Species and genetic diversity of notorious dinoflagellates Pfiesteria piscicida, Luciella masanensis, and relatives in marine sediments of China

The dinoflagellate genus Pfiesteria, encompassing Pfiesteria piscicida and P. shumwayae, became a hot topic in HABs research in the early 2000s due to numerous but controversial reports regarding life cycle and toxicity. While Pfiesteria presence has been reported from all continents, surprisingly, there has been no documented presence in China to date. Here, we report our findings on the presence, species, and genetic diversity of Pfiesteria and its phylogenetic relatives (particularly Luciella masanensis) using a combined approach. First, we demonstrated the presence of P. piscicida and L. masanensis using metabarcoding analysis of >320 sediment samples. Pfiesteria piscicida was identified in 32 sampling sites across all four seas of China, with rDNA sequences exhibiting considerable differences from the type strain (up to 3.83 %), while Luciella masanensis presented in 212 sites from all four seas and included only ribotypes 1 and 3 among the four known ribotypes. Second, based on the metabarcoding detections, our application of FISH with species-specific probes and subsequent single-cyst PCR sequencing to the “positive” sediments confirmed, both morphologically and molecularly, the existence of P. piscicida and L. masanensis cysts in the sediments. Finally, individual cysts were isolated using the sodium polytungstate protocol and sequenced targeting 28S rDNA D1–D6 domains. Phylogenetic analysis revealed that 35 resting cysts were relatives of Pfiesteria, likely belonging to either new species or novel genera and family because they formed at least two distinct clades in the phylogenetic tree. Pfiesteria shumwayae was not detected from any sample, suggesting its absence in Chinese waters. We believe this work provides important factual basis for the global biogeography of these species and future HABs monitoring of in China.