PCR protocols for genetic identification of dinoflagellates directly from single cysts and plankton cells

Abstract

A simple preparation method and PCR protocol are described which allow successful PCR amplification of partial ribosomal RNA gene sequences from as little as one dinoflagellate cyst or vegetative cell. Amplification from single or small numbers of cysts can be applied to a range of morphologically identifiable cyst species and produces identical rDNA sequence data to those obtained from DNA extractions from cultured vegetative cells. Applications of the approach have the potential to aid phylogenetic studies of dinoflagellates and other microalgae by: (1) improving taxonomic sampling of unculturable and heterotrophic species; (2) providing data to link cysts of unknown affinity with their potential planktonic cell counterparts; and (3) confirming the identification of cysts that cannot be germinated or are non-viable. Examples are presented where this method was used to confirm the identity and distribution of nonviable microreticulate cysts in coastal marine sediment samples, such as those of the recently described species Gymnodinium microreticulatum.