The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS‐1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS‐1 (WKS‐I type) or A. tamarense CU‐15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS‐1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS‐1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150‐177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28‐bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.

BackgroundA dinoflagellate genus Ostreopsis is known as a potential producer of Palytoxin derivatives. Palytoxin is the most potent non-proteinaceous compound reported so far. There has been a growing number of reports on palytoxin-like poisonings in southern areas of Japan; however, the distribution of Ostreopsis has not been investigated so far. Morphological plasticity of Ostreopsis makes reliable microscopic identification difficult so the employment of molecular tools was desirable.Methods/Principal FindingIn total 223 clones were examined from samples mainly collected from southern areas of Japan. The D8–D10 region of the nuclear large subunit rDNA (D8–D10) was selected as a genetic marker and phylogenetic analyses were conducted. Although most of the clones were unable to be identified, there potentially 8 putative species established during this study. Among them, Ostreopsis sp. 1–5 did not belong to any known clade, and each of them formed its own clade. The dominant species was Ostreopsis sp. 1, which accounted for more than half of the clones and which was highly toxic and only distributed along the Japanese coast. Comparisons between the D8–D10 and the Internal Transcribed Spacer (ITS) region of the nuclear rDNA, which has widely been used for phylogenetic/phylogeographic studies in Ostreopsis, revealed that the D8–D10 was less variable than the ITS, making consistent and reliable phylogenetic reconstruction possible.Conclusions/SignificanceThis study unveiled a surprisingly diverse and widespread distribution of Japanese Ostreopsis. Further study will be required to better understand the phylogeography of the genus. Our results posed the urgent need for the development of the early detection/warning systems for Ostreopsis, particularly for the widely distributed and strongly toxic Ostreopsis sp. 1. The D8–D10 marker will be suitable for these purposes.

Species in the epi-benthic dinoflagellate genus Gambierdiscus produce ciguatoxins (CTXs) and maitotoxins (MTXs), which are among the most potent marine toxins known. Consumption of fish contaminated with sufficient quantities of CTXs causes Ciguatera Fish Poisoning (CFP), the largest cause of non-bacterial food poisoning worldwide. Maitotoxins, which can be found in the digestive system of fish, could also contribute to CFP if such tissues are consumed. Recently, an increasing number of Gambierdiscus species have been identified; yet, little is known about the variation in toxicity among Gambierdiscus strains or species. This study is the first assessment of relative CTX- and MTX-toxicity of Gambierdiscus species from areas as widespread as the North-Eastern Atlantic Ocean, Pacific Ocean and the Mediterranean Sea. A total of 13 strains were screened: (i) seven Pacific strains of G. australes, G. balechii, G. caribaeus, G. carpenteri, G. pacificus, G. scabrosus and one strain of an undetermined species (Gambierdiscus sp. Viet Nam), (ii) five strains from the North-Eastern Atlantic Ocean (two G. australes, a single G. excentricus and two G. silvae strains), and (iii) one G. carolinianus strain from the Mediterranean Sea. Cell pellets of Gambierdiscus were extracted with methanol and the crude extracts partitioned into a CTX-containing dichloromethane fraction and a MTX-containing aqueous methanol fraction. CTX-toxicity was estimated using the neuro-2a cytoxicity assay, and MTX-toxicity via a human erythrocyte lysis assay. Different species were grouped into different ratios of CTX- and MTX-toxicity, however, the ratio was not related to the geographical origin of species (Atlantic, Mediterranean, Pacific). All strains showed MTX-toxicity, ranging from 1.5 to 86pg MTX equivalents (eq) cell. All but one of the strains showed relatively low CTX-toxicity ranging from 0.6 to 50 fg CTX3C eq cell. The exception was the highly toxic G. excentricus strain from the Canary Islands, which produced 1426 fg CTX3C eq cell. As was true for CTX, the highest MTX-toxicity was also found in G. excentricus. Thus, the present study confirmed that at least one species from the Atlantic Ocean demonstrates similar toxicity as the most toxic strains from the Pacific, even if the metabolites in fish have so far been shown to be more toxic in the Pacific Ocean.

The 5.8S ribosomal RNA (rDNA) gene and flanking internal transcribed spacers (ITS1 and ITS2)from 9 isolates of Alexandrium catenella (Whedon and Kofoid) Taylor, 11 isolates of A. tamarense (Lebour) Taylor, and single isolates of A. affine (Inoue et Fukuyo) Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from various locations in Japan were amplified using the polymerase chain reaction (PCR) and subjected to restriction fragment‐length polymorphism (RFLP) analysis. PCR products from all strains were approximately 610 bp, inclusive of a limited region of the 18S and 28S rRNA coding regions. RFLP analysis using four restriction enzymes revealed six distinct classes of rDNA (“ITS types”). Restriction patterns of A. catenella were uniform at the intra‐specific level and clearly distinguishable from those of A. tamarense. The patterns associated with A. tamarense (“tamarense group”) were also uniform except for one strain, WKS‐1. Some restriction fragments from WKS‐1 were in common with those of A. catenella or A. tamarense, whereas some were distinct from all Alexandrium species tested. Alexandrium affine, A. insuetum, and A. pseudogonyaulax carry unique ITS types. The ITSs of the “tamarense group” exhibit sequence heterogeneity. In contrast, the ITSs of all other isolates (including WKS‐1) appear homogeneous. RFLP analysis of the 5.8S rDNA and flanking ITSs regions from Alexandrium species reveals useful taxonomic and genetic markers at the species and/or population levels.

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