Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing

Abstract

Very small eukaryotic organisms (picoeukaryotes) are fundamental components of marine planktonic systems, often accounting for a significant fraction of the biomass and activity in a system. Their identity, however, has remained elusive, since the small cells lack morphological features for identification. We determined the diversity of marine picoeukaryotes by sequencing cloned 18S rRNA genes in five genetic libraries from North Atlantic, Southern Ocean, and Mediterranean Sea surface waters. Picoplankton were obtained by filter size fractionation, a step that excluded most large eukaryotes and recovered most picoeukaryotes. Genetic libraries of eukaryotic ribosomal DNA were screened by restriction fragment length polymorphism analysis, and at least one clone of each operational taxonomic unit (OTU) was partially sequenced. In general, the phylogenetic diversity in each library was rather great, and each library included many different OTUs and members of very distantly related phylogenetic groups. Of 225 eukaryotic clones, 126 were affiliated with algal classes, especially the Prasinophyceae, the Prymnesiophyceae, the Bacillariophyceae, and the Dinophyceae. A minor fraction (27 clones) was affiliated with clearly heterotrophic organisms, such as ciliates, the chrysomonad Paraphysomonas, cercomonads, and fungi. There were two relatively abundant novel lineages, novel stramenopiles (53 clones) and novel alveolates (19 clones). These lineages are very different from any organism that has been isolated, suggesting that there are previously unknown picoeukaryotes. Prasinophytes and novel stramenopile clones were very abundant in all of the libraries analyzed. These findings underscore the importance of attempts to grow the small eukaryotic plankton in pure culture.

FIG. 1

DGGE gel separating eukaryotic 18S rDNA fragments from the populations retained on prefilters and from the populations appearing in filtrates from the five samples used to generate genetic libraries. The filtrate samples analyzed by using genetic libraries are circled.

FIG. 2

Numbers of bands that were unique to the filtrates, that were shared, and that were unique to the prefilters for the five samples used to generate genetic libraries after quantitative analysis of the DGGE gel shown in Fig. 1. The values above the bars for unique bands are the percentages of band intensity accounted for by these bands in the DGGE profile.

FIG. 3

Phylogenetic tree for partial sequences of environmental clones and the most closely related cultured organisms. Environmental clones are indicated by boldface type and each clone is designated by the library designation followed by a number. One clone representing each different OTU detected by the RFLP analysis of the five genetic libraries is included. The bar indicates 10% estimated sequence divergence. The number of clones in each genetic library belonging to each phylogenetic group is indicated on the right.