Description: The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.

Description: Azadinium poporum produces a variety of azaspiracids and consists of several ribotypes, but information on its biogeography is limited. A strain of A. poporum (GM29) was incubated from a Gulf of Mexico sediment sample. Strain GM29 was characterized by a plate pattern of po, cp, x, 4′, 3a, 6″, 6C, 5S, 6‴, 2⁗, a distinct ventral pore at the junction of po and the first two apical plates, and a lack of an antapical spine, thus fitting the original description of A. poporum. The genus Azadinium has not been reported in waters of the United States of America before this study. Molecular phylogeny, based on large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences, reveals that strain GM29 is nested within the well-resolved A. poporum complex, but forms a sister clade either to ribotype B (ITS) or ribotype C (LSU). It is, therefore, designated as a new ribotype, termed as ribotype D. LSU and ITS sequences similarity among different ribotypes of A. poporum ranges from 95.4% to 98.2%, and from 97.1% to 99.2% respectively, suggesting that the LSU fragment is a better candidate for molecular discrimination. Azaspiracid profiles were analyzed using LC–MS/MS and demonstrate that strain GM29 produces predominantly AZA-2 with an amount of 45 fg/cell. The results suggest that A. poporum has a wide distribution and highlights the risk potential of azaspiracid intoxication in the United States.

Description: The marine planktonic dinophyceaen genus Azadinium is a primary source of azaspiracids, but due to their small size its diversity may be underestimated and information on its biogeography is still limited. A new Azadinium species, A. zhuanum was obtained from the East China Sea and Yellow Sea of China by incubating surface sediments. Five strains were established by isolating single germinated cells and their morphology was examined with light microscopy and scanning electron microscopy. Azadinium zhuanum was characterized by a plate pattern of Po, cp, X, 4′, 2a, 6′′, 6C, 5S, 6′′′, 2′′′′, by a distinct ventral pore at the junction of Po, the first and fourth apical plates, and a conspicuous antapical spine. Moreover, Azadinium poporum was obtained for the first time from the Mediterranean by incubating surface sediment collected from Diana Lagoon (Corsica) and a new strain of Azadinium dalianense was isolated from the French Atlantic. The morphology of both strains was examined. Small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer (ITS) sequences were obtained from cultured strains. In addition, LSU sequences were obtained by single cell sequencing of two presumable A. poporum cells collected from the French Atlantic. Molecular phylogeny based on concatenated SSU, LSU and ITS sequences revealed that A. zhuanum was closest to A. polongum. French A. poporum from Corsica (Mediterranean) and from the Atlantic showed some genetic differences but were nested within one of the A. poporum ribotypes together with other European strains. Azadinium dalianense from France together with the type strain of the species from China comprised a well resolved clade now consisting of two ribotypes. Azaspiracid profiles were analyzed for the cultured Azadinium strains using LC–MS/MS and demonstrate that the Mediterranean A. poporum strain produced AZA-2 and AZA-2 phosphate with an amount of 0.44 fg cell−1. Azadinium zhuanum and A. dalianense did not produce detectable AZA. Results of the present study support the view of a high diversity and wide distribution of species belonging to Azadinium. The first record of AZA-2 producing A. poporum from the Mediterranean suggests that this species may be responsible for azaspiracid contaminations in shellfish from the Mediterranean Sea.

Description: A recently published study analyzed the phylogenetic relationship between the genera Centrodinium and Alexandrium, confirming an earlier publication showing the genus Alexandrium as paraphyletic. This most recent manuscript retained the genus Alexandrium, introduced a new genus Episemicolon, resurrected two genera, Gessnerium and Protogonyaulax, and stated that: “The polyphyly [sic] of Alexandrium is solved with the split into four genera”. However, these reintroduced taxa were not based on monophyletic groups. Therefore this work, if accepted, would result in replacing a single paraphyletic taxon with several non-monophyletic ones. The morphological data presented for genus characterization also do not convincingly support taxa delimitations. The combination of weak molecular phylogenetics and the lack of diagnostic traits (i.e., autapomorphies) render the applicability of the concept of limited use. The proposal to split the genus Alexandrium on the basis of our current knowledge is rejected herein. The aim here is not to present an alternative analysis and revision, but to maintain Alexandrium. A better constructed and more phylogenetically accurate revision can and should wait until more complete evidence becomes available and there is a strong reason to revise the genus Alexandrium. The reasons are explained in detail by a review of the available molecular and morphological data for species of the genera Alexandrium and Centrodinium. In addition, cyst morphology and chemotaxonomy are discussed, and the need for integrative taxonomy is highlighted.