Analysis of photosynthetic picoeukaryote diversity at open ocean sites in the Arabian Sea using a PCR biased towards marine algal plastids

Marine photosynthetic picoeukaryotes (PPEs), representing organisms < 3 mu m in size, are major contributors to global carbon cycling. However, the key members of the PPE community and hence the major routes of carbon fixation, particularly in the open ocean environment, are poorly described. Here, we have accessed PPE community structure using the plastid encoded 16S rRNA gene. Plastid 16S rRNA genes were sequenced from 65 algal cultures, about half being PPEs, representing 14 algal classes. These included sequences from 5 classes where previously no such sequences from cultured representatives had been available (Bolidophyceae, Dictyochophyceae, Eustigmatophyceae, Pelagophyceae and Pinguiophyceae). Sequences were also obtained for 6 of the 7 (according to 18S rRNA gene sequence) prasinophyte clades. Phylogenetic analysis revealed plastids from the same class as clustering together. Using all the obtained sequences, as well as plastid sequences currently in public databases, a non-degenerate marine algal plastid-biased PCR primer, PLA491F, was developed to minimize amplification of picocyanobacteria, which often dominate numerically environmental samples. Clone libraries subsequently constructed from the pico-sized fraction from 2 open ocean sites in the Arabian Sea, revealed an abundance of 16S rRNA gene clones phylogenetically related to chrysophytes, whilst prymnesiophyte, clade II prasinophyte (Ostreococcus-like) and pelagophyte clones were also well represented. The finding of a wealth of novel clones related to the Chrysophyceae highlights the utility of a PCR biased towards marine algal plastids as a valuable complement to 18S rDNA based studies of PPE diversity