First report of Alexandrium (Dinophyceae) associated with marine macroalgae off Japan: Diversity, distribution, and toxicity.

ABSTRACT

Macroalgal samples were collected from coastal waters in subboreal to subtropical zones in Japan (< 3-30 m depths) and 32 clonal strains of non-motile dinoflagellate-like protists were established. Molecular phylogenetic analyses of the LSU rDNA D1/D2, SSU rDNA, ITS region, and concatenated SSU rDNA + LSU rDNA D1/D2 sequences revealed that the strains nested within the genus Alexandrium. They were separated into three novel phylotypes Alexandrium spp. type 1, type 2, and type 3. Analysis of the concatenated sequences revealed that the most closely related species for the three phylotypes was A. ostenfeldii. Most cells from strains of the three phylotypes were non-motile and hemispherical to spherical in shape. The average diameters of the non-motile cells were between 35 and 39 µm. Type 1 and type 2 were widely distributed in Japan from the temperate to subtropical zones, whereas type 3 was restricted to the temperate zone. Furthermore, type 2 was widespread from shallow to deep waters, whereas type 1 and type 3 were restricted to deep waters. Growth experiments in strains belonging to the three phylotypes revealed that the occurrence ratios of motile cells were very low (≤ 1.1% of the total cells). The production of paralytic shellfish poisoning toxins, tetrodotoxin, and cyclic imines was assessed in strains belonging to the three phylotypes by LC/MS/MS analysis. The strains did not produce any of the toxins tested. The strains of the three phylotypes showed lethal toxicity to mice by intraperitoneal administration. To the best of our knowledge, this is the first study to report the existence of Alexandrium associated with marine macroalgae from Japan.