Diversity and seasonal occurrence of haptophyta in northern South China Sea through size-fractionated metabarcoding.

Haptophyta plays a key role in marine pico-nanoeukaryote communities but information on their diversity and ecology is extremely limited. A total of 103 water samples were collected in northern South China Sea to assess the diversity of haptophyta through metabarcoding targeting 18S V4 rDNA. Furthermore, we investigated the potential genetic differentiation among seasonal occurring Phaeocystis globosa using the high resolution molecular marker pgcp1. 18S V4 rDNA metabarcoding dataset revealed 41 species of haptophytes, with 16 of them as the first record in this region. Notably, six harmful species were detected, including Chrysochromulina leadbeateri, Phaeocystis globosa, and Prymnesium parvum. The pgcp1 marker revealed two clades of Phaeocystis globosa and both of them were present around the year. Clade I was found to predominate in warm season, while Clade III tended to bloom in cold waters. Our results highlight the risk potential of harmful haptophytes in the northern South China Sea.

Kre6 (yeast 1,6-ß-transglycosylase) homolog, PhTGS, is essential for ß-glucan synthesis in the haptophyte Pleurochrysis haptonemofera.

Haptophytes synthesize unique ß-glucans containing more ß-1,6-linkages than ß-1,3 linkages, as a storage polysaccharide. To understand the mechanism of the synthesis, we investigated the roles of Kre6 (yeast 1,6-ß-transglycosylase) homologs, PhTGS, in the haptophyte Pleurochrysis haptonemofera. RNAi of PhTGS repressed ß-glucan accumulation and simultaneously induced lipid production, suggesting that PhTGS is involved in ß-glucan synthesis and that the knockdown leads to the alteration of the carbon metabolic flow. PhTGS was expressed more in light, where ß-glucan was actively produced by photosynthesis, than in the dark. The crude extract of E. coli expressing PhKre6 demonstrated its activity to incorporate 14C-UDP-glucose into ß-glucan of P. haptonemofera. These findings suggest that PhTGS functions in storage ß-glucan synthesis specifically in light, probably by producing the ß-1,6-branch.

Bioprospecting for new exopolysaccharide-producing microalgae of marine origin.

Microalgae are photosynthetic organisms that can produce biomolecules with industrial interest, including exopolysaccharides (EPS). Due to their structural and compositional diversity, microalgae EPS present interesting properties that can be considered in cosmetic and/or therapeutic areas. Seven microalgae strains from three different lineages, namely Dinophyceae (phylum Miozoa), Haptophyta, and Chlorophyta, were investigated as EPS producers. All strains were found to be EPS producers, though the highest EPS yield was obtained for Tisochrysis lutea, followed by Heterocapsa sp. (126.8 and 75.8 mg L-1, respectively). Upon assessment of the polymers' chemical composition, significant contents of unusual sugars, including fucose, rhamnose, and ribose, were found. Heterocapsa sp. EPS stood out due to its high content of fucose (40.9 mol%), a sugar known to confer biological properties to polysaccharides. The presence of sulfate groups (10.6-33.5 wt%) was also noticed in the EPS produced by all microalgae strains, thus contributing to the possibility that these EPS might have biological activities worth exploring.

Haptophyte communities along the Kuroshio current reveal their geographical sources and ecological traits.

Haptophytes are one of the most ecologically successful phytoplankton groups in the modern ocean and tend to maintain balanced and stable communities across various environments. However, little is known about the mechanisms that enable community stability and ecological success. To reveal the community characteristics and interactions among haptophytes, we conducted comprehensive observations from the upstream to downstream regions of the Kuroshio Current. Haptophyte abundance and taxonomy were assessed using quantitative polymerase chain reaction and metabarcoding of 18S rRNA sequences, respectively. The haptophyte community structure changed abruptly at sites on the shelf-slope of the East China Sea, indicating the strong influence of shelf waters with high phytoplankton biomass on downstream communities. Correlation network analysis combined with the phylogeny suggested that haptophytes can coexist with their close relatives, possibly owing to their nutritional flexibility, thereby escaping from resource competition. Consistently, some noncalcifying haptophyte genera with high mixotrophic capacities such as Chrysochromulina constituted a major component of the co-occurrence network, whereas coccolithophores such as Emiliania/Gephyrocapsa were rarely observed. Our study findings suggest that noncalcifying haptophytes play crucial roles in community diversity and stability, and in sustaining the food web structure in the Kuroshio ecosystems.

Large Differences in the Haptophyte Phaeocystis globosa Mitochondrial Genomes Driven by Repeat Amplifications.

The haptophyte Phaeocystis globosa is a well-known species for its pivotal role in global carbon and sulfur cycles and for its capability of forming harmful algal blooms (HABs) with serious ecological consequences. Its mitochondrial genome (mtDNA) sequence has been reported in 2014 but it remains incomplete due to its long repeat sequences. In this study, we constructed the first full-length mtDNA of P. globosa, which was a circular genome with a size of 43,585 bp by applying the PacBio single molecular sequencing method. The mtDNA of this P. globosa strain (CNS00066), which was isolated from the Beibu Gulf, China, encoded 19 protein-coding genes (PCGs), 25 tRNA genes, and two rRNA genes. It contained two large repeat regions of 6.7 kb and ∼14.0 kb in length, respectively. The combined length of these two repeat regions, which were missing from the previous mtDNA assembly, accounted for almost half of the entire mtDNA and represented the longest repeat region among all sequenced haptophyte mtDNAs. In this study, we tested the hypothesis that repeat unit amplification is a driving force for different mtDNA sizes. Comparative analysis of mtDNAs of five additional P. globosa strains (four strains obtained in this study, and one strain previously published) revealed that all six mtDNAs shared identical numbers of genes but with dramatically different repeat regions. A homologous repeat unit was identified but with hugely different numbers of copies in all P. globosa strains. Thus, repeat amplification may represent an important driving force of mtDNA evolution in P. globosa.

Regulation of Phagotrophy by Prey, Low Nutrients, and Low Light in the Mixotrophic Haptophyte Isochrysis galbana.

Mixotrophy combines autotrophy and phagotrophy in the same cell. However, it is not known to what extent the phagotrophy influences metabolism, cell composition, and growth. In this work, we assess, on the one hand (first test), the role of phagotrophy on the elemental and biochemical composition, cell metabolism, and enzymes related to C, N, and S metabolism of Isochrysis galbana Parke, 1949. On the other hand, we study how a predicted increase of phagotrophy under environmental conditions of low nutrients (second test) and low light (third test) can affect its metabolism and growth. Our results for the first test revealed that bacterivory increased the phosphorous and iron content per cell, accelerating cell division and improving the cell fitness; in addition, the stimulation of some C and N enzymatic routes help to maintain, to some degree, compositional homeostasis. Under nutrient or light scarcity, I. galbana grew more slowly despite greater bacterial consumption, and the activities of key enzymes involved in C, N, and S metabolism changed according to a predominantly phototrophic strategy of nutrition in this alga. Contrary to recent studies, the stimulation of phagotrophy under low nutrient and low irradiance did not imply greater and more efficient C flux.

Large-scale genome sequencing reveals the driving forces of viruses in microalgal evolution.

Being integral primary producers in diverse ecosystems, microalgal genomes could be mined for ecological insights, but representative genome sequences are lacking for many phyla. We cultured and sequenced 107 microalgae species from 11 different phyla indigenous to varied geographies and climates. This collection was used to resolve genomic differences between saltwater and freshwater microalgae. Freshwater species showed domain-centric ontology enrichment for nuclear and nuclear membrane functions, while saltwater species were enriched in organellar and cellular membrane functions. Further, marine species contained significantly more viral families in their genomes (p = 8e-4). Sequences from Chlorovirus, Coccolithovirus, Pandoravirus, Marseillevirus, Tupanvirus, and other viruses were found integrated into the genomes of algal from marine environments. These viral-origin sequences were found to be expressed and code for a wide variety of functions. Together, this study comprehensively defines the expanse of protein-coding and viral elements in microalgal genomes and posits a unified adaptive strategy for algal halotolerance.

The Polar Lipidome of Cultured Emiliania huxleyi: A Source of Bioactive Lipids with Relevance for Biotechnological Applications.

Polar lipids from microalgae have aroused greater interest as a natural source of omega-3 (n-3) polyunsaturated fatty acids (PUFA), an alternative to fish, but also as bioactive compounds with multiple applications. The present study aims to characterize the polar lipid profile of cultured microalga Emiliania huxleyi using hydrophilic interaction liquid chromatography coupled with high-resolution mass spectrometry (HILIC-MS) and fatty acids (FA) analysis by gas chromatography (GC-MS). The lipidome of E. huxleyi revealed the presence of distinct n-3 PUFA (40% of total FA), namely docosahexaenoic acid (22:6n-3) and stearidonic acid (18:4n-3), which give this microalga an increased commercial value as a source of n-3 PUFA present in the form of polar lipids. A total of 134 species of polar lipids were identified and some of these species, particularly glycolipids, have already been reported for their bioactive properties. Among betaine lipids, the diacylglyceryl carboxyhydroxymethylcholine (DGCC) class is the least reported in microalgae. For the first time, monomethylphosphatidylethanolamine (MMPE) has been found in the lipidome of E. huxleyi. Overall, this study highlights the potential of E. huxleyi as a sustainable source of high-value polar lipids that can be exploited for different applications, namely human and animal nutrition, cosmetics, and pharmaceuticals.

Deciphering Patterns of Adaptation and Acclimation in the Transcriptome of Phaeocystis antarctica to Changing Iron Conditions1.

The haptophyte Phaeocystis antarctica is endemic to the Southern Ocean, where iron supply is sporadic and its availability limits primary production. In iron fertilization experiments, P. antarctica showed a prompt and steady increase in cell abundance compared to heavily silicified diatoms along with enhanced colony formation. Here we utilized a transcriptomic approach to investigate molecular responses to alleviation of iron limitation in P. antarctica. We analyzed the transcriptomic response before and after (14 h, 24 h and 72 h) iron addition to a low-iron acclimated culture. After iron addition, we observed indicators of a quick reorganization of cellular energetics, from carbohydrate catabolism and mitochondrial energy production to anabolism. In addition to typical substitution responses from an iron-economic toward an iron-sufficient state for flavodoxin (ferredoxin) and plastocyanin (cytochrome c6 ), we found other genes utilizing the same strategy involved in nitrogen assimilation and fatty acid desaturation. Our results shed light on a number of adaptive mechanisms that P. antarctica uses under low iron, including the utilization of a Cu-dependent ferric reductase system and indication of mixotrophic growth. The gene expression patterns underpin P. antarctica as a quick responder to iron addition.

The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae.

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.

Seasonal Dynamics of Haptophytes and dsDNA Algal Viruses Suggest Complex Virus-Host Relationship.

Viruses influence the ecology and diversity of phytoplankton in the ocean. Most studies of phytoplankton host-virus interactions have focused on bloom-forming species like Emiliania huxleyi or Phaeocystis spp. The role of viruses infecting phytoplankton that do not form conspicuous blooms have received less attention. Here we explore the dynamics of phytoplankton and algal viruses over several sequential seasons, with a focus on the ubiquitous and diverse phytoplankton division Haptophyta, and their double-stranded DNA viruses, potentially with the capacity to infect the haptophytes. Viral and phytoplankton abundance and diversity showed recurrent seasonal changes, mainly explained by hydrographic conditions. By 454 tag-sequencing we revealed 93 unique haptophyte operational taxonomic units (OTUs), with seasonal changes in abundance. Sixty-one unique viral OTUs, representing Megaviridae and Phycodnaviridae, showed only distant relationship with currently isolated algal viruses. Haptophyte and virus community composition and diversity varied substantially throughout the year, but in an uncoordinated manner. A minority of the viral OTUs were highly abundant at specific time-points, indicating a boom-bust relationship with their host. Most of the viral OTUs were very persistent, which may represent viruses that coexist with their hosts, or able to exploit several host species.

Emerging Interaction Patterns in the Emiliania huxleyi-EhV System.

Viruses are thought to be fundamental in driving microbial diversity in the oceanic planktonic realm. That role and associated emerging infection patterns remain particularly elusive for eukaryotic phytoplankton and their viruses. Here we used a vast number of strains from the model system Emiliania huxleyi/Emiliania huxleyi Virus to quantify parameters such as growth rate (µ), resistance (R), and viral production (Vp) capacities. Algal and viral abundances were monitored by flow cytometry during 72-h incubation experiments. The results pointed out higher viral production capacity in generalist EhV strains, and the virus-host infection network showed a strong co-evolution pattern between E. huxleyi and EhV populations. The existence of a trade-off between resistance and growth capacities was not confirmed.

Pleurochrysome: A Web Database of Pleurochrysis Transcripts and Orthologs Among Heterogeneous Algae.

Pleurochrysis is a coccolithophorid genus, which belongs to the Coccolithales in the Haptophyta. The genus has been used extensively for biological research, together with Emiliania in the Isochrysidales, to understand distinctive features between the two coccolithophorid-including orders. However, molecular biological research on Pleurochrysis such as elucidation of the molecular mechanism behind coccolith formation has not made great progress at least in part because of lack of comprehensive gene information. To provide such information to the research community, we built an open web database, the Pleurochrysome (http://bioinf.mind.meiji.ac.jp/phapt/), which currently stores 9,023 unique gene sequences (designated as UNIGENEs) assembled from expressed sequence tag sequences of P. haptonemofera as core information. The UNIGENEs were annotated with gene sequences sharing significant homology, conserved domains, Gene Ontology, KEGG Orthology, predicted subcellular localization, open reading frames and orthologous relationship with genes of 10 other algal species, a cyanobacterium and the yeast Saccharomyces cerevisiae. This sequence and annotation information can be easily accessed via several search functions. Besides fundamental functions such as BLAST and keyword searches, this database also offers search functions to explore orthologous genes in the 12 organisms and to seek novel genes. The Pleurochrysome will promote molecular biological and phylogenetic research on coccolithophorids and other haptophytes by helping scientists mine data from the primary transcriptome of P. haptonemofera.

Complex Ancestries of Isoprenoid Synthesis in Dinoflagellates.

Isoprenoid metabolism occupies a central position in the anabolic metabolism of all living cells. In plastid-bearing organisms, two pathways may be present for de novo isoprenoid synthesis, the cytosolic mevalonate pathway (MVA) and nuclear-encoded, plastid-targeted nonmevalonate pathway (DOXP). Using transcriptomic data we find that dinoflagellates apparently make exclusive use of the DOXP pathway. Using phylogenetic analyses of all DOXP genes we inferred the evolutionary origins of DOXP genes in dinoflagellates. Plastid replacements led to a DOXP pathway of multiple evolutionary origins. Dinoflagellates commonly referred to as dinotoms due to their relatively recent acquisition of a diatom plastid, express two completely redundant DOXP pathways. Dinoflagellates with a tertiary plastid of haptophyte origin, by contrast, express a hybrid pathway of dual evolutionary origin. Here, changes in the targeting motif of signal/transit peptide likely allow for targeting the new plastid by the proteins of core isoprenoid metabolism proteins. Parasitic dinoflagellates of the Amoebophyra species complex appear to have lost the DOXP pathway, suggesting that they may rely on their host for sterol synthesis.

Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion.

Isolation and characterization of a virus infecting the freshwater algae Chrysochromulina parva.

Water samples from Lake Ontario, Canada were tested for lytic activity against the freshwater haptophyte algae Chrysochromulina parva. A filterable lytic agent was isolated and identified as a virus via transmission electron microscopy and molecular methods. The virus, CpV-BQ1, is icosahedral, ca. 145nm in diameter, assembled within the cytoplasm, and has a genome size of ca. 485kb. Sequences obtained through PCR-amplification of DNA polymerase (polB) genes clustered among sequences from the family Phycodnaviridae, whereas major capsid protein (MCP) sequences clustered among sequences from either the Phycodnaviridae or Mimiviridae. Based on quantitative molecular assays, C. parva׳s abundance in Lake Ontario was relatively stable, yet CpV-BQ1׳s abundance was variable suggesting complex virus-host dynamics. This study demonstrates that CpV-BQ1 is a member of the proposed order Megavirales with characteristics of both phycodnaviruses and mimiviruses indicating that, in addition to its complex ecological dynamics, it also has a complex evolutionary history.

Seasonal diversity and dynamics of haptophytes in the Skagerrak, Norway, explored by high-throughput sequencing.

Microalgae in the division Haptophyta play key roles in the marine ecosystem and in global biogeochemical processes. Despite their ecological importance, knowledge on seasonal dynamics, community composition and abundance at the species level is limited due to their small cell size and few morphological features visible under the light microscope. Here, we present unique data on haptophyte seasonal diversity and dynamics from two annual cycles, with the taxonomic resolution and sampling depth obtained with high-throughput sequencing. From outer Oslofjorden, S Norway, nano- and picoplanktonic samples were collected monthly for 2 years, and the haptophytes targeted by amplification of RNA/cDNA with Haptophyta-specific 18S rDNA V4 primers. We obtained 156 operational taxonomic units (OTUs), from c. 400.000 454 pyrosequencing reads, after rigorous bioinformatic filtering and clustering at 99.5%. Most OTUs represented uncultured and/or not yet 18S rDNA-sequenced species. Haptophyte OTU richness and community composition exhibited high temporal variation and significant yearly periodicity. Richness was highest in September-October (autumn) and lowest in April-May (spring). Some taxa were detected all year, such as Chrysochromulina simplex, Emiliania huxleyi and Phaeocystis cordata, whereas most calcifying coccolithophores only appeared from summer to early winter. We also revealed the seasonal dynamics of OTUs representing putative novel classes (clades HAP-3-5) or orders (clades D, E, F). Season, light and temperature accounted for 29% of the variation in OTU composition. Residual variation may be related to biotic factors, such as competition and viral infection. This study provides new, in-depth knowledge on seasonal diversity and dynamics of haptophytes in North Atlantic coastal waters.

Phylogenomic analysis of Emiliania huxleyi provides evidence for haptophyte-stramenopile association and a chimeric haptophyte nuclear genome.

Emiliania huxleyi is a haptophyte alga of uncertain phylogenetic affinity containing a secondarily derived, chlorophyll c containing plastid. We sought to characterize its relationships with other taxa by quantifying the bipartitions in which it was included from a group of single protein phylogenetic trees in a way that allowed for variation in taxonomic content and accounted for paralogous sequences. The largest number of sequences supported a phylogenetic relationship of E. huxleyi with the stramenopiles, in particular Aureococcus anophagefferens. Far fewer nuclear sequences gave strong support to the placement of this coccolithophorid with the cryptophyte, Guillardia theta. The majority of the sequences that did support this relationship did not have plastid related functions. These results suggest that the haptophytes may be more closely allied with the heterokonts than with the cryptophytes. Another small set of genes associated E. huxleyi with the Viridiplantae with high support. While these genes could have been acquired with a plastid, the lack of plastid related functions among the proteins for which they code and the lack of other organisms with chlorophyll c containing plastids within these bipartitions suggests other explanations may be possible. This study also identified several genes that may have been transferred from the haptophyte lineage to the dinoflagellates Karenia brevis and Karlodinium veneficum as a result of their haptophyte derived plastid, including some with non-photosynthetic functions.

Characterisation of three novel giant viruses reveals huge diversity among viruses infecting Prymnesiales (Haptophyta).

We have isolated three novel lytic dsDNA-viruses from Raunefjorden (Norway) that are putative members of the Mimiviridae family, namely Haptolina ericina virus RF02 (HeV RF02), Prymnesium kappa virus RF01 (PkV RF01), and Prymnesium kappa virus RF02 (PkV RF02). Each of the novel haptophyte viruses challenges the common conceptions of algal viruses with respect to host range, phylogenetic affiliation and size. PkV RF01 has a capsid of ~310 nm and is the largest algal virus particle ever reported while PkV RF01 and HeV RF02 were able to infect different species, even belonging to different genera. Moreover, PkV RF01 and HeV RF02 infected the same hosts, but phylogenetic analysis placed them in different groups. Our results reveal large variation among viruses infecting closely related microalgae, and challenge the common conception that algal viruses have narrow host range, and phylogeny reflecting their host affiliation.

Deep-branching novel lineages and high diversity of haptophytes in the Skagerrak (Norway) uncovered by 454 pyrosequencing.

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high-throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano- and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta-specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep-branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity.