Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Polyphyletic origin of saxitoxin biosynthesis genes in the marine dinoflagellate Alexandrium revealed by comparative transcriptomics.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms, and at least 14 species within the genus can produce saxitoxins (STXs). STX biosynthesis genes (sxt) are individually revealed in toxic dinoflagellates; however, the evolutionary history remains controversial. Herein, we determined the transcriptome sequences of toxic Alexandrium (A. catenella and A. pacificum) and non-toxic Alexandrium (A. fraterculus and A. fragae) and characterized their sxt by focusing on evolutionary events and STX production. Comparative transcriptome analysis revealed higher homology of the sxt in toxic Alexandrium than in non-toxic species. Notably, non-toxic Alexandrium spp. were found to have lost two sxt core genes, namely sxtA4 and sxtG. Expression levels of 28 transcripts related to eight sxt core genes showed that sxtA, sxtG, and sxtI were relatively high (>1.5) in the toxic group compared to the non-toxic group. In contrast, the non-toxic group showed high expression levels in sxtU (1.9) and sxtD (1.7). Phylogenetic tree comparisons revealed distinct evolutionary patterns between 28S rDNA and sxtA, sxtB, sxtI, sxtD, and sxtU. However, similar topology was observed between 28S rDNA, sxtS, and sxtH/T. In the sxtB and sxtI phylogeny trees, toxic Alexandrium and cyanobacteria were clustered together, separating from non-toxic species. These suggest that Alexandrium may acquire sxt genes independently via horizontal gene transfer from toxic cyanobacteria and other multiple sources, demonstrating monocistronic transcripts of sxt in dinoflagellates.

DNA and RNA Stability of Marine Microalgae in Cold-Stored Sediments and Its Implications in Metabarcoding Analyses.

The ever-increasing applications of metabarcoding analyses for environmental samples demand a well-designed assessment of the stability of DNA and RNA contained in cells that are deposited or buried in marine sediments. We thus conducted a qPCR quantification of the DNA and RNA in the vegetative cells of three microalgae entrapped in facsimile marine sediments and found that >90% of DNA and up to 99% of RNA for all microalgal species were degraded within 60 days at 4 °C. A further examination of the potential interference of the relic DNA of the vegetative cells with resting cyst detection in sediments was performed via a metabarcoding analysis in artificial marine sediments spiked with the vegetative cells of two Kareniaceae dinoflagellates and the resting cysts of another three dinoflagellates. The results demonstrated a dramatic decrease in the relative abundances of the two Kareniaceae dinoflagellates in 120 days, while those of the three resting cysts increased dramatically. Together, our results suggest that a positive detection of microalgae via metabarcoding analysis in DNA or RNA extracted from marine sediments strongly indicates the presence of intact or viable cysts or spores due to the rapid decay of relic DNA/RNA. This study provides a solid basis for the data interpretation of metabarcoding surveys, particularly in resting cyst detection.

Toxic dinoflagellate Karenia mikimotoi induces apoptosis in Neuro-2a cells through an oxidative stress-mediated mitochondrial pathway.

The dinoflagellate Karenia mikimotoi is a toxic bloom-forming species that threatens aquaculture and public health worldwide. Previous studies showed that K. mikimotoi induces neurotoxicity; however, the underlying mechanism is poorly understood. In this study, three neural cell lines were used to investigate the potential neurotoxicity of K. mikimotoi. The tested cells were exposed to a ruptured cell solution (RCS) of K. mikimotoi at different concentrations (0.5 × 105, 1.0 × 105, 2.0 × 105, 4.0 × 105, and 6 × 105 cells mL-1) for 24 h, and the RCS decreased cell viabilities and promoted Neuro-2a (N2A) cell apoptosis in a dose-dependent manner. The underlying mechanism was further investigated in N2A cells. At the biochemical level, the RCS stimulated reactive oxygen species (ROS) and malondialdehyde (MDA) formation, decreased SOD activity, and reduced mitochondrial membrane potential (MMP). At the gene level, the moderate RCS treatment (2.0 × 105 cells mL-1) upregulated antioxidant response genes (e.g., nrf-2, HO-1, NQO-1, and cat) to alleviate RCS-induced oxidative stress, while the high RCS treatment (4.0 × 105 cells mL-1) downregulated these genes, thereby aggravating oxidative stress. Meanwhile, apoptosis-related genes (e.g., p53, caspase 3, and bax2) were significantly upregulated and the anti-apoptotic gene bcl2 was suppressed after RCS treatment. Western blotting results for Caspase 3, Bax2 and Bcl2 were consistent with the mRNA trends. These results revealed that K. mikimotoi RCS can induce neural cell apoptosis via the oxidative stress-mediated mitochondrial pathway, providing novel insights into the neurotoxicity of K. mikimotoi.

Variation in intracellular polyphosphate and associated gene expression in response to different phosphorus conditions in the dinoflagellate Karenia mikimotoi.

Polyphosphate (polyP) has long been recognized as a crucial intracellular reservoir for phosphorus in microorganisms. However, the dynamics of polyP and its regulatory mechanism in eukaryotic phytoplankton in response to variations in external phosphorus conditions remain poorly understood. A comprehensive investigation was conducted to examine the intracellular polyP-associated metabolic response of the dinoflagellate Karenia mikimotoi, a harmful algal bloom species, through integrated physiological, biochemical, and transcriptional analyses under varying external phosphorus conditions. Comparable growth curves and Fv/Fm between phosphorus-replete conditions and phosphorus-depleted conditions suggested that K. mikimotoi has a strong capability to mobilize the intracellular phosphorus pool for growth under phosphorus deficiency. Intracellular phosphate (IPi) and polyP contributed approximately 6-23 % and 1-3 %, respectively, to the overall particulate phosphorus (PP) content under different phosphorus conditions. The significant decrease in PP and increase in polyP:PP suggested that cellular phosphorus components other than polyP are preferred for utilization under phosphorus deficiency. Genes involved in polyP synthesis and hydrolysis were upregulated to maintain phosphorus homeostasis in K. mikimotoi. These findings provide novel insights into the specific cellular strategies for phosphorus storage and the transcriptional response in intracellular polyP metabolism in K. mikimotoi. Additionally, these results also indicate that polyP may not play a crucial role in cellular phosphorus storage in phytoplankton, at least in dinoflagellates.

Establishment of a New Model Sea Anemone for Comparative Studies on Cnidarian-Algal Symbiosis.

Frequent coral bleaching has drawn attention to the mechanisms of coral dinoflagellate endosymbiosis. Owing to the difficulty of rearing corals in the laboratory, model symbiosis systems are desired. The sea anemone Exaiptasia diaphana, hosting clade B1 of the genus Breviolum, has long been studied as a model system; however, a single species is insufficient for comparative studies and thus provides only limited resources for symbiosis research, especially regarding the specificity of host-symbiont associations. We established a clonal strain of the sea anemone Anthopleura atodai, whose symbiont was identified as a novel subclade of Symbiodinium (clade A) using a novel feeding method. We also developed a method to efficiently bleach various sea anemone species using a quinoclamine-based herbicide. Bleached A. atodai polyps were vital and able to reproduce asexually, exhibiting no signs of harmful effects of the drug treatment. Pilot studies have suggested that host-symbiont specificity is influenced by multiple steps differently in A. atodai and E. diaphana. RNAseq analyses of A. atodai showed that multiple NPC2 genes were expressed in the symbiotic state, which have been suggested to function in the transport of sterols from symbionts to host cells. These results reveal the usefulness of A. atodai in comparative studies of cnidarian-algal symbiosis.

Identification and genomic analysis of Pseudosulfitobacter koreense sp. nov. isolated from toxin-producing dinoflagellate Alexandrium pacificum.

The bacterial strain AP-MA-4T isolated from the marine dinoflagellate Alexandrium pacificum (KCTC AG60911), was subjected to a taxonomic analysis. Cells of strain AP-MA-4T were Gram-stain-negative, aerobic, rod-shaped, optimum growth at 20 °C, pH 7.0, in the presence of 5% (w/v) NaCl. Strain AP-MA-4T shared the highest 16S rRNA gene sequence similarity to Pseudosulfitobacter pseudonitzschiae DSM 26824T (98.5%), followed by Ascidiaceihabitans donghaensis RSS1-M3T (96.3%), Pseudoseohaeicola caenipelagi BS-W13T (95.7%), and Sulfitobacter pontiacus CHLG 10T (95.3%). Based on 16S rRNA phylogeny, strain AP-MA-4T is phylogenetically closely related to Pseudosulfitobacter pseudonitzschiae (type species of Pseudosulfitobacter) and could be distinguished from the type species based on their phenotypic properties. The genome length of strain AP-MA-4T was 3.48 Mbp with a 62.9% G + C content. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain AP-MA-4 T and its closely related type strains were 72.2-83.3 and 18.2-27.6%, respectively. Summed feature 8 (C18:1ω7c and/or C18:1ω6c) was identified the major fatty acids (> 10%). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and phospholipid (PL) were demonstrated as the major polar lipids. The major respiratory quinone is ubiquinone-10 (Q-10). Based on genotypic and phenotypic features, strain AP-MA-4T (= KCTC 92289T = GDMCC 1.3585T) represents a new Pseudosulfitobacter species, in which the name Pseudosulfitobacter koreense sp. nov. is proposed.

Metabarcoding revealed a high diversity of Amphidomataceae (Dinophyceae) and the seasonal distribution of their toxigenic species in the Taiwan Strait.

The dinophyte family Amphidomataceae includes the genera Azadinium and Amphidoma. Four of these species are known to produce azaspiracids, which are lipophilic phycotoxins accumulating in shellfish. The diversity and biogeography of Amphidomataceae is far from yet resolved. Here we performed a time series sampling of both water and sediments in the Taiwan Strait from Nov. 2018 to April 2021. Metabarcoding was performed to unveil the diversity of Amphidomataceae targeting internal transcribed spacer (ITS1) region and partial large subunit ribosomal DNA (LSU rDNA D1-D3), followed by quantitative PCR (qPCR) with modified primers for Az. poporum ribotypes. The diversity of Amphidomataceae was revealed from the water samples with the aid of ITS1 and LSU based molecular phylogeny. The LSU based approach detected only a few species. In contrast, ITS1 based dataset showed eight new Azadinium clades and several ZOTUs (zero-radius operational taxonomic units) grouping together with Am. languida. Moreover, eleven known Azadinium species including three ribotypes of Az. poporum and Az. dexteroporum, and two ribotypes of Az. spinosum, were detected. The latter two species have not been reported in China before. Among these toxigenic species, Az. poporum was relevantly abundant whereas others were rare. The maximum of 209 cells L -1 of Az. poporum ribotype A was estimated using qPCR nearby Quanzhou in Nov. 2018 and 172 cells L 1 of Az. poporum ribotype B was detected far off coast in Apr. 2021. Metabarcoding on sediment samples revealed Az. poporum ribotypes B and C, but strains obtained with sediment incubation experiments yielded only ribotype B. Using qPCR about 0.2 cysts g -1 of Az. poporum ribotype B were quantified in May 2019 but cysts of Az. poporum ribotype C were not detected. Our results suggest that metabarcoding targeting ITS1 region is powerful to uncover the diversity of harmful dinophytes. Our results also highlight the rich diversity of Amphidomataceae and risk potential of azaspiracids in the Taiwan Strait and surrounding waters.

Morphology and molecular phylogeny of the benthic dinoflagellates (Dinophyceae, Peridiniales) Amphidiniopsis crumena n. sp. and Amphidiniopsis nileribanjensis n. sp.

Amphidiniopsis is a benthic, heterotrophic and thecate dinoflagellate genus that has a smaller epitheca and larger hypotheca. The genus contains 24 described species, but is considered to be polyphyletic based on morphological characters and molecular phylogenetics. In this study, two new species were discovered from two distant sampling localities, Amphidiniopsis crumena sp. nov. from Japan, and Amphidiniopsis nileribanjensis sp. nov., from Australia. These species have a uniquely shaped, additional second postcingular plate. Both species are dorsoventrally flattened, an apical hook is present, and have six postcingular plates. The plate formula is: APC 4' 3a 7″ ?C 4?S 6″' 2″″. The cells of these species were examined with LM and SEM, and molecular phylogenic analyses were performed using 18S and 28S rDNA. These species are distinguished by the presence of spines on the hypotheca and touching of the sixth postcingular plate and the anterior sulcal plate. Their shape and disposition of several thecal plates also differ. Molecular phylogenetic analyses showed that the two new species formed a monophyletic clade and did not belong to any morphogroup proposed by previous studies. Considering the morphological features and the molecular phylogenetic results, a new morphogroup is proposed, Amphidiniopsis morphogroup VI ('crumena group').

Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements.

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent. In this study, using the cosmopolitan HABs-causing species Scrippsiella acuminata as a representative, we measured the transcriptional activity of the most efficient pathway of the energy catabolism tricarboxylic acid (TCA) cycle, cell viability (via neutral red staining), and the cellular ATP content of resting cysts under a set of mock conditions in marine sediments (e.g., 4 °C, darkness, and anoxia) for a maximum period of one year. Based on the correlation analyses among the expression levels of genes, cyst viability, and ATP content, we revealed that the TCA cycle was still a crucial pathway of energetic catabolism for resting cysts under aerobic conditions, and its expression was elevated at higher temperatures, light irradiation, and the early stage of dormancy. Under anaerobic conditions, however, the TCA cycle pathway ceased expression in resting cysts, as also supported by ATP measurements. Our results have laid a cornerstone for the comprehensive revelation of the energetic metabolism and biochemical processes of dormancy of resting cysts in marine sediments.

Active meiosis during dinoflagellate blooms: A 'sex for proliferation' hypothesis.

In dinoflagellates, sexual reproduction is best known to be induced by adverse environmental conditions and culminate in encystment for survival ('sex for encystment'). Although increasing laboratory observations indicate that sex can lead to production of vegetative cells bypassing encystment, the occurrence of this alternative pathway in natural populations and its ecological roles remain poorly understood. Here we report evidence that sex in dinoflagellates can potentially be an instrument for bloom proliferation or extension. By bloom metatranscriptome profiling, we documented elevated expression of meiosis genes in two evolutionarily distinct species (Prorocentrum shikokuense and Karenia mikimotoi) during bloom, a timing unexpected of the 'sex for encystment' scenario. To link these genes to meiosis, we induced encystment and cyst germination in the cyst-forming species Scrippsiella acuminata, and found that five of these genes were upregulated during cyst germination, when meiosis occurs. Integrating data from all three species revealed that SPO11, MND1, and DMC1 were likely common between cyst-forming and non-encysting sex in dinoflagellates. Furthermore, flow cytometric analyses revealed consecutive rounds of DNA halving during blooms of P. shikokuense and K. mikimotoi, evidencing meiosis. These data provided novel evidence that sexual reproduction in dinoflagellates might serve to promote cell proliferation, and along with the consequent enhancement of genetic diversity facilitating resistance against pathogens and environmental stress, to boost or extend a bloom ('sex for proliferation'). The putative meiosis-specific genes and insights reported here will prove to be helpful for rigorously testing the hypothesis and addressing whether the two modes of sex are genetically predisposed (i.e. species-specific) or environmentally induced (switchable within species), and if the latter what triggers the switch.

The coral Acropora loripes genome reveals an alternative pathway for cysteine biosynthesis in animals.

The metabolic capabilities of animals have been derived from well-studied model organisms and are generally considered to be well understood. In animals, cysteine is an important amino acid thought to be exclusively synthesized through the transsulfuration pathway. Corals of the genus Acropora have lost cystathionine ß-synthase, a key enzyme of the transsulfuration pathway, and it was proposed that Acropora relies on the symbiosis with dinoflagellates of the family Symbiodiniaceae for the acquisition of cysteine. Here, we identify the existence of an alternative pathway for cysteine biosynthesis in animals through the analysis of the genome of the coral Acropora loripes. We demonstrate that these coral proteins are functional and synthesize cysteine in vivo, exhibiting previously unrecognized metabolic capabilities of animals. This pathway is also present in most animals but absent in mammals, arthropods, and nematodes, precisely the groups where most of the animal model organisms belong to, highlighting the risks of generalizing findings from model organisms.

Coral larvae suppress heat stress response during the onset of symbiosis decreasing their odds of survival.

The endosymbiosis between most corals and their photosynthetic dinoflagellate partners begins early in the host life history, when corals are larvae or juvenile polyps. The capacity of coral larvae to buffer climate-induced stress while in the process of symbiont acquisition could come with physiological trade-offs that alter behaviour, development, settlement and survivorship. Here we examined the joint effects of thermal stress and symbiosis onset on colonization dynamics, survival, metamorphosis and host gene expression of Acropora digitifera larvae. We found that thermal stress decreased symbiont colonization of hosts by 50% and symbiont density by 98.5% over 2 weeks. Temperature and colonization also influenced larval survival and metamorphosis in an additive manner, where colonized larvae fared worse or prematurely metamorphosed more often than noncolonized larvae under thermal stress. Transcriptomic responses to colonization and thermal stress treatments were largely independent, while the interaction of these treatments revealed contrasting expression profiles of genes that function in the stress response, immunity, inflammation and cell cycle regulation. The combined treatment either cancelled or lowered the magnitude of expression of heat-stress responsive genes in the presence of symbionts, revealing a physiological cost to acquiring symbionts at the larval stage with elevated temperatures. In addition, host immune suppression, a hallmark of symbiosis onset under ambient temperature, turned to immune activation under heat stress. Thus, by integrating the physical environment and biotic pressures that mediate presettlement event in corals, our results suggest that colonization may hinder larval survival and recruitment under projected climate scenarios.

Transcriptomic response of the harmful algae Heterosigma akashiwo to polyphosphate utilization and phosphate stress.

Phosphorus (P) is one of the major macronutrients necessary for phytoplankton growth. In some parts of the ocean, however, P is frequently scarce, hence, there is limited phytoplankton growth. To cope with P deficiency, phytoplankton evolved a variety of strategies, including, utilization of different P sources. Polyphosphate (polyP) is ubiquitously present and serves an essential function in aquatic environments, but it is unclear if and how this polymer is utilized by phytoplankton. Here, we examined the physiological and molecular responses of the widely present harmful algal bloom (HAB) species, Heterosigma akashiwo in polyP utilization, and in coping with P-deficiency. Our results revealed that two forms of inorganic polyP, namely, sodium tripolyphosphate and sodium hexametaphosphate, support H. akashiwo growth as efficiently as orthophosphate. However, few genes involved in polyP utilization have been identified. Under P-deficient conditions, genes associated with P transport, dissolved organic P utilization, sulfolipid synthesis, and energy production, were markedly elevated. In summary, our results indicate that polyP is bioavailable to H. akashiwo, and this HAB species have evolved a comprehensive strategy to cope with P deficiency.

Energy metabolism and genetic information processing mark major transitions in the life history of Scrippsiella acuminata (Dinophyceae).

Many dinoflagellates perform sexual reproduction and form cysts as a life history strategy to survive adverse environmental conditions and seed annual harmful algal blooms (HABs). The molecular mechanisms underpinning the life stage transitions can provide clues about how key environmental factors induce encystment and initiation of a HAB but are still poorly understood. Here, we conducted an integrated physiological and transcriptomic study to unravel the mechanisms in Scrippsiella acuminata. We established a culture from a bloom, induced cyst formation, and divided the process into four life stages. Transcriptomic analysis of these stages revealed 19,900 differentially expressed genes (DEGs). The expression of genes related to photosynthesis was significantly up-regulated from vegetative stage to immature cyst stage, consistent with the marked increase in cell contents of energy-storing macromolecules (carbohydrates and lipids). When proceeding to resting cysts, most photosynthesis genes were down-regulated while "genetic information processing" related genes were up-regulated. Comparing germinating cysts with resting cysts revealed 100 DEGs involved in energy metabolism, indicating a high energy requirement of germination. In addition, the transition from germinating cysts to vegetative cells featured up-regulation of photosynthesis. Our results demonstrate that energy storage and consumption play a pivotal role in cyst formation and germination respectively and genetic information processing is crucial in cyst dormancy.

On the Hunt for New Toxin Families Produced by a Mediterranean Strain of the Benthic Dinoflagellate Ostreopsis cf. ovata.

Ostreopsis cf. ovata is a benthic dinoflagellate known to produce palytoxin (PLTX) and its analogues. Recent investigations suggested the production of unknown toxins by a Mediterranean strain. In the present work, two new families of toxins, potentially novel in their structures, were purified from this same Mediterranean strain of Ostreopsis cf. ovata. The low amount of material isolated only allowed for acquisition of high-resolution mass spectrometry data and the evaluation of their cytotoxicity to human lung cancer cells. Based on their HRMS data, none of these new compounds appear to be close PLTX analogues, although their mass spectra suggest poly-hydroxylated long chain compounds of high molecular weight (1370-2143 Da). The cell cytotoxicity concentrations (CC50) of these new purified toxins ranged between 0.68 and 3.12 µg/mL, and this was enhanced when they were tested as mixtures, suggesting synergistic effects of Ostreopsis toxins. The two families of compounds were named the liguriatoxins (LGTX) and rivieratoxins (RVTX), with each family containing three members. Additional work on purification is needed to fully characterize the structures of these six new dinoflagellate toxins.

Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves.

The purpose of this study was to survey the eukaryotic microbiome of two karst caves in the Valley and Ridge physiographic region of the Appalachian Mountains. Caves are known to harbour eukaryotic microbes but their very low densities and small cell size make them difficult to collect and identify. Microeukaryotes were surveyed using two methodologies, filtering water and submerging glass microscope slides mounted in periphytometers in cave pools. The periphyton sampling yielded 13.5 times more unique amplicon sequence variants (ASVs) than filtered water. The most abundant protist supergroup was Alveolata with large proportions of the ASVs belonging to dinoflagellate, ciliate and apicomplexan clades. The next most abundant were Rhizarians followed by Stramenopiles (diatoms and chrysophytes) and Ameobozoans. Very few of the ASVs, 1.5%, matched curated protist sequences with greater than 99% identity and only 2.5% could be identified from surface plankton samples collected in the same region. The overall composition of the eukaryotic microbiome appears to be a combination of bacterial grazers and parasitic species that could possibly survive underground as well as cells, cysts and spores probably transported from the surface.

Unraveling the Gonyaulax baltica Species Complex: Cyst-theca Relationship of Impagidinium variaseptum, Spiniferites pseudodelicatus sp. nov. and S. ristingensis (Gonyaulacaceae, Dinophyceae), With Descriptions of Gonyaulax bohaiensis sp. nov, G. amoyensis sp. nov. and G. portimonensis sp. nov.

The taxonomy of the extant dinoflagellate genus Gonyaulax is challenging since its thecate morphology is rather conservative. In contrast, cysts of Gonyaulax are varied in morphology and have been related with the fossil-based genera Spiniferites and Impagidinium. To better understand the systematics of Gonyaulax species, we performed germination experiments on cysts that can be identified as S. ristingensis, an unidentified Spiniferites with petaloid processes here described as Spiniferites pseudodelicatus sp. nov. and Impagidinium variaseptum from Chinese and Portuguese waters. Despite marked differences in cyst morphology, motile cells of S. pseudodelicatus and I. variaseptum are indistinguishable from Gonyaulax baltica. Motile cells hatched from S. ristingensis are morphologically similar to G. baltica as well but differ in the presence of one pronounced antapical spine. Three new species, Gonyaulax amoyensis (cyst equivalent S. pseudodelicatus), Gonyaulax bohaiensis (cyst equivalent I. variaseptum), and Gonyaulax portimonensis (cyst equivalent S. ristingensis), were erected. In addition, a new ribotype (B) of G. baltica was reported from South Korea and a bloom of G. baltica ribotype B is reported from New Zealand. Molecular phylogeny based on LSU and SSU rRNA gene sequences revealed that Gonyaulax species with minute or short antapical spines formed a well-resolved clade, whereas species with two pronounced antapical spines or lack of antapical spines formed the sister clade. Six strains of four above species were examined for yessotoxin production by liquid chromatography coupled with tandem mass spectrometry, and very low concentrations of yessotoxin were detected for one G. bohaiensis strain.

Gymnodinialimonas ceratoperidinii gen. nov., sp. nov., isolated from rare marine dinoflagellate Ceratoperidinium margalefii.

A bacterial strain, designated J12C1-MA-4T, was isolated from liquid culture of the dinoflagellate Ceratoperidinium margalefii. The bacterium was Gram-negative, aerobic, and rod-shaped. Oxidase and catalase were positive. Optimal growth was observed at 30 °C, pH 7.0, in the presence of 1% (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene and a 92 core gene set suggested that the strain J12C1-MA-4T belongs to the family Rhodobacteraceae in the class Alphaproteobacteria and represents a taxon separated from other genera. 16S rRNA gene sequence of the strain J12C1-MA-4T showed high similarities to Loktanella ponticola KCTC 42133T (95.7%), Pseudooctadecabacter jejudonensis KCTC 32525T (95.5%) and Jannaschia helgolandensis KCTC 12191T (95.3%). The genome length of strain J12C1-MA-4T was 3,621,968 bp with a DNA G + C content of 64.48 mol%. The major cellular fatty acids of strain J12C1-MA-4T were summed feature 8 (comprising C18:1ω7c and/or C18:1ω6c) (> 10%). Phosphatidylglycerol (PG), phosphatidylcholine (PC), phospholipids (PL), lipids 1 (L1) and aminolipid (AL) were shown to be the major polar lipids. The sole predominant isoprenoid quinone was Q-10. Based on phylogenetic, phenotypic, chemotaxonomic and genomic features, we propose that strain J12C1-MA-4T represent a novel species in the novel genus of the family Rhodobacteraceae, with the proposed name Gymnodinialimonas ceratoperidinii gen. nov., sp. nov.. The type strain is J12C1-MA-4T (=KCTC 82770T =GDMCC 1.2729T).

Coral symbionts evolved a functional polycistronic flavodiiron gene.

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.