Spatial fragmentation in the distribution of diatom endosymbionts from the taxonomically clarified dinophyte Kryptoperidinium triquetrum (= Kryptoperidinium foliaceum, Peridiniales).

Among the photosynthetically active dinophytes, the Kryptoperidiniaceae are unique in having a diatom as endosymbiont instead of the widely present peridinin chloroplast. Phylogenetically, it is unresolved at present how the endosymbionts are inherited, and the taxonomic identities of two iconic dinophyte names, Kryptoperidinium foliaceum and Kryptoperidinium triquetrum, are also unclear. Multiple strains were newly established from the type locality in the German Baltic Sea off Wismar and inspected using microscopy as well as molecular sequence diagnostics of both host and endosymbiont. All strains were bi-nucleate, shared the same plate formula (i.e., po, X, 4', 2a, 7'', 5c, 7s, 5''', 2'''') and exhibited a narrow and characteristically L-shaped precingular plate 7''. Within the molecular phylogeny of Bacillariaceae, endosymbionts were scattered over the tree in a highly polyphyletic pattern, even if they were gained from different strains of a single species, namely K. triquetrum. Notably, endosymbionts from the Baltic Sea show molecular sequences distinct from the Atlantic and the Mediterranean Sea, which is the first report of such a spatial fragmentation in a planktonic species of dinophytes. The two names K. foliaceum and K. triquetrum are taxonomically clarified by epitypification, with K. triquetrum having priority over its synonym K. foliaceum. Our study underlines the need of stable taxonomy for central questions in evolutionary biology.

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.