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: It is well known that modern resting cysts with morphologies matching those of species of the fossil genus Spiniferites germinate into motile cells of the genus Gonyaulax. Different Spiniferites species have been connected to a single Gonyaulax species, raising the question of whether they are over-classified. Through germination experiments of cysts with the morphological features of four species of Spiniferites, viz. S. bentorii, S. hyperacanthus, S. ramosus and S. scabratus, we established cyst-theca relationships. Cysts with the morphology of S. bentorii gave rise to vegetative, motile cells of Gonyaulax nezaniae sp. nov., which is characterized by two stout antapical spines. Cysts with S. hyperacanthus and S. ramosus morphologies germinated into Gonyaulax whaseongensis and G. spinifera, respectively. Cysts with S. scabratus morphology lacked a ventral pore and were attributed to Gonyaulax cf. spinifera. Gene sequences for SSU, LSU and/or ITS-5.8S rRNA were obtained from these four species, and from cysts with the morphology of Spiniferites belerius, S. mirabilis, S. lazus, Spiniferites cf. bentorii and Tectatodinium pellitum. The maximum likelihood and Bayesian inference analyses based on LSU and SSU rRNA gene sequences revealed that cysts assignable to Spiniferites formed a polyphyletic group, intermingled with Tectatodinium, Bitectatodinium, Ataxiodinium and Impagidinium, whereas Gonyaulax species appeared as monophyletic. From our results we inferred the phylogenetic positions of S. bentorii, S. mirabilis, S. lazus, S. scabratus, Tectatodinium pellitum and Gonyaulax digitale for the first time, supporting the idea that Spiniferites species are not over-classified and each of them may correspond to different Gonyaulax species.