Microtubule reorganization during mitotic cell division in the dinoflagellate Ostreospis cf. ovata.

Dinoflagellates are marine organisms that undergo seasonal proliferation events known as algal blooms. Vegetative cell proliferation is a main contributing factor in these events. However, mechanistical understanding of mitosis and cytokinesis in dinoflagellates remains rudimentary. Using an optimized immunofluorescence protocol, we analysed changes in microtubule organization occurring during the mitotic cycle of the toxic dinoflagellate Ostreopsis cf. ovata. We find that the flagella and the cortical microtubule array persist throughout the mitotic cycle. Two cytoplasmic microtubule bundles originate from the ventral area, where the basal bodies are located - a cortical bundle and a cytoplasmic bundle. The latter associates with the nucleus in the cell centre before mitosis and with the acentrosomal extranuclear spindle during mitosis. Analysis of tubulin post-translational modifications identifies two populations of spindle microtubules - polar acetylated microtubules, whose length is constant, and central tyrosinated microtubules, which elongate during chromosome segregation. During cell division a microtubule-rich structure forms along the dorsal-ventral axis, associated with the site of cytokinesis, consistent with a cytokinetic mechanism that is independent of the actomyosin ring typical of animal and yeast cells.

Fensomea setacea, gen. & sp. nov. (Cladopyxidaceae, Dinophyceae), is neither gonyaulacoid nor peridinioid as inferred from morphological and molecular data.

Dinophyte evolution is essentially inferred from the pattern of thecal plates, and two different labelling systems are used for the important subgroups Gonyaulacales and Peridiniales. The partiform hypotheca of cladopyxidoid dinophytes fits into the morphological concepts of neither group, although they are assigned to the Gonyaulacales. Here, we describe the thecate dinophyte Fensomea setacea, gen. & sp. nov., which has a cladopyxidoid tabulation. The cells displayed a Kofoidean plate formula APC, 3', 4a, 7″, 7C, 6S, 6''', 2'''', and slender processes were randomly distributed over the echinate or baculate surface. In addition, we obtained rRNA sequences of F. setacea, gen. & sp. nov., but dinophytes that exhibit a partiform hypotheca did not show a close relationship to Gonyaulacales. Character evolution of thecate dinophytes may have progressed from the ancestral state of six postcingular plates, and two more or less symmetrically arranged antapical plates, towards patterns of only five postcingular plates (Peridiniales) or more asymmetrical configurations (Gonyaulacales). Based on our phylogenetic reconsiderations the contact between the posterior sulcal plate and the first postcingular plate, as well as the contact between an antapical plate and the distalmost postcingular plate, do not represent a rare, specialized gonyaulacoid plate configuration (i.e., the partiform hypotheca of cladopyxidoid dinophytes). Instead, these contacts correspond to the common and regular configuration of peridinioid (and other) dinophytes.

N-Acetyl-d-Glucosamine-Binding Lectin in Acropora tenuis Attracts Specific Symbiodiniaceae Cell Culture Strains.

Many corals establish symbiosis with Symbiodiniaceae cells from surrounding environments, but very few Symbiodiniaceae cells exist in the water column. Given that the N-acetyl-d-glucosamine-binding lectin ActL attracts Symbiodiniaceae cells, we hypothesized that corals must attract Symbiodiniaceae cells using ActL to acquire them. Anti-ActL antibody inhibited acquisition of Symbiodiniaceae cells, and rearing seawater for juvenile Acropora tenuis contained ActL, suggesting that juvenile A. tenuis discharge ActL to attract these cells. Among eight Symbiodiniaceae cultured strains, ActL attracted NBRC102920 (Symbiodinium tridacnidorum) most strongly followed by CS-161 (Symbiodinium tridacnidorum), CCMP2556 (Durusdinium trenchii), and CCMP1633 (Breviolum sp.); however, it did not attract GTP-A6-Sy (Symbiodinium natans), CCMP421 (Effrenium voratum), FKM0207 (Fugacium sp.), and CS-156 (Fugacium sp.). Juvenile polyps of A. tenuis acquired limited Symbiodiniaceae cell strains, and the number of acquired Symbiodiniaceae cells in a polyp also differed from each other. The number of Symbiodiniaceae cells acquired by juvenile polyps of A. tenuis was correlated with the ActL chemotactic activity. Thus, ActL could be used to attract select Symbiodiniaceae cells and help Symbiodiniaceae cell acquisition in juvenile polyps of A. tenuis, facilitating establishment of symbiosis between A. tenuis and Symbiodiniaceae cells.

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp.

BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

Lens eyes in protists.

Eyes are not unique to animals. As described by Nilsson and Marshall, prominent eyes, complete with retina and lens, have unexpectedly evolved in single cell dinoflagellates.

Host and Symbiont Cell Cycle Coordination Is Mediated by Symbiotic State, Nutrition, and Partner Identity in a Model Cnidarian-Dinoflagellate Symbiosis.

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia (Exaiptasia pallida) and its native microalgal photosymbionts (Breviolum minutum and Breviolum psygmophilum). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G2/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate.IMPORTANCE Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.

Transcriptomic responses of Artemia salina exposed to an environmentally relevant dose of Alexandrium minutum cells or Gonyautoxin2/3.

Toxicities of the marine algae Alexandrium minutum and its excreted gonyautoxins (GTXs) to the marine crustacean Artemia salina were investigated. Mortality was observed for neither larvae nor adult A. salina exposed to A. minutum at a density of 5000 cells/mL or 0.5 µM GTX2/3. After exposure, the full transcriptome of adult A. salina was assembled and functionally annotated. A total of 599,286 transcripts were obtained, which were clustered into 515,196 unigenes. Results of the transcriptional effect level index revealed that direct exposure to the toxic algae A. minutum caused greater alterations in the transcriptome than did exposure to the extracellular product GTX2/3. Mechanisms of effects were different between exposure of A. salina to A. minutum cells or GTX2/3. Exposure to A. minutum modulated formation of the ribonucleoprotein complex and metabolism of amino acids and lipids in A. salina. Exposure to GTX2/3 exposure inhibited expression of genes related to metabolism of chitin, which might result in disruption of molting process or disturbed sheath morphogenesis. Overall, effects on transcription observed in this study represent the first report based on application of next generation sequencing techniques to investigate the transcriptomic response of A. salina exposed to an environmentally realistic level of A. minutum or GTX2/3.

Morphology and Phylogenetics of Benthic Prorocentrum Species (Dinophyceae) from Tropical Northwestern Australia.

Approximately 70 species of Prorocentrum are known, of which around 30 species are associated with benthic habitats. Some produce okadaic acid (OA), dinophysistoxin (DTX) and their derivatives, which are involved in diarrhetic shellfish poisoning. In this study, we isolated and characterized Prorocentrum concavum and P. malayense from Broome in north Western Australia using light and scanning electron microscopy as well as molecular sequences of large subunit regions of ribosomal DNA, marking the first record of these species from Australian waters. The morphology of the motile cells of P. malayense was similar to P. concavum in the light microscopy, but differed by the smooth thecal surface, the pore pattern and the production of mucous stalk-like structures and a hyaline sheath around the non-motile cells. P. malayense could also be differentiated from other closely related species, P. leve and P. foraminosum, despite the similarity in thecal surface and pore pattern, by its platelet formula and morphologies. We tested the production of OA and DTXs from both species, but found that they did not produce detectable levels of these toxins in the given culturing conditions. This study aids in establishing more effective monitoring of potential harmful algal taxa in Australian waters for aquaculture and recreational purposes.

New morphological data and molecular phylogeny of the benthic dinoflagellate Pseudothecadinium campbellii (Dinophyceae, Gonyaulacales).

Pseudothecadinium campbellii, a phototrophic, thecate, marine benthic species, has been found in the Sea of Japan, Russia. The morphological description of the species has been emended, and the thecal tabulation pattern is now APC 4' 2a 4″ 6c 6 s 5‴ 1⁗. Our study indicates that P. campbellii is related to Thecadinium kofoidii and Thecadiniopsis tasmanica, based on a unique morphological feature: incomplete precingular plate series. Previously, molecular data was not available for the taxa, and thus the phylogenetic position of P. campbellii within the Dinophyceae remained obscure. In this study, analyses of the rRNA gene sequences (partial 18S and 28S) revealed that unexpectedly, P. campbellii is most closely related to Halostylodinium arenarium. It formed a common clade with the Thecadinium sensu stricto clade comprising T. kofoidii and T. pseudokofoidii. This clade was placed within the order Gonyaulacales. However, almost no similarity in morphology was found between the two genetically closest species. In addition, they have different lifestyles: unlike P. campbellii, the nonmotile stage is dominant in the life cycle of H. arenarium. It has been shown that other genetically similar species (T. kofoidii, T. pseudokofoidii, T. yashimaense) exhibited some morphological features that unite them with gonyaulacoids.

Gertia stigmatica gen. et sp. nov. (Kareniaceae, Dinophyceae), a New Marine Unarmored Dinoflagellate Possessing the Peridinin-type Chloroplast with an Eyespot.

Marine unarmored dinoflagellates in the family Kareniaceae are known to possess chloroplasts of haptophyte origin, which contain fucoxanthin and its derivatives as major carotenoids, and lack peridinin. In the present study, the first species with the peridinin-type chloroplast in this family, Gertia stigmatica gen. et sp. nov., is described on the basis of ultrastructure, photosynthetic pigment composition, and molecular phylogeny inferred from nucleus- and chloroplast-encoded genes. Cells of G. stigmatica were small and harboring a chloroplast with an eyespot and two pyrenoids. The apical structure complex was straight, similar to Karenia and Karlodinium. Under transmission electron microscopy, the chloroplast was surrounded by two membranes, and the eyespot was composed of a single layer of osmiophilic globules (eyespot type A); this was never previously reported from the Kareniaceae. High performance liquid chromatography demonstrated the chloroplast contains peridinin, and neither fucoxanthin nor 19'-acyloxyfucoxanthins was identified. A phylogeny based on nucleus-encoded rDNAs suggested a position of G. stigmatica in the Kareniaceae, but not clustered within the previously described genera, i.e., Karenia, Karlodinium and Takayama. A phylogeny of chloroplast-encoded psbA, psbC and psbD indicated the chloroplast is of peridinin-type typical of dinoflagellates, but the most related species remains unclear.

Acidification-induced cellular changes in Symbiodinium isolated from Mussismilia braziliensis.

Dinoflagellates from the Symbiodiniaceae family and corals have an ecologically important endosymbiotic relationship. Scleractinian corals cannot survive for long periods without their symbionts. These algae, also known as zooxanthellae, on the other hand, thrives outside the coral cells. The free-living populations of zooxanthellae are essential for the resilience of the coral to environmental stressors such as temperature anomalies and ocean acidification. Yet, little is known about how ocean acidification may affect the free-living zooxanthellae. In this study we aimed to test morphological, physiological and biochemical responses of zooxanthellae from the Symbiodinium genus isolated from the coral Mussismilia braziliensis, endemic to the Brazilian coast, to acidification led by increased atmospheric CO2. We tested whether photosynthetic yield, cell ultrastructure, cell density and lipid profile would change after up to 16 days of exposure to pH 7.5 in an atmospheric pCO2 of 1633 µatm. Photosynthetic yield and cell density were negatively affected and chloroplasts showed vesiculated thylakoids, indicating morphological damage. Moreover, Symbiodinium fatty acid profile drastically changed in acidified condition, showing lower polyunsaturated fatty acids and higher saturated fatty acids contents, when compared to the control, non-acidified condition. These results show that seawater acidification as an only stressor causes significant changes in the physiology, biochemistry and ultrastructure of free-living Symbiodinium.

Cryopreservation of a Thermotolerant Lineage of the Coral Reef Dinoflagellate Symbiodinium.

Dinoflagellates of the genus Symbiodinium form symbiotic relationships with corals, other marine invertebrates, and protists; thus, they are considered as important species in coral reef ecosystems. If Symbiodinium could be successfully cryopreserved, the cell bank generated could prove to be a valuable resource for researchers interested in basic biological research of Symbiodinium-invertebrate symbioses. Herein, successful cryopreservation of clade D Symbiodinium was achieved using a two-step freezing protocol. Symbiodinium cells were exposed to cryoprotectants (CPAs) for 30 minutes before being vapor frozen for 20 minutes in liquid nitrogen (LN2); afterward, cells were immediately immersed in LN2 for 2 hours or 10 days. The initial experiment was conducted with the following CPAs at 1, 2, and 3 M concentrations: methanol, dimethyl sulfoxide, glycerol, ethylene glycol (EG), and propylene glycol (PG). It was found that infiltration with 2 M EG and PG yielded cells with the highest percentage viability. Upon thawing, culture of these Symbiodinium was carried out for 2 months in a growth chamber, and cells continued to grow and proliferate over this period. This represents successful cryopreservation of a dominant reef coral symbiont, a feat that will ideally aid in future research of this important lineage of dinoflagellate.

A widespread coral-infecting apicomplexan with chlorophyll biosynthesis genes.

Apicomplexa is a group of obligate intracellular parasites that includes the causative agents of human diseases such as malaria and toxoplasmosis. Apicomplexans evolved from free-living phototrophic ancestors, but how this transition to parasitism occurred remains unknown. One potential clue lies in coral reefs, of which environmental DNA surveys have uncovered several lineages of uncharacterized basally branching apicomplexans1,2. Reef-building corals have a well-studied symbiotic relationship with photosynthetic Symbiodiniaceae dinoflagellates (for example, Symbiodinium3), but the identification of other key microbial symbionts of corals has proven to be challenging4,5. Here we use community surveys, genomics and microscopy analyses to identify an apicomplexan lineage-which we informally name 'corallicolids'-that was found at a high prevalence (over 80% of samples, 70% of genera) across all major groups of corals. Corallicolids were the second most abundant coral-associated microeukaryotes after the Symbiodiniaceae, and are therefore core members of the coral microbiome. In situ fluorescence and electron microscopy confirmed that corallicolids live intracellularly within the tissues of the coral gastric cavity, and that they possess apicomplexan ultrastructural features. We sequenced the genome of the corallicolid plastid, which lacked all genes for photosystem proteins; this indicates that corallicolids probably contain a non-photosynthetic plastid (an apicoplast6). However, the corallicolid plastid differs from all other known apicoplasts because it retains the four ancestral genes that are involved in chlorophyll biosynthesis. Corallicolids thus share characteristics with both their parasitic and their free-living relatives, which suggests that they are evolutionary intermediates and implies the existence of a unique biochemistry during the transition from phototrophy to parasitism.

On the morphology and predatory behavior of the dinoflagellate Oxyrrhis marina exposed to reduced salinity.

Changes in salinity are known to alter the morphology of protists, and we hypothesized that these changes subsequently alter also the predatory behavior of the dinoflagellate Oxyrrhis marina. Oxyrrhis was grown in media of 33, 25, 20, and 10% of the regular salinity of f/2 medium (31-32‰). In all cases, the cells discharged trichocysts and swelled. Cell surfaces and volumes increased with decreasing salinity, such that cell surface area at least doubled at 10% and the cell volume increased approximately fourfold. After 1 h, the cells started to regain their regular shape, which was almost completed after 24 h. Oxyrrhis immediately regained its regular shape when culture medium was added 5-10 min after the osmotic stress. When incubated with Pyramimonas grossii as prey, those short-term stressed cells showed no significant different prey uptake in comparison to non-stressed cells. In contrast, 24 h after the addition of prey, short-term stressed Oxyrrhis cells had, with weak statistical significance, more Pyramimonas cells engulfed than non-stressed cells. These results indicated that (1) trichocysts were most likely not involved in prey capture and (2) salinity-stressed Oxyrrhis either enhanced its capability to capture more prey, or its digestion apparatus was hampered.

Tuberlatum coatsi gen. n., sp. n. (Alveolata, Perkinsozoa), a New Parasitoid with Short Germ Tubes Infecting Marine Dinoflagellates.

Perkinsozoa is an exclusively parasitic group within the alveolates and infections have been reported from various organisms, including marine shellfish, marine dinoflagellates, freshwater cryptophytes, and tadpoles. Despite its high abundance and great genetic diversity revealed by recent environmental rDNA sequencing studies, Perkinsozoa biodiversity remains poorly understood. During the intensive samplings in Korean coastal waters during June 2017, a new parasitoid of dinoflagellates was detected and was successfully established in culture. The new parasitoid was most characterized by the presence of two to four dome-shaped, short germ tubes in the sporangium. The opened germ tubes were biconvex lens-shaped in the top view and were characterized by numerous wrinkles around their openings. Phylogenetic analyses based on the concatenated SSU and LSU rDNA sequences revealed that the new parasitoid was included in the family Parviluciferaceae, in which all members were comprised of two separate clades, one containing Parvilucifera species (P. infectans, P. corolla, and P. rostrata), and the other containing Dinovorax pyriformis, Snorkelia spp., and the new parasitoid from this study. Based on morphological, ultrastructural, and molecular data, we propose to erect a new genus and species, Tuberlatum coatsi gen. n., sp. n., from the new parasitoid found in this study. Further, we examined and discussed the validity of some diagnostic characteristics reported for parasitoids in the family Parviluciferaceae at both the genus and species levels.

First Report of Okadaic Acid and Pectenotoxins in Individual Cells of Dinophysis and in Scallops Argopecten purpuratus from Perú.

Causative species of Harmful Algal Bloom (HAB) and toxins in commercially exploited molluscan shellfish species are monitored weekly from four classified shellfish production areas in Perú (three in the north and one in the south). Okadaic acid (OA) and pectenotoxins (PTXs) were detected in hand-picked cells of Dinophysis (D. acuminata-complex and D. caudata) and in scallops (Argopecten purpuratus), the most important commercial bivalve species in Perú. LC-MS analyses revealed two different toxin profiles associated with species of the D. acuminata-complex: (a) one with OA (0.3⁻8.0 pg cell-1) and PTX2 (1.5⁻11.1 pg cell-1) and (b) another with only PTX2 which included populations with different toxin cell quota (9.3⁻9.6 pg cell-1 and 5.8⁻9.2 pg cell-1). Toxin results suggest the likely presence of two morphotypes of the D. acuminata-complex in the north, and only one of them in the south. Likewise, shellfish toxin analyses revealed the presence of PTX2 in all samples (10.3⁻34.8 µg kg-1), but OA (7.7⁻15.2 µg kg-1) only in the northern samples. Toxin levels were below the regulatory limits established for diarrhetic shellfish poisoning (DSP) and PTXs (160 µg OA kg-1) in Perú, in all samples analyzed. This is the first report confirming the presence of OA and PTX in Dinophysis cells and in shellfish from Peruvian coastal waters.

Gonyaulax whaseongensis sp. nov. (Gonyaulacales, Dinophyceae), a new phototrophic species from Korean coastal waters.

The planktonic phototrophic dinoflagellate Gonyaulax whaseongensis sp. nov., isolated from coastal waters of western Korea, was described from living and fixed cells under light and scanning electron microscopy, and its rDNA was sequenced. Gonyaulax whaseongensis had a plate formula of 2pr, 4', 6'', 6c, 6''', 1p, and 1'''' with S-type ventral organization like the other species in the genus. However, this dinoflagellate had a narrow cingulum (ca. 2.6 µm), small displacement of the cingulum, slight overhang and steep angle between the ends of the cingulum, quadrangular sixth precingular plate, reticulated cell surface without longitudinal lines or ridges, and two unequal antapical spines, together which distinguish this from all other reported Gonyaulax species. In addition, the SSU and LSU rDNA sequences were 8%-12% and 11%-24%, respectively, different from those of Gonyaulax polygramma, Gonyaulax spinifera, Gonyaulax fragilis, Gonyaulax membranacea, and Gonyaulax digitale, the putatively closest related species in the phylogenetic analysis.

The toxic benthic dinoflagellate Prorocentrum maculosum Faust is a synonym of Prorocentrum hoffmannianum Faust.

Three strains of the toxic benthic dinoflagellate Prorocentrum hoffmannianum were isolated in the Canary Islands (north-east Atlantic Ocean, Spain). The identity of the strains was determined by phylogenetic analyses of partial LSU rDNA (D1-D2 regions) but their morphology based on SEM images corresponded to P. maculosum. Their toxin profiles were analyzed by liquid chromatography and high resolution mass spectrometry analysis (LC-HRMS) on cell extracts and culture media. Okadaic acid and three analogs were detected in all strains. Rather, in culture media the detected compounds were variable among strains, two of them being okadaic acid analogs not found on cell extracts. As a result, the taxonomy of the species was revised and P. maculosum is proposed as a junior synonym of P. hoffmannianum whose description is emended.

Morphology, ultrastructure, and molecular phylogeny of Wangodinium sinense gen. et sp. nov. (Gymnodiniales, Dinophyceae) and revisiting of Gymnodinium dorsalisulcum and Gymnodinium impudicum.

The genus Gymnodinium includes many morphologically similar species, but molecular phylogenies show that it is polyphyletic. Eight strains of Gymnodinium impudicum, Gymnodinium dorsalisulcum and a novel Gymnodinium-like species from Chinese and Malaysian waters and the Mediterranean Sea were established. All of these strains were examined with light microscopy, scanning electron microscopy and transmission electron microscopy. SSU, LSU and internal transcribed spacers rDNA sequences were obtained. A new genus, Wangodinium, was erected to incorporate strains with a loop-shaped apical structure complex (ASC) comprising two rows of amphiesmal vesicles, here referred to as a new type of ASC. The chloroplasts of Wangodinium sinense are enveloped by two membranes. Pigment analysis shows that peridinin is the main accessory pigment in W. sinense. Wangodinium differs from other genera mainly in its unique ASC, and additionally differs from Gymnodinium in the absence of nuclear chambers, and from Lepidodinium in the absence of Chl b and nuclear chambers. New morphological information was provided for G. dorsalisulcum and G. impudicum, e.g., a short sulcal intrusion in G. dorsalisulcum; nuclear chambers in G. impudicum and G. dorsalisulcum; and a chloroplast enveloped by two membranes in G. impudicum. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference with independent SSU and LSU rDNA sequences. Our results support the classification of Wangodinium within the Gymnodiniales sensu stricto clade and it is close to Lepidodinium. Our results also support the close relationship among G. dorsalisulcum, G. impudicum, and Barrufeta. Further research is needed to assign these Gymnodinium species to Barrufeta or to erect new genera.

Seasonal distribution of Gambierdiscus spp. in Wakasa Bay, the Sea of Japan, and antagonistic relationships with epiphytic pennate diatoms.

The occurrence of the ciguatera fish poisoning (CFP) causative Gambierdiscus spp. was confirmed in the Sea of Japan for the first time in 2009. This paper reports seasonal distribution of Gambierdiscus spp. and epiphytic diatoms in the Sea of Japan. Monitoring results suggested an antagonistic interaction in abundances between epiphytic diatoms and the dinoflagellate Gambierdiscus spp. Allelopathic effects of diatoms were considered to be involved in the competitive phenomenon. Therefore it is hypothesized that cell densities of epiphytic pennate diatoms on macroalgae are a novel determinant affecting the abundance of Gambierdiscus spp. other than sea water temperature, salinity and nutrients. Monitorings of the abundance of epiphytic diatoms would lead us to predict the occurrences of Gambierdiscus spp. blooms in the CFP area, and thereby the CFP risk assessments would be developed. Phylogenetic analyses indicated that Gambierdiscus spp. in the Sea of Japan belonged to Gambierdiscus sp. type 2 which was reported to be non-toxic. Nevertheless, based on morphological characteristics, at least two types of Gambierdiscus spp. were found in the Sea of Japan. It is needed to test the toxicity of the both types of Gambierdiscus recognized in the present study for evaluation of the probability of CFP outbreak risks in the Sea of Japan in the future.