Protoperidinium minutum (Dinophyceae) from Portugal: Cyst-theca relationship and phylogenetic position on the basis of single-cell SSU and LSU rDNA sequencing

Round brown spiny cysts are common elements of Recent and Quaternary dinoflagellate cyst records and are often used to infer past climate conditions. Echinidinium and Islandinium, two cyst-based genera composed of round brown spiny cysts, are believed to have affinities within the Protoperidiniaceae. However, their biological counterparts are still virtually unknown. In this study, we examined the cyst-theca relationship of an Echinidinium-like cyst isolated from recent sediments of the Portuguese coast. The cysts (25-34 μm) had an intercalary theropylic archeopyle and numerous processes (4-9 μm) with tapered stems and minutely expanded tips. Germinated cells were identified as Protoperidinium minutum on the basis of theca morphology and tabulation. This taxon has a complicated taxonomic history and most likely represents a complex of species with very similar thecae but different cyst morphologies. To provide a first step in elucidating the phylogeny of P. minutum and its evolutionary relationship among the Protoperidiniaceae, we undertook the first molecular study of this taxon on the basis of small-subunit (SSU) and large-subunit (LSU) ribosomal (r)DNA genetic sequences obtained through single-cell polymerase chain reaction. On the basis of SSU rDNA analysis, P. minutum formed a clade together with the Diplopsaloideae, not grouping together with the other Protoperidinium species. LSU rDNA-based phylogenies indicate P. minutum as early divergent within the Protoperidiniaceae. The evolutionary significance of round brown spiny cysts produced by P. minutum-like species and diplopsalids is discussed.