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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]

• 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]