Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean

Abstract

Despite the fact that the smallest eukaryotes (cells less than 5 micro m in diameter) play key roles in marine food webs, particularly in open oligotrophic areas, the study of their in situ diversity started just one year ago. Perhaps the most remarkable finding of the most recent studies has been the discovery of completely new phylogenetic lineages, such as novel clades belonging to the stramenopile and alveolate phyla. The two new groups account for a significant fraction of clones in genetic libraries from North Atlantic, equatorial Pacific, Antarctic, and Mediterranean Sea waters. However, the identities and ecological relevance of these organisms remain unknown. Here we investigate the phylogenetic relationships, morphology, in situ abundance, and ecological role of novel stramenopiles. They form at least eight independent clades within the stramenopile basal branches, indicating a large phylogenetic diversity within the group. Two lineages were visualized and enumerated in field samples and enrichments by fluorescent in situ hybridization using specific rRNA-targeted oligonucleotide probes. The targeted organisms were 2- to 3- micro m-diameter, round-shaped, nonpigmented flagellates. Further, they were found to be bacterivorous. One lineage accounted for up to 46% (average during an annual cycle, 19%) of heterotrophic flagellates in a coastal environment, providing evidence that novel stramenopiles are important and unrecognized components of the total stock of bacterial grazers.

FIG. 1.

Maximum-likelihood phylogenetic tree with complete 18S rDNA sequences of novel stramenopiles. The scale bar indicates 0.1% sequence divergence. Neighbor-joining and maximum-parsimony bootstrap values (left to right, respectively) are shown at the internal branches (100 replicates; values >70% shown). Bold numbers indicate identical bootstrap values in both analyses. Novel stramenopile lineages are marked with roman numerals (from I to VIII). The right box shows subtrees of several lineages with additional partial sequences. The position of lineage VIII is intermediate between lineages IV and VII. The code of the clones indicates their origin as follows: Mediterranean Sea (ME), Equatorial Pacific (OLI), North Atlantic (NA), and surface (ANT) and deep (DH) Antarctic waters.

FIG. 2.

Epifluorescence micrographs of novel stramenopiles. (a and c) DAPI-stained cells and the corresponding microscopic field showing NS3 (b) and NS4 (d) cells after FISH. Panels a and b also include insets showing a different microscopic field. The scale bar is 10 μm. Two of the five eukaryotes were NS3 cells (compare a and b), and two of the four eukaryotes were NS4 cells (compare c and d). The nuclear region, brightest by DAPI staining, is dimmer by FISH fluorescence, which is consistent with the cytoplasmic localization of ribosomes. The inset in panel d shows a NS4 cell (magnified three times) with one ingested FLB.

FIG. 3.

Counts of HF and PE and NS3 and NS4 cells in an enrichment culture, which was started with 2-μm-diameter-pore-filtered Blanes seawater in September 2001. Average values and standard errors from two replicates are shown. Note that both NS3 and NS4 numbers became higher than the total number of PE.

FIG. 4.

In situ abundance of HF and PE and of NS3 and NS4 cells (expressed as percentages of HF numbers) in samples collected in Blanes Bay during an annual cycle from March 2001 to February 2002.