Description of a New Planktonic Mixotrophic Dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. from the Coastal Waters off Western Korea: Morphology,Pigments, and Ribosomal DNA Gene Sequence

ABSTRACT. The mixotrophic dinoflagellate Paragymnodinium shiwhaense n. gen., n. sp. is described from living cells and from cells prepared by light, scanning electron, and transmission electron microscopy. In addition, sequences of the small subunit (SSU) and large subunit (LSU) rDNA and photosynthetic pigments are reported. The episome is conical, while the hyposome is hemispherical. Cells are covered with polygonal amphiesmal vesicles arranged in 16 rows and containing a very thin plate‐like component. There is neither an apical groove nor apical line of narrow plates. Instead, there is a sulcal extension‐like furrow. The cingulum is as wide as 0.2–0.3 × cell length and displaced by 0.2–0.3 × cell length. Cell length and width of live cells fed Amphidinium carterae were 8.4–19.3 and 6.1–16.0 μm, respectively. Paragymnodinium shiwhaense does not have a nuclear envelope chamber nor a nuclear fibrous connective (NFC). Cells contain chloroplasts, nematocysts, trichocysts, and peduncle, though eyespots, pyrenoids, and pusules are absent. The main accessory pigment is peridinin. The sequence of the SSU rDNA of this dinoflagellate (GenBank AM408889) is 4% different from that of Gymnodinium aureolum, Lepidodinium viride, and Gymnodinium catenatum, the three closest species, while the LSU rDNA was 17–18% different from that of G. catenatum, Lepidodinium chlorophorum, and Gymnodinium nolleri. The phylogenetic trees show that this dinoflagellate belongs within the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers, NFC, and an apical groove. Unlike Polykrikos spp., which have a taeniocyst–nematocyst complex, P. shiwhaense has nematocysts without taeniocysts. In addition, P. shiwhaense does not have ocelloids in contrast to Warnowia spp. and Nematodinium spp. Therefore, based on morphological and molecular analyses, we suggest that this taxon is a new species, also within a new genus.     

Cellular and nuclear morphological variability within a single species of the toxigenic dinoflagellate genus Gambierdiscus: Relationship to life-cycle processes

Dinoflagellates belonging to the genus Gambierdiscus are the causative agent of ciguatera fish poisoning (CFP). This syndrome, which is widespread in tropical and subtropical regions, has recently been reported also in temperate latitudes. Taxonomic studies of Gambierdiscus have yet to completely couple the morphological features of member species with their genetics. In this study, the cellular and nuclear morphology of a single strain of one species of Gambierdiscus was determined in cells grown under different culture conditions. The results showed a wide-ranging variability of cell sizes, together with a clear relationship between cell size and nuclear morphology. Thus, small cells were associated with round to oval or slightly U-shaped nuclei and large cells with obviously U-shaped nuclei. Most cells exhibited the typical anterio-posteriorly compressed lenticular, shape of Gambierdiscus, with the exception of a few small globular-shaped specimens. In all cells, regardless of their size, the arrangement of the thecal plates was typical of lenticular Gambierdiscus. Dividing cells were consistently the largest. In these cells, nuclear morphology, karyokinesis, and cytokinesis were characterized. Cells underwent division only during the dark period, thus demonstrating their spontaneous synchronized division. Cellular forms related to the sexual cycle were also present in the cultures and included gamete pairs and putative meiotic planozygotes. The effect of the culture medium was studied by means of principal component analyses, which showed a positive correlation between the medium used and nuclear size and shape but not cell size.     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.     

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2′, and G. belizeanus by the asymmetrical Plate 3′. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell⁻¹, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.     

First report of the epiphytic benthic dinoflagellates Coolia canariensis and Coolia malayensis in the waters off Jeju Island, Korea: morphology and rDNA sequences

Coolia spp. are epiphytic and benthic dinoflagellates. Herein, we report for the first time, the occurrence of Coolia canariensis and Coolia malayensis in Korean waters. The morphology of the Korean strains of C. canariensis and C. malayensis isolated from the waters off Jeju Island, Korea was similar to that of the original Canary lslands strains and Malaysian strains, respectively. We found several pores and a line of small knobs on the pore plate, and perforations within the large pores of both C. canariensis and C. malayensis. The plates of the Korean strains of C. canariensis and C. malayensis were arranged in a Kofoidian series of Po, 3′, 7′′, 6c, 6s, 5′′′, and 2′′′′, and Po, 3′, 7′′, 7c, 6–7s, 5′′′, and 2′′′′, respectively. When properly aligned, the large subunit (LSU) rDNA sequence of the Korean strain of C. canariensis was identical to that of the Biscayan strains, but it was 2–3% different from the Canary lslands strain VGO0775 and the Australian strain. In addition, the sequences of small subunit (SSU) and/or LSU rDNA from the two Korean strains of C. malayensis were < 1% different from the Malaysian strains of C. malayensis and the Florida strain CCMP1345 and New Zealand strain CAWD39 (“Coolia monotis”). In phylogenetic trees based on LSU rDNA sequences, the Korean strains of C. malayensis belonged to a clade including the Malaysian strains and these two strains. Therefore, based on genealogical analyses, we suggest that the Korean strain of C. canariensis is closely related to two Atlantic strains and the Australian strain, whereas the Korean strains of C. malayensis are related to the Malaysian strains of C. malayensis and the Florida and New Zealand strains.     

First report of three benthic dinoflagellates,Gambierdiscus pacificus,G. australes and G. caribaeus (Dinophyceae), from Hainan Island,South China Sea

Species of the marine benthic dinoflagellate genus Gambierdiscus are the principal cause of Ciguatera fish poisoning. This genus has been recorded from tropical to temperate oceans, although Gambierdiscus species have rarely been found in Chinese waters. Our work revealed the morphological and genetic characteristics of three potentially toxic Gambierdiscus species observed in the temperate to subtropical waters of China. The fine thecal morphology was determined based on light microscopy and scanning electron microscopy analyses, and these species were also characterized by sequencing the D1–D3 and D8–D10 regions of the LSU rDNA. The morphological and genetic data indicated that these three Gambierdiscus species were G. pacificus, G. australes and G. caribaeus. This work provides the first report of these species in Chinese waters, which increases the known species distribution of this genus.     

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 μm long and 6.6-15.7 μm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.     

Gambierdiscus (Gonyaulacales,Dinophyceae) diversity in Vietnamese waters with description of G. vietnamensis sp. nov.

Viet Nam has a coastline of 3200 km with thousands of islands providing diverse habitats for benthic harmful algal species including species of Gambierdiscus. Some of these species produce ciguatera toxins, which may accumulate in large carnivore fish potentially posing major threats to public health. This study reports five species of Gambierdiscus from Vietnamese waters, notably G. australes, G. caribaeus, G. carpenteri, G. pacificus, and G. vietnamensis sp. nov. All species are identified morphologically by LM and SEM, and identifications are supported by molecular analyses of nuclear rDNA (D1–D3 and D8–D10 domains of LSU, SSU, and ITS1-5.8S-ITS2 region) based on cultured material collected during 2010–2021. Statistical analyses of morphometric measurements may be used to differentiate some species if a sufficiently large number of cells are examined. Gambierdiscus vietnamensis sp. nov. is morphologically similar to other strongly reticulated species, such as G. belizeanus and possibly G. pacificus; the latter species is morphologically indistinguishable from G. vietnamensis sp. nov., but they are genetically distinct, and molecular analysis is deemed necessary for proper identification of the new species. This study also revealed that strains denoted G. pacificus from Hainan Island (China) should be included in G. vietnamensis sp. nov.     

Morphology and Phylogenetic Analyses of Three Novel Naegleria Isolated from Freshwaters on Jeju Island,Korea, During the Winter Period

The genus Naegleria is one of the best known heterolobosean groups, and is the causative agent of primary amoebic meningoencephalitis. This group is rarely studied in temperate regions during winter. Here, three novel Naegleria were isolated from freshwaters on Jeju Island, Korea, during winter. Two isolates were amoeboflagellates, and one of the three amoebae did not undergo enflagellation. All amoebae had eruptive pseudopodia, and the layer of refractile granules around a large nucleus. They formed a cyst with ~2 pores in the cyst stage. The amoeboflagellate form had two flagella and no division in the flagellate stage, and no cytostome. These features are very similar to typical Naegleria. Furthermore, our isolates were able to grow at > 30 °C, suggesting that they had different thermophilicity from Naegleria in polar regions. All amoebae were largely encysted at 5 or 10 °C, indicating that they were likely encysted during winter. Based on the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequences, the phylogenetic analyses consistently revealed that the isolates are members of the Naegleria group. However, the isolates differ from other species in both phylogenetic trees. Thus, Naegleria in cold habitats appeared to have a high degree of novelty, but their thermophilicity may be dependent on locality.     

Distribution and Diversity of Microbial Eukaryotes in Bathypelagic Waters of the South China Sea

Little is known about the biodiversity of microbial eukaryotes in the South China Sea, especially in waters at bathyal depths. Here, we employed SSU rDNA gene sequencing to reveal the diversity and community structure across depth and distance gradients in the South China Sea. Vertically, the highest alpha diversity was found at 75‐m depth. The communities of microbial eukaryotes were clustered into shallow‐, middle‐, and deep‐water groups according to the depth from which they were collected, indicating a depth‐related diversity and distribution pattern. Rhizaria sequences dominated the microeukaryote community and occurred in all samples except those from less than 50‐m deep, being most abundant near the sea floor where they contributed ca. 64–97% and 40–74% of the total sequences and OTUs recovered, respectively. A large portion of rhizarian OTUs has neither a nearest named neighbor nor a nearest neighbor in the GenBank database which indicated the presence of new phylotypes in the South China Sea. Given their overwhelming abundance and richness, further phylogenetic analysis of rhizarians were performed and three new genetic clusters were revealed containing sequences retrieved from the deep waters of the South China Sea. Our results shed light on the diversity and community structure of microbial eukaryotes in this not yet fully explored area.     

Temporal and spatial dynamics of phytoplankton diversity in the East China Sea near Jeju Island (Korea): A pyrosequencing-based study

The East China Sea (ECS) has long been considered an important monitoring site for oceanic ecosystem changes because many water currents and river discharges constantly influence this area. In this study, the community structure and diversity of phytoplankton in the northern part of the ECS adjacent to Jeju Island were explored using small subunit ribosomal RNA (SSU) pyrosequencing. We analysed samples collected from four stations from the surface and at 30-m and 50-m depths during April and September 2011. We observed spatial and temporal variations in the phytoplankton community. Among phytoplankton, diatoms and dinoflagellates constituted a major portion at all stations (60–90%). However, comparison of the April and September samples showed seasonal variation and shifts in the dispersion of diatom and dinoflagellates among stations. Among stations, diatoms dominated St. 1 and others were dominated by dinoflagellates. Furthermore, phylotypes of potentially toxin-producing genera such as Karlodinium, Heterocapsa, Gymnodinium, Gyrodinium, and Pseudo-nitzschia were dominant in this area.     

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales,Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food‐borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8‐D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography‐Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7‐8s, 5?, 0p, 2″″ and distinctive by size and hatchet‐shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal‐derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.     

Gambierdiscus (Dinophyceae) species diversity in the Flower Garden Banks National Marine Sanctuary,Northern Gulf of Mexico,USA

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45 m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40 m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10 μmol photons m⁻² s⁻¹. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50 m.     

First record of Grammatodinium (Dinophyceae) for the American Eastern Pacific coast: Morphological,molecular and ecological confirmation

Grammatodinium Li & Shin is a monospecific genus described from the Tongyeong Bay area in Korea. In the current study, we describe its presence in the American Eastern Pacific coast for the first time, particularly in Acapulco Bay, Mexico, using morphological, molecular and environmental data. Sequences generated in this study with SSU and LSU formed a monophyletic group with other sequences from GenBank belonging to Gr. tongyeonginum, the only species known for the genus; however, genetic distance values between this species and our specimens (8.5% SSU; 2.8% LSU) were equivalent or even greater than those reported in other genera of dinoflagellates. Our phylogeny clearly showed the relationship of Grammatodinium with the families Pyrocystaceae and Gonyaulacaceae. In our specimens, cells appeared individually and in colonies of up to 16 cells, which were observed mainly during the dry season, so they could be confused with Gymnodinium catenatum, a common dinoflagellate in Acapulco with which they can coexist and share their general appearance, but they are clearly differentiated by the presence of longitudinal furrows throughout the body and a yellowish-green coloration, both absent in Gymnodinium catenatum. Although our evidence strongly suggests the presence of a new species for the region, more detailed morphological examinations are needed to confirm this statement.     

Molecular Phylogeny of the Ocelloid-Bearing Dinoflagellates Erythropsidinium and Warnowia (Warnowiaceae, Dinophyceae)

ABSTRACT. Members of the family Warnowiaceae are unarmored phagotrophic dinoflagellates that possess an ocelloid. The genus Erythropsidinium (= Erythropsis ) has also developed a unique dynamic appendage, the piston, which is able to independently retract and extend for at least 2 min after the cell lyses. We provide the first small subunit ribosomal RNA gene sequences of warnowiid dinoflagellates, those of the type Erythropsidinium agile and one species of Warnowia . Phylogenetic analyses show that warnowiid dinoflagellates branch within the Gymnodinium sensu stricto group, forming a cluster separated from the Polykrikos clade and with autotrophic Pheopolykrikos beauchampii as closest relative. This reinforces their classification as unarmored dinoflagellates based on the shape of the apical groove, despite the strong ecological and ultrastructural diversity of the Gymnodinium s.s. group. Other structures, such as the ocelloid and piston, have no systematic value above the genus level.     

First Record of the Blue-banded Sea Krait(Laticauda laticaudata,Reptilia: Squamata: Elapidae: Laticaudinae) on Jeju Island,South Korea

We report the first recorded capture of a blue-banded sea snake(Laticauda laticaudata Linnaeus, 1758, Jobeuntti Kun Badabam in Korean) in South Korea based on one male specimen collected from Marado-ri, Seogwiposi, Jeju-do on 20 October 2016. The morphological features of the lateral nostrils, the much wider ventrals than adjacent dorsals, the horizontally undivided rostral, the two prefrontals, and the uniform black bands on the body indicate that the specimen is L. laticaudata. An analysis of the partial mitochondrial cytochrome b gene sequence indicated that the specimen fits well into the known L. laticaudata phylogenetic group, which confirms that the sea krait is L. laticaudata. Including this report, five species of sea snakes(L. laticaudata, L. semifasciata, Hydrophis platurus, H. cyanocinctus, and H. melanocephalus) have now been reported in Korean waters.