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.     

Morphology and phylogeny of Prorocentrum caipirignum sp. nov. (Dinophyceae), a new tropical toxic benthic dinoflagellate

A new species of toxic benthic dinoflagellate is described based on laboratory cultures isolated from two locations from Brazil, Rio de Janeiro and Bahia. The morphology was studied with SEM and LM. Cells are elliptical in right thecal view and flat. They are 37–44 μm long and 29–36 μm wide. The right thecal plate has a V shaped indentation where six platelets can be identified. The thecal surface of both thecal plates is smooth and has round or kidney shaped and uniformly distributed pores except in the central area of the cell, and a line of marginal pores. Some cells present an elongated depression on the central area of the apical part of the right thecal plate. Prorocentrum caipirignum is similar to Prorocentrum lima in its morphology, but can be differentiated by the general cell shape, being elliptical while P. lima is ovoid. In the phylogenetic trees based on ITS and LSU rDNA sequences, the P. caipirignum clade appears close to the clades of P. lima and Prorocentrum hoffmannianum. The Brazilian strains of P. caipirignum formed a clade with strains from Cuba, Hainan Island and Malaysia and it is therefore likely that this new species has a broad tropical distribution. Prorocentrum caipirignum is a toxic species that produces okadaic acid and the fast acting toxin prorocentrolide.     

Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3′, 7″, 6c, 7s, 5?, 2′′′′, is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1′ and 3′ makes them approximately hexagonal, (ii) the 1′ plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 μm, AP: 25.66 ± 2.97 μm, W: 39.81 ± 5.05 μm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX‐a and structural isomers OVTX‐d and ‐e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin‐like compounds (ovatoxin‐i, ovatoxin‐j₁, ovatoxin‐j₂, and ovatoxin‐k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28–0.94 pg · cell^?1. On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C–31.5°C), with peaks in June and August.     

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.     

Laciniporus arabicus gen. et sp. nov. (Dinophyceae,Peridiniales), a new thecate,marine, sand‐dwelling dinoflagellate from the northern Indian Ocean (Arabian Sea)1

A new thecate, photosynthetic, sand‐dwelling marine dinoflagellate, Laciniporus arabicus gen. et sp. nov., is described from the subtidal sediments of the Omani coast in the Arabian Sea, northern Indian Ocean, based on detailed morphological and molecular data. Cells of L. arabicus are small (16.2–30.1 μm long and 13.1–23.2 μm wide), dorsoventrally compressed, with a small apical flap‐shaped projection pointing to the left. The thecal plate pattern is distinguished by minute first precingular plate and sulcus, which extends into the epitheca, with large anterior and right sulcal plates. The Kofoidian thecal tabulation is Po, X, 4′, 2a, 7′′, 6c, 6s, 5′′′, 2′′′′. Morphologically, the revealed plate pattern has an affinity to the Peridiniales, and LSU rDNA based phylogenetic analyses placed L. arabicus within the Thoracosphaeraceae, close to calcareous‐cyst producing scrippsielloids, predatory pfiesteriaceans, and photosynthetic freshwater peridinioids Chimonodinium lomnickii and Apocalathium spp. However, the thecal plate arrangement of L. arabicus differs noticeably from any currently described dinoflagellates, and the species stands out from closely related taxa by extensive differences in physiology and ecology.     

Scrippsiella hexapraecingula sp. nov. (Dinophyceae), a tida pool dinoflagellate from the Northwest Pacific

Specimens of dinoflagellate collected in tide pools along the Pacific coast of central and southern Japan are described as a new species,Scrippsiella hexapraecingula Horiguchi et Chihara, of the Peridiniaceae (Class Dinophyceae). The plate formula is pp, x, 4′, 3a, 6″, 6c, 5‴, 2″" and, 5s, the same as that of other species ofScrippsiella, except in lacking one precingular plate. The genus must be emended, therefore, as having either six or seven precingular plates. This dinoflagellate migrates diurnally. In the morning motile cells are released from non-motile cells attached to the substrate and in the evening the motile cells swim down to settle on the bottom of the tide pool. Attached non-motile cells form either motile mono- or bispores. Sexual reproduction was not observed.     

Molecular and phylogenetic characterization of Ostreopsis (Dinophyceae) and the description of a new species,Ostreopsis rhodesae sp. nov., from a subtropical Australian lagoon

Cryptic and pseudo-cryptic species are common amongst marine phytoplankton, and may cause misleading inferences of ecological and physiological data of plankton community studies. Deciphering the diversity and distribution of species of the benthic dinoflagellate Ostreopsis is one example, as there are many morphologically indistinct clades that differ greatly genetically and toxicologically from one another. In this study, a new species, Ostreopsis rhodesae from the southern Great Barrier Reef was described. While it initially appeared to be highly similar to several other Ostreopsis species, we found O. rhodesae can be distinguished based on the relative size of the second apical plate (2′), which is twice as long as the APC plate, and separates the third apical (3′) from the third precingular (3′′) plate. Phylogenetic trees based on the SSU, ITS/5.8S and D1-D2 and D8-D10 regions of the LSU rRNA were well supported, and showed a clear difference to other Ostreopsis clades. Compensatory base changes (CBCs) were identified in helices of the ITS2 between O. rhodesae and O. cf. ovata and O. cf. siamensis, which were also present in the same habitat. Fish gill cell lines were toxic to O. rhodesae, cell extracts but no palytoxin-like analogues were found in them. The findings highlight a case of pseudo-cryptic speciation, found in sympatry with closely related and morphologically similar species, but biologically and functionally distinct.     

Amphidiniopsis uroensis sp. nov. and Amphidiniopsis pectinaria sp. nov. (Dinophyceae): Two new benthic dinoflage llates from Japan

Two new armoured, heterotrophic sand‐dwelling marine dinoflagellates, Amphidiniopsis uroensis Toriumi, Yoshimatsu et Dodge sp. nov. and Amphidiniopsis pectinaria Toriumi, Yoshimatsu et Dodge sp. nov. were collected from Japanese sandy beaches, and their morphological features observed by light microscopy and scanning electron microscopy (SEM). The cell size of A. uroensis is 28–31 μm in length and 23–28 μm in width. The plate formula is Po 3′, 3a, 6″, 3c, 4s (+1 acc.), 5″′, 2″″. The thecal surface is ornamented with small processes, pores and spines, however, the surface of plate 2a is smooth. The epitheca possesses a narrow ridge that is extended along on the suture between 1′ and 3′. Plate 1″ connects with the right sulcal (Sd) and right sulcal accessory (Sda) plates, so the cingulum is incomplete. A nucleus is situated in the central part of the cell. There are a few small spines at the antapex. There are no stigma or chloroplasts. Amphidiniopsis pectinaria cells are 33–40 urn in length and 29–35 μm in width. The plate formula is Po 4′, 3a, 7″, 3c, 4s (+1 acc.), 5″′, 2″″. Plate 1″ connects directly with Sd and Sda plates, so the cingulum is incomplete. The thecal surface is ornamented with small processes, spines and pores. The epitheca is provided with a narrow ridge that is extended along on the suture between plates 1′, 4′ and 7″. The ornamentation on the antapical plates is unique. It is arranged in 10 straight rows on the hypotheca; each row has a strong spine at its posterior end. In addition, there is a long spine at the antapex. There are no stigma or chloroplasts. A nucleus is located in the central part of the cell.     

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.     

Plagiodinium ballux sp. nov. (Dinophyceae), a deep (36 m) sand dwelling dinoflagellate from subtropical Japan

A new species of marine sand‐dwelling dinoflagellate, Plagiodinium ballux N. Yamada, Dawut, R. Terada & T. Horiguchi is described from a deep (36 m) seafloor off Takeshima Island, Kagoshima Prefecture, Japan in the subtropical region of the northwest Pacific. The species is thecate and superficially resembles species of Prorocentrum, but possesses an extremely small epitheca. The cell varies from ovoid to a rounded square, and is small (15.0–22.5 μm in length) and laterally compressed. The thecal plates are smooth and the thecal plate arrangement (Po, 1′, 0a, 5″, 5C, 2S, 5?, 0p, 1″″) is similar to that of Plagiodinium belizeanum, the type species of the genus. Molecular phylogenetic analyses based on SSU rDNA and partial LSU rDNA reveal that the dinoflagellate is closely related to P. belizeanum, but it can be clearly distinguished by its size and cell shape. This suite of morphological and molecular differences leads to the conclusion that this deep benthic dinoflagellate represents a new species of the genus Plagiodinium.     

Gymnodinium litoralis sp. nov. (Dinophyceae), a newly identified bloom-forming dinoflagellate from the NW Mediterranean Sea

Recurrent high-biomass blooms of a gymnodinioid species have been periodically recorded at different sites in the NW Mediterranean Sea (Catalan and Sardinian coast), causing intense discolorations of the water. In this study, several strains of the causative organism were isolated and subsequently studied with respect to the morphology of the vegetative cells and different life cycle stages, pigments profile, and molecular phylogeny. Based on phylogenetic analyses, the strains were placed within the Gymnodinium sensu stricto clade. The species possessed a horseshoe-shaped apical groove running anticlockwise around the apex and the major accessory pigment was identified as peridinin. These characteristics place the organism within the Gymnodinium genus, as defined today, although some other characteristics, such as vesicular chambers in the nuclear envelope and a nuclear fibrous connective were not observed. Morphologically, the isolates highly resemble Gyrodinium vorax (Biecheler) but major differences with the latter suggest that they comprise a new species, Gymnodinium litoralis sp. nov. The resting cyst of this species is described herein from field samples of the Catalan and Sardinian coast; pellicle cysts were observed in field samples and also in cultures. This species recurrently produces high biomass blooms (>10⁶ cell L⁻¹) in summer along several beaches and coastal lagoons in the NW Mediterranean Sea (L’Estartit, La Muga River mouth, and Corru S’Ittiri). Knowledge about its geographic distribution is limited, since the precise identification of G. litoralis from the field or fixed samples can be difficult. Therefore we expect that molecular studies will reveal a much wider distribution of the species.     

Morphology of Sinophysis minima sp. nov. and three Sinophysis species (Dinophyceae, Dinophysiales) from the Sea of Japan

Four sand‐dwelling species of the marine dinoflagellate genus Sinophysis, including one new species, have been examined from intertidal and subtidal sand, from the Sea of Japan. The morphological features of these species were observed by light and scanning electron microscopy. Sinophysis minima sp. nov. is flattened laterally and is 17.5–35.0 μm in length and 15.0–27.5 μm in depth, with a length/depth ratio of 1.1–1.4, an epitheca depth of 5.0–7.5 μm, and a sulcus length of about three‐quarters the hypotheca length. Sinophysis ebriola (Herdman) Balech, Sinophysis grandis Hoppenrath and Sinophysis stenosoma Hoppenrath were recorded for the first time in the seas of Russia. Sinophysis stenosoma is the most common species in the Sea of Japan. All species usually occurred together in the region investigated. Additional information on the known species is provided.     

Madanidinium loirii gen. et sp. nov. (Dinophyceae), a new marine benthic dinoflagellate from Martinique Island,Eastern Caribbean

A new benthic phototrophic dinoflagellate is described from sediments of a tropical marine cove at Martinique Island and its micromorphology is studied by means of light and electron microscopy. The cell contains small golden-brown chloroplasts and the oval nucleus is posterior. It is laterally compressed, almost circular in shape when viewed laterally. It consists of a small epitheca tilted toward the right lateral side and a larger hypotheca. In the left view, the cingulum is more anterior and the epitheca is reduced. The cingulum is displaced and left-handed. This organism is peculiar in having no apical pore and its thecal plate arrangement is 2′ 1a 7′′ 5c 3s 5′′′ 1′′′′. The plates are smooth with small groups of pores scattered on their surface. An area with 60–80 densely arranged pores is found near the centre of the 2′′′ plate, on the left lateral side. Morphologically, these features are different from all other laterally compressed benthic genera. In addition, molecular genetic sequences of SSU and partial LSU form a distinct and well-supported clade among dinoflagellates and support the erection of a new genus. However, molecular phylogenies inferred from ribosomal genes failed to confirm any clear relationship with other benthic taxa and affinity with other laterally compressed dinoflagellates has not been demonstrated. Hence, the taxonomic affinity of Madanidinium loirii with a defined order and family is unclear at the moment.     

Amphidiniella sedentaria gen. et sp. nov. (Dinophyceae), a new sand-dwelling dinoflagellate from Japan

A new sand-dwelling dinoflagellate is described from Sesoko Beach, Okinawa Island, subtropical Japan and its micromorphology is studied by means of light and electron microscopy. The cell consists of a small epitheca and a large hypothecs superficially resembling members of the unarmored genus Amphidinium. The cell is dorso-ventrally flattened and possesses a single chloroplast with a large conspicuous pyrenoid. Transmission electron microscopy revealed that the dinoflagellate possesses typical dinoflagellate cellular organization. Scanning electron microscopy demonstrated that the organism is thecate and the thecal plate arrangement is Po, 4′, 1a, 7″, 5c, 4s, 6″′, 2″″. Most of the characteristics suggest gonyaulacalean affinity of the new species. These are the presence of ventral pore, lack of canal plate, direct contact between the sulcal anterior plate and the flagellar pore, possession of six postcingular plates and asymmetrical arrangement of the antapical plates. Affinity to existing families of the order Gonyaulacales has not been determined. Based on the unique cell shape, thecal plate arrangement and the presence of ventral pore, a new genus, Amphidiniella, is established for this organism and the species is named A. sedentaria Horiguchi gen. et sp. nov.     

Morphology of Gambierdiscus scabrosus sp. nov. (Gonyaulacales): a new epiphytic toxic dinoflagellate from coastal areas of Japan

A new epiphytic dinoflagellate is described, G ambierdiscus scabrosus sp. nov., from tidal pools and rocky shores along the coastal areas of Japan. Cells are 63.2 ± 5.7 μm in depth, 58.2 ± 5.7 μm in width, and 37.3 ± 3.5 μm in length. The plate formula of G . scabrosus is Po, 4′, 0a, 6′′, 6c, ?s, 5′′′, 0p, and 2′′′′. Morphologically, G . scabrosus resembles G . belizeanus as follows: anterioposteriorly compressed cell shape, narrow 2′′′′ plate, and areolated surface. Despite this similarity, the cells of G . scabrosus can be distinguishable by the presence of the asymmetric shaped 3′′ plate and the rectangular shaped 2′ plate.     

Heterocapsa psammophila sp. nov. (Peridiniales, Dinophyceae), a new sand-dwelling marine dinoflagellate

A new armored dinoflagellate species, Heterocapsa psammophila Tamura, Iwataki et Horiguchi sp. nov. is described from Kenmin‐no‐hama beach, Hiroshima, Japan using light and electron microscopy. This dinoflagellate possesses the typical thecal plate arrangement of the genus Heterocapsa, Po, cp, 5′, 3a, 7′′, 6c, 5s, 5′′′, 2′′′′; and the 3‐D body scales of Heterocapsa on the plasma membrane. The cell shape is ovoidal. The spherical nucleus and the pyrenoid are situated in the hypotheca and the epitheca, respectively. The ultrastructure of H. psammophila is typical of dinoflagellates and the pyrenoid is invaginated by cytoplasmic tubules. H. psammophila is distinguished from all other hitherto‐described Heterocapsa species by the cell shape, the relative position of the nucleus and pyrenoid and the structure of the body scale. The habitat and behavior of this new species in culture suggest that the organism is truly a sand‐dwelling species.     

A new potentially toxic Azadinium species (Dinophyceae) from the Mediterranean Sea,A. dexteroporum sp. nov.

A new photosynthetic planktonic marine dinoflagellate, Azadinium dexteroporum sp. nov., is described from the Gulf of Naples (South Tyrrhenian Sea, Mediterranean Sea). The plate formula of the species, Po, cp, X, 4′, 3a, 6″, 6C, 5?S, 6? and 2″″, is typical for this recently described genus. Azadinium dexteroporum is the smallest rep‐resentative of the genus (8.5 μm average length, 6.2 μm average width) and shares the presence of a small antapical spine with the type species A. spinosum and with A. polongum. However, it differs from all other Azadinium species for the markedly asymmetrical Po plate and the position of the ventral pore, which is located at the right posterior end of the Po plate. Another peculiarity of A. dexteroporum is the pronounced concavity of the second intercalary plate (2a), which appears collapsed with respect to the other plates. Phylogenetic analyses based on the large subunit 28S rDNA (D1/D2) and the internal transcribed spacer (ITS rDNA) support the attribution of A. dexteroporum to the genus Azadinium and its separation from the other known species. LC/MS‐TOF analysis shows that Azadinium dex‐teroporum produces azaspiracids in low amounts. Some of them have the same molecular weight as known compounds such as azaspiracid‐3 and ‐7 and Compound 3 from Amphidoma languida, as well as similar fragmentation patterns in some cases. This is the first finding of a species producing azapiracids in the Mediterranean Sea.