Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological, molecular and ecophysiological data.

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.

High abundance of Amphidomataceae (Dinophyceae) during the 2015 spring bloom of the Argentinean Shelf and a new, non-toxigenic ribotype of Azadinium spinosum.

Azaspiracids (AZA) are the most recently discovered group of lipophilic marine biotoxins of microalgal origin, and associated with human incidents of shellfish poisoning. They are produced by a few species of Amphidomataceae, but diversity and occurrence of the small-sized dinophytes remain poorly explored for many regions of the world. In order to analyze the presence and importance of Amphidomataceae in a highly productive area of Argentinean coastal waters (El Rincón area, SW Atlantic), a scientific cruise was performed in 2015 to sample the early spring bloom. In a multi-method approach, light microscopy was combined with real-time PCR molecular detection of Amphidomataceae, with chemical analysis of AZA, and with the establishment and characterization of amphidomatacean strains. Both light microscopy and PCR revealed that Amphidomataceae were widely present in spring plankton communities along the El Rincón area. They were particularly abundant offshore at the shelf front, reaching peak densities of 2.8 × 105 cells L-1, but no AZA were detected in field samples. In total, 31 new strains were determined as Az. dalianense and Az. spinosum, respectively. All Az. dalianense were non-toxigenic and shared the same rRNA sequences. The large majority of the new Az. spinosum strains revealed for the first time the presence of a non-toxigenic ribotype of this species, which is otherwise the most important AZA producer in European waters. One of the new Az. spinosum strains, with a particular slender shape and some other morphological peculiarities, clustered with toxigenic strains of Az. spinosum from Norway and, exceptionally for the species, produced only AZA-2 but not AZA-1. Results indicate a wide diversity within Az. spinosum, both in terms of sequence data and toxin profiles, which also will affect the qualitative and quantitative performance of the specific qPCR assay for this species. Overall, the new data provide a more differentiated perspective of diversity, toxin productivity and occurrence of Amphidomataceae in a poorly explored region of the global ocean.