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Acoels in the family Convolutidae are commonly found with microalgal symbionts. Convolutids can host green algal Tetraselmis and dinoflagellates within the family Symbiodiniaceae and the genus Amphidinium. The diversity of these microalgae has not been well surveyed. In this study, we used PCR and culture techniques to demonstrate the biodiversity of Tetraselmis and dinoflagellates in symbiosis with meiofaunal acoels. Here, 66 acoels were collected from seven localities around Okinawa, Ishigaki, and Kochi, Japan. While convolutids were heavily represented in this sampling, some acoels formed a clade outside Convolutidae and are potentially a new family of acoels harboring symbiotic microalgae. From the acoels collected, a total of 32 Tetraselmis and 26 Symbiodiniaceae cultures were established. Molecular phylogenies were constructed from cultured material (and from total host DNA) using the 18S rRNA gene (Tetraselmis) and 28S rRNA gene (dinoflagellates). The majority of Tetraselmis sequences grouped within the T. astigmatica clade but strains closely related to T. convolutae, T. marina, and T. gracilis were also observed. This is the first report of Tetraselmis species, other than T. convolutae, naturally associating with acoels. For dinoflagellates, members of Cladocopium and Miliolidium were observed, but most Symbiodiniaceae sequences formed clusters within Symbiodinium, grouping with S. natans, or sister to S. tridacnidorum. Several new Symbiodinium sequences from this study may represent novel species. This is the first molecular record of Miliolidium and Symbiodinium from acoels. Microalgal strains from this study will provide a necessary framework for future taxonomic studies and research on symbiotic relationships between acoels and microalgae. [ABSTRACT FROM AUTHOR]

Marine benthic dinoflagellates within the genus Amphidinium were isolated from Guam and Okinawa. Isolated strains were identified to species-level using phylogenetic analyses of 28S rRNA and ITS-5.8S rRNA genes as well as microscopy. Of the six isolated strains, two were new species: A. pagoense sp. nov. and A. uduigamense sp. nov. Other isolates included strains of A. massartii and A. operculatum from Guam, and two strains of A. operculatum from Okinawa. Both new species were described using light and electron microscopy (SEM and TEM). The combination of characteristics that make A. pagoense sp. nov. unique includes a pair of centrally-located pyrenoids, variable cell shape, absence of scales and a long, curved ventral ridge. For A. uduigamense sp. nov., a combination of several morphological features distinguishes it from other species. These include a constriction near the anterior of the hypocone, two centrally located pyrenoids, a longitudinal flagellum inserted in the posterior one-third of the cell, cell size, cell division in the motile stage and the absence of scales. Toxicity was confirmed in these two novel species by testing methanol extracts in an Artemia bioassay. Previously unrecorded ITS rRNA gene sequences from A. operculatum were also sequenced from both locations. Species identified and newly described in this study expand the taxonomic knowledge of Amphidinium in the Pacific. [ABSTRACT FROM AUTHOR]

This study describes two novel parasitic dinoflagellates: Haplozoon gracile sp. nov. isolated from a bamboo worm (Maldanidae), 'cf. Petaloclymene sp.' sensu Kobayashi et al. 2018; and, H. pugnus sp. nov. isolated from Nicomache sp. and Nicomache personata (Maldanidae). Trophonts (feeding stages) were observed with light, scanning, and transmission electron microscopy. Molecular phylogenetic analyses were performed based on 18S rDNA. COI sequences were obtained for host organisms. Trophonts of H. gracile were linear (single longitudinal row) and relatively slender with a mean length of 190 μm, and consisted of a long and narrow trophocyte, rectangular gonocytes (mean width = 10 μm), and slightly rounded sporocytes. Trophonts of H. pugnus were pectinate (1–8 rows of sporocytes in one plane), with a mean length of 179 μm, consisting of a bulbous trophocyte, rectangular gonocytes (mean width = 25 μm), and rounded sporocytes. The body of both species was covered with many depressions that overlaid the amphiesmal vesicles. TEM observations of trophocytes in H. gracile revealed a stylet with a central dense core and rich mitochondria subtending the amphiesma. Furthermore, amphiesmal vesicles appeared to contain thecal plates in both species. Phylogenetic analyses generally resolved a Haplozoon clade, and H. gracile and H. pugnus were clearly distinguished from other species for which molecular data are available. Based on the morphological and host comparisons with all described species and their molecular phylogeny, we conclude that these two isolates are new species of Haplozoon, H. gracile sp. nov. and H. pugnus sp. nov. [ABSTRACT FROM AUTHOR]

Cabra is a genus of marine heterotrophic thecate dinoflagellates with a peculiar cell shape and thecal plate tabulation. Previous studies suggested it was related to the benthic genus Roscoffia based on morphological features. A previous molecular phylogenetic study using 18S rDNA showed that Roscoffia capitata and members of the family Podolampadaceae formed a well-supported monophyletic group; however, the classification of Cabra remains uncertain. In this study, the first molecular phylogenetic analysis based on 18S rDNA sequence for the genus was performed using specimens of Cabra matta collected in Okinawa, Japan. Molecular phylogeny showed that C. matta formed a clade with R. capitata and the planktonic oceanic members of Podolampadaceae including Podolampas spp. and Blepharocysta sp. This result supported previous studies that inferred the close affinity between Cabra, podolampadaceans and R. capitata based on a very narrow and long 1′ plate, and the diminutive 2′ and 3′ plates, the larger precingular plates, smaller apical plates, the presence of three cingular plates, the hypothecal plate pattern of five postcingular plates and one antapical plate. In contrast, there are clear morphological differences between the planktonic podolampadaceans and the benthic genera Roscoffia and Cabra: the planktonic members have a cryptic cingulum and lack an apparent sulcus, while the benthic members have an apparent cingulum and sulcus. Molecular phylogeny in the present study did not resolve the evolutionary branching order for Cabra, Roscoffia, Podolampas and Blepharocysta. [ABSTRACT FROM AUTHOR]

This study describes a novel species of Haplozoon , H . ezoense n. sp., a dinoflagellate parasite isolated from the intestines of Praxillella pacifica (Polychaeta). Trophonts (feeding stages) of H . ezoense n. sp. were isolated and studied with scanning and transmission electron microscopy, and molecular phylogenetic analyses was performed using 18S rDNA and 28S rDNA. Trophonts had an average length of 120 μm, and were linear, forming a single longitudinal row comprising a trophocyte with a stylet, an average of 14 gonocytes (width = 10 μm), and bulbous cells that we concluded were likely sporocytes. The surface of H. ezoense n. sp. was covered with projections of the amphiesma. Sections viewed under TEM revealed multiple triple membrane-bound organelles reminiscent of relic non-photosynthetic plastids within the gonocytes. Haplozoon ezoense n. sp., H . praxillellae , and H. axiothellae formed a well-supported clade in the 18S rDNA datasets. The sequences of H . ezoense n. sp. differed from H . praxillellae , a species of Haplozoon isolated from the same host species in the Northeast Pacific, at 88/1,748 bases; and 155/1,752 bases from H . axiothellae . Concatenated 18S rDNA and 28S rDNA datasets were unable to resolve the deeper relationships of Haplozoon in the context of dinoflagellates. [ABSTRACT FROM AUTHOR]

We isolated Plagiodinium belizeanum into clonal culture from the Pacific island of Okinawa (Japan) and characterized it using a combination of light microscopy, scanning electron microscopy, transmission electron microscopy and 18S/28S ribosomal (r) gene sequences. Although molecular phylogenetic analyses of 18S rDNA and 28S rDNA sequences were unable to resolve the phylogenetic position of P. belizeanum within dinoflagellates, the ultrastructural data provided some new traits for the species. For instance, double-membrane-bound vesicles, distinct from the mitochondria, were interpreted as autolysosomes containing electron-dense virus particles. The thecal plate pattern was Po 1' 0a 5''5(6)C 4S 5'p 1', which is slightly different from the original description in having an additional epithecal plate and four sulcal plates. The laterally flattened cells were 22-34 μm long, 11-13 μm deep, and 15-18 μm wide and contained a peridinin-type plastid with lobes radiating from a central pyrenoid that lacked starch sheaths and was traversed by stacks of thylakoids. This isolate represents the first record of the species in Japan, and the new ultrastructural and DNA sequence data were used to emend the species description. [ABSTRACT FROM AUTHOR]

Variations in toxicity of the benthic dinoflagellate Ostreopsis Schmidt 1901 have been attributed to specific molecular clades, biogeography of isolated strains, and the associated bacterial community. Here, we attempted to better understand the biodiversity and the basic biology influencing toxin production of Ostreopsis. Nine clonal cultures were established from Okinawa, Japan, and identified using phylogenetic analysis of the ITS-5.8S rRNA and 28S rRNA genes. Morphological analysis suggests that the apical pore complex L/W ratio could be a feature for differentiating Ostreopsis sp. 2 from the O. ovata species complex. We analyzed the toxicity and bacterial communities using liquid chromatography-mass spectrometry, and PCR-free metagenomic sequencing. Ovatoxin was detected in three of the seven strains of O. cf. ovata extracts , highlighting intraspecies variation in toxin production. Additionally, two new potential analogs of ovatoxin-a and ostreocin-A were identified. Commonly associated bacteria clades of Ostreopsis were identified from the established cultures. While some of these bacteria groups may be common to Ostreopsis (Rhodobacterales, Flavobacteria-Sphingobacteria, and Enterobacterales) , it was not clear from our analysis if any one or more of these plays a role in toxin biosynthesis. Further examination of biosynthetic pathways in metagenomic data and additional experiments isolating specific bacteria from Ostreopsis would aid these efforts. [ABSTRACT FROM AUTHOR]

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'). [ABSTRACT FROM AUTHOR]

The genus Amphidiniopsis is a benthic (sand-dwelling) lineage of thecate dinoflagellates, containing 19 morphologically diverse species. Past work has shown that some Amphidiniopsis species form a clade with the sand-dwelling Herdmania litoralis as well as some planktonic species in the family Protoperidiniaceae (i.e. the Monovela group). Still, our contemporary knowledge regarding Amphidiniopsis is limited, compared to the Protoperidiniaceae. To this end, we obtained 18S rDNA data from seven Amphidiniopsis species and a part of the 28S rDNA from four Amphidiniopsis species, with the goal of improving our understanding of phylogenetic relationships among Amphidiniopsis and the Monovela group. Results from the molecular phylogenetic analyses showed that Amphidiniopsis spp., with the exception of A. cf. arenaria , H. litoralis, and members within the Monovela group formed a single clade. Within the clade, relationships among Amphidiniopsis spp. and the Monovela group were more complicated — some subclades contained both representatives of Amphidiniopsis and the Monovela group. Our study suggests that habitat (benthic or planktonic), as well as traditionally used, general morphological characteristics, do not reflect molecular phylogenetic relationships, and that the taxonomy of the sand-dwelling genus Amphidiniopsis, and the planktonic family Protoperidiniaceae, should be reconsidered simultaneously. [ABSTRACT FROM AUTHOR]

The dinoflagellate genera Oxytoxum and Corythodinium that account for more than fifty species are widespread in warm oceans. These genera have been considered synonyms and thecal plate designations varied among authors. Several planktonic and sand-dwelling genera have been placed within the Oxytoxaceae. We obtained the first molecular data based on small subunit (SSU) rRNA gene sequences of Oxytoxum and Corythodinium, including the type species (O. scolopax and C. tessellatum) and C. frenguellii and C. cristatum. The three species of Corythodinium branched together a strong support [bootstrap (BP) of 98%]. This formed a sister clade with moderate support (BP 75%) with O. scolopax that supported the generic split. Oxytoxaceae should exclusively remain for Oxytoxum and Corythodinium, as an independent group, unrelated to any other known dinoflagellate. Oxytoxum was characterized by spindle-shaped cells with an anterior narrow epitheca, an apical spine and little cingular displacement. Corythodinium exhibits relatively broad cell shapes, with wider epitheca and greater cingular displacement, and an obovate or pentangular anterior sulcal plate that noticeably indented the epitheca. This suggested the need of new combinations for species that were described as Oxytoxum and possessed the characteristics of Corythodinium. [ABSTRACT FROM AUTHOR]

Marine benthic dinoflagellates within the genus Coolia have been reported to produce natural products, some of which are known to be toxic (i.e., cooliatoxin). To date, five species of Coolia have been reported in tropical and temperate waters around the world; however, very few studies have combined detailed morphological and molecular data with chemical analyses. In this study, a clonal culture of Coolia malayensis was isolated and mass cultivated from a coral reef on the island of Okinawa, Japan. Analysis of the thecal plate morphology and molecular phylogeny from 28S rDNA strongly supported the close relationship between this new isolate of C. malayensis from Okinawa and other isolates of C. malayensis from around the world. Following methanol extraction of 250 L of mass culture, chemical analyses using NanoLiquid chromatography mass spectrometry revealed the mass profiles of water-soluble and ethyl acetate-soluble parts. High-resolution mass spectrometry derived the molecular formulas of three novel disulphated polyether analogs of yessotoxin (C 56 H 78 O 18 S 2 1102.4 (Compound 1 ), C 57 H 80 O 18 S 2 1116.4 (Compound 2 ), and C 57 H 78 O 19 S 2 1130.4 (Compound 3 )); two potential homologous compounds (Compounds 4 and 5 ) were also observed on the high-resolution mass, albeit with low signal intensity. The five compounds in the C. malayensis from Okinawa are composed of less oxygen, compared to cooliatoxin and other analogs of yessotoxin, suggesting the metabolites produced by C. malayensis are unique to those previously reported from other strains of Coolia . [ABSTRACT FROM AUTHOR]

• High diversity of Coolia found in Guam. • New lineage of C. canariensis , phylogroup IV, displaying cryptic diversity. • Extracts from C. malayensis toxic to Artemia sp. • C. palmyrensis, C. canariensis, C. tropicalis extracts are nontoxic to Artemia sp. • Chemical analysis of six strains of Coolia showing variation in toxin production. Coolia Meunier 1919 from benthic assemblages of Hawai'i and Guam were isolated and clonal cultures were established from single cells. Cultures were identified to species-level based on 28S rRNA and ITS-5.8S rRNA genes and tested for toxicity. In Hawai'i, two strains of C. malayensis were isolated. In Guam, a high biodiversity was identified: four strains of C. malayensis , one strain of C. palmyrensis , one strain of C. tropicalis , one strain of C. canariensis phylogroup III, and two strains forming a new phylogroup (phylogroup IV) of nontoxic C. canariensis. Morphology of the new C. canariensis phylogroup was described using light microscopy and scanning electron microscopy. Mass cultures and methanol extracts of representative cultures (C. malayensis, C. palmyrensis, C. canariensis, C. tropicalis) from Guam were prepared for liquid chromatography-mass spectrometry analysis. Chemical analyses revealed yessotoxin analogue C 56 H 78 O 18 S 2 is produced by C. malayensis, C. canariensis phylogroup IV and C. palmyrensis , but other analogues, C 57 H 80 O 18 S 2 and C 58 H 86 O 18 S 2 , were only found in C. malayensis (Okinawa) and C. canariensis phylogroup IV. Individual toxin profiles were also different over time for an Okinawa strain of C. malayensis (NIES-3637), highlighting intra and inter-species variation in Yessotoxin-analogue expression. Biological activity was tested using Artemia bioassay and toxicity was observed in Guam and Okinawa strains of C. malayensis. Strong support of four distinct clades within the C. canariensis species complex was recovered in phylogenetic analyses, despite morphological similarities. [ABSTRACT FROM AUTHOR]