Abundance and distribution of Ostreococcus sp. in the San Pedro Channel, California, as revealed by quantitative PCR.

Picoeukaryotes (algae and protozoa of <2 μm in diameter) from marine and freshwater ecosystems have increasingly become the focus of ecological, physiological, and genomic studies. The discovery of their widespread distribution, large genetic diversity, and periodically high abundance has caused a reassessment of their importance in microbial food webs. In particular, picoeukaryotic phototrophs contribute significantly to microbial biomass and total primary productivity (39, 40, 70). As a consequence, phototrophic picoeukaryotes (PPE) are an important component of the base of pelagic microbial food webs. Picoeukaryotes serve as prey for nanoplanktonic (2 to 20 μm) phagotrophic protists (14), whose grazing activities provide a link to higher trophic levels (57) and a mechanism for nutrient remineralization (56). Many picoeukaryotes have only recently been described in the scientific literature. These discoveries include both phototrophic (3, 16, 18, 31) and heterotrophic (30) taxa. The global distribution of many picoeukaryotes remains to be determined. However, Ostreococcus isolates have been documented at numerous sites around the world, including the Mediterranean Sea (22, 67), Long Island Sound, New York (48), the English Channel (32, 54), the Arabian Sea (9), and the North Pacific Ocean (13, 70; S. Suda et al., unpublished data). The discovery of many picoeukaryotes has been aided by advances in instrumentation (e.g., flow cytometry [FCM]), improved culturing techniques, and the development of culture-independent molecular assays. Culture-independent molecular tools such as environmental cloning/sequencing of rRNA genes (49) and DNA fragment-based techniques such as denaturing gradient gel electrophoresis (DGGE) (45) and terminal restriction fragment length polymorphism analysis (41) have become particularly important for revealing the presence of previously unknown taxa (21, 23, 30, 43) and documenting the geographic ranges of both newly and previously described taxa (42, 53). In addition, the accumulation of DNA sequence information in public databases has facilitated the development of taxon-directed approaches to identify and count species of interest. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) have proven effective for the detection and quantification of specific microbial eukaryotes from complex natural assemblages (47, 71). The use of qPCR for targeting eukaryotic microbes in environmental samples has received increased attention in recent years. A number of studies employing quantitative genetic methods for the study of bloom-forming algae that pose a threat to the health of humans or marine organisms have recently emerged (5, 8, 15, 28, 29, 50, 55). These approaches have detection accuracies over a large dynamic range and have substantially shorter sample processing times than more traditional techniques. Increasingly, qPCR methods have been developed for carrying out ecological studies of cryptic marine microbes. We designed and validated a qPCR assay specific to members of the genus Ostreococcus and applied this technique to samples collected at the University of Southern California (USC) Microbial Observatory site, located in coastal waters of the eastern North Pacific. Subsurface samples from 5 m and the deep chlorophyll maximum (DCM) were analyzed for a 2-year period (September 2000 to August 2002) to reveal Ostreococcus dynamics. Our estimates constitute some of the highest abundances ever reported for Ostreococcus (up to 3.2 × 105 cells ml−1) at a coastal oceanic site. Additionally, Ostreococcus comprised a substantial proportion of the total number of phototrophic picoeukaryotes in March 2005. Overall, this minute prasinophyte was almost always present and occasionally abundant at our study site off the coast of Southern California.