Sequence-based diagnostics and phylogenetic approach of uncultured freshwater dinoflagellate Peridinium (Dinophyceae) species, based on single-cell sequencing of rDNA

The armoured dinoflagellate Peridinium is widely distributed in freshwater environments worldwide and contains a large number of species. Their identity, however, has remained elusive, since the small cells tend to be morphologically similar. To help resolve this, a sequence-based diagnostics for uncultured Peridinium cells from field samples was applied, using single-cell PCR and direct DNA sequencing of the PCR products. Single cells were isolated randomly from field samples, and PCR successfully amplified the target rDNA regions from the crude lysates. Phylogenetic trees showed that all the cells were strongly grouped into the same clade (> 99% bootstrap value), including the previously identified P. bipes f. occultatum, and apparently separated from relatives such as P. cinctum, P. volzii and P. willei. All 17 isolates were genotypically identified as P. bipes f. occultatum, based on over than 99% of sequence similarities, and the organism was responsible for water blooms at different seasons in Korean waters. The sequence-based typing could clearly resolve P. bipes f. occultatum from the various Peridinium cells, and that the method is accurate and more labor-saving than the conventional method to monitor Peridinium species. This protocol may be useful for the application of molecular tools to uncultured Peridinium cells.