferens expansion in polluted estuaries.

Summary Oceanic photosynthetic picoeukaryotes ( 40% of total primary production at low latitudes such as the North-Eastern tropical Atlantic. In the world ocean, warmed by climate changes, the expected gradual shift towards smaller primary producers could render the role of photosynthetic picoeukaryotes even more important than they are today. Little is still known, however, about how the taxonomic composition of this highly diverse group affects primary production at the basin scale. Here, we combined flow cytometric cell sorting, NaH14CO3 radiotracer incubations and class-specific fluorescence in situ hybridization (FISH) probes to determine cell- and biomass-specific inorganic carbon fixation rates and taxonomic composition of two major photosynthetic picoeukaryote groups on a ∼7500-km-long latitudinal transect across the Atlantic Ocean (Atlantic Meridional Transect, AMT19). We show that even though larger cells have, on average, cell-specific CO2 uptake rates ∼5 times higher than the smaller ones, the average biomass-specific uptake is statistically similar for both groups. On the other hand, even at a high taxonomic level, i.e. class, the contributions to both groups by Prymnesiophyceae, Chrysophyceae and Pelagophyceae are significantly different (P < 0.001 in all cases). We therefore conclude that these group's carbon fixation rates are independent of the taxonomic composition of photosynthetic picoeukaryotes across the Atlantic Ocean. Because the above applies across different oceanic regions the diversity changes seem to be a secondary factor determining primary production.

Photosynthetic picoeukaryotes (PPEs) of a size < 3 µm play a crucial role in oceanic primary production. However, little is known of the structure of the PPE community over large spatial scales. Here, we investigated the distribution of various PPE classes along an Atlantic Meridional Transect sampled in boreal autumn 2004 that encompasses a range of ocean provinces (gyres, upwelling, temperate regions), using dot blot hybridization technology targeting plastid 16S rRNA gene amplicons. Two algal classes, Prymnesiophyceae and Chrysophyceae, dominated the PPE community throughout the Atlantic Ocean, over a range of water masses presenting different trophic profiles. However, these classes showed strongly complementary distributions with Chrysophyceae dominating northern temperate waters, the southern gyre and equatorial regions, while prymnesiophytes dominated the northern gyre. Phylogenetic analyses using both plastid and nuclear rRNA genes revealed a high diversity among members of both classes, including sequences contained in lineages with no close cultured counterpart. Other PPE classes were less prevalent along the transect, with members of the Cryptophyceae, Pelagophyceae and Eustigmatophyceae essentially restricted to specific regions. Multivariate statistical analyses revealed strong relationships between the distribution patterns of some of these latter PPE classes and temperature, light intensity and nutrient concentrations. Cryptophyceae, for example, were mostly found in the upwelling region and associated with higher nutrient concentrations. However, the key classes of Prymnesiophyceae and Chrysophyceae were not strongly influenced by the variables measured. Although there appeared to be a positive relationship between Chrysophyceae distribution and light intensity, the complementary distributions of these classes could not be explained by the variables recorded and this requires further explanation.

• By comparative analyses we identify lineage-specific diversity in transcription factors (TFs) from stramenopile (or heterokont) genome sequences. We compared a pennate (Phaeodactylum tricornutum) and a centric diatom (Thalassiosira pseudonana) with those of other stramenopiles (oomycetes, Pelagophyceae, and Phaeophyceae (Ectocarpus siliculosus)) as well as to that of Emiliania huxleyi, a haptophyte that is evolutionarily related to the stramenopiles. • We provide a detailed description of diatom TF complements and report numerous peculiarities: in both diatoms, the heat shock factor (HSF) family is overamplified and constitutes the most abundant class of TFs; Myb and C2H2-type zinc finger TFs are the two most abundant TF families encoded in all the other stramenopile genomes investigated; the presence of diatom and lineage-specific gene fusions, in particular a class of putative photoreceptors with light-sensitive Per-Arnt-Sim (PAS) and DNA-binding (basic-leucine zipper, bZIP) domains and an HSF-AP2 domain fusion. • Expression data analysis shows that many of the TFs studied are transcribed and may be involved in specific responses to environmental stimuli. • Evolutionary and functional relevance of these observations are discussed.

Heterokont members of the Pelagophyceae form the massive brown tides that have continually plagued the coastal regions of the eastern U.S. seaboard and the Gulf of Mexico. To gain a better understanding of the photosynthetic competence that may be linked to their success in forming massive blooms, we sequenced the chloroplast genomes of two pelagophytes: Aureococcus anophagefferens Hargraves et Sieburth and Aureoumbra lagunensis D. A. Stockw., DeYoe, Hargraves et P. W. Johnson. The chloroplast genomes of A. anophagefferens (89,599 bp) and Ar. lagunensis (94,346 bp) are significantly smaller than those of six other stramenopiles sequenced to date. The structure (or configuration) is partially due to the absence of the large inverted repeats common in chloroplast genomes. Eight of 10 small and tandem repeats from the A. anophagefferens and Ar. lagunensis genomes are adjacent to genes coding for photosynthetic or energy production functions, implying that these domains may have functional constraints. High genomic synteny, a multigene phylogenetic analysis, and a synapomorphic change in the form of an attenuated psbA gene confirm that A. anophagefferens and Ar. lagunensis are closely related taxa. Finally, the presence of three light-independent chl-biosynthesis genes in the chloroplast of Ar. lagunensis, but absence in the chloroplast and nuclear genomes of A. anophagefferens, suggests the persistence of a more ancient (i.e., dark-adaptive) potential in Ar. lagunensis but not in A. anophagefferens. Whether the presence of both chl-biosynthesis pathways in Ar. lagunensis contributes to the ability of this organism to sustain prolonged bloom (continuously for ∼8 years) under reduced light conditions, but not A. anophagefferens (a few months), remains an open question.

The new type blue light (BL) receptor aureochrome (AUREO) was recently discovered in a stramenopile alga, Vaucheria (Takahashi et al. Proc Natl Acad Sci USA 104(49):19625-19630, 2007). AUREO has a bZIP (basic region/leucine zipper) and BL-sensing light-oxygen-voltage (LOV) domain and functions as a BL-activated transcription factor. It mediates BL-induced branching and regulates the development of the sex organ in V. frigida. Although AUREO sequences have previously been found in Fucus and some diatoms, here we report that AUREO orthologs are commonly conserved in photosynthetic stramenopiles. Five AUREO orthologs were isolated from three stramenopile genera (Fucus, Ochromonas, and Chattonella). By BLAST search, several AUREO sequences were also detected in genomes in Aureococcus anophagefferens (Pelagophyceae). However, AUREO was not found in heterotrophic stramenopiles or in closely related phyla, such as haptophytes and cryptophytes, or in green plants. Stramenopiles do not possess phototropin, the well-known BL receptor for phototropism of green plants. From comparative analysis of LOV domains, together with kinship analysis of AUREO bZIP domains, AUREO can be regarded as the BL receptor specific to phototrophic stramenopiles. The evolution of AUREO and the phylogeny of LOV domains in stramenopiles and green plants are discussed.

Brown tides of the marine pelagophyte Aureococcus anophagefferens Hargraves et Sieburth have been investigated extensively for the past two decades. Its growth is fueled by a variety of nitrogen (N) compounds, with dissolved organic nitrogen (DON) being particularly important during blooms. Characterization of a cDNA library suggests that A. anophagefferens can assimilate eight different forms of N. Expression of genes related to the sensing, uptake, and assimilation of inorganic and organic N, as well as the catabolic process of autophagy, was assayed in cells grown on different N sources and in N-limited cells. Growth on nitrate elicited an increase in the relative expression of nitrate and ammonium transporters, a nutrient stress-induced transporter, and a sensory kinase. Growth on urea increased the relative expression of a urea and a formate/nitrite transporter, while growth on ammonium resulted in an increase in the relative expression of an ammonium transporter, a novel ATP-binding cassette (ABC) transporter and a putative high-affinity phosphate transporter. N limitation resulted in a 30- to 110-fold increase in the relative expression of nitrate, ammonium, urea, amino acid/polyamine, and formate/nitrite transporters. A. anophagefferens demonstrated the highest relative accumulation of a transcript encoding a novel purine transporter, which was highly expressed across all N sources. This finding suggests that purines are an important source of N for the growth of this organism and could possibly contribute to the initiation and maintenance of blooms in the natural environment.

Eukaryotic ultraphytoplankton (