Picoeukaryotes

The least well-studied and understood component of the photosynthetic picoplankton is a diverse array of small eukaryotic algae collectively referred to as picoeukaryotes. These small algal cells may be the most abundant eukaryotes on earth and occur at concentrations between 102 to 104 cell ml-1 in the photic zone throughout the oceans (Diez et al. 2001). Their diversity, distribution as well as their ecology however remain largely unknown (Partensky et al. 1997). Much of the difficulty associated with studying these algae results from the lack of morphological variation when viewed under conventional light microscopy. Cells often merely appear as little green balls (Potter et al. 1997) and display few distinguishing features (Thomsen 1986, Simon et al. 1994, Caron et al. 1999). Often cells are referred to as LRGTs (Little Round Green Things). Epi-fluorescence microscopy yields little additional clues and cells appear as red-fluorescing spheres with no distinguishing features. Electron microscopy and cell culture may alleviate some of these limitations, but are too expensive, too difficult and too time consuming to be feasible as routine oceanographic techniques. Similarly flow cytometry can be used to estimate bulk cell abundance, but completely lacks phylogenetic resolution.

Despite limitations progress has been made in several ways over the past decade. At least three novel algal classes have been identified and newly described. Moestrup (Moestrup 1991) described the new class Pedinophyceae composed of the two green algae Pedinomonas micron and Pedinomonas minor in 1991. Shortly thereafter Pelagomonas calceolata was first identified and described in the marine ultraplankton (Andersen et al. 1993). 18S rDNA sequence data placed this species at an unresolved position among other chromophytic algae illustrating at the time how little was known regarding the phylogenetic diversity of heterokont algae (yellow-green algae with flagella of unequal length) in the environment. Pelagophytes are now thought to be an important and prolific component of the autotrophic picoplankton. Most recently the algal class Bollidophyceae, has been identified and described in cultures and the environment (Guillou et al. 1999a, Guillou et al. 1999b). Although the Bollidophyceae are not thought to be a major component of the picoplankton their discovery nonetheless illustrates that numerous novel lineages of picoeukaryotes may yet be identified.

To circumvent some of the limitations of traditional microscope-dependent techniques for the description of picoplankton communities, molecular techniques have been widely used. Using group specific 18S rDNA probes in order to probe for the prymnesiophyte fraction of an 18S rDNA PCR (Polymerase Chain Reaction) amplicon generated using universal eukaryotic 18S primers, Moon-van der Staay (Moon-van der Staay et al. 2000) found that this group accounted for less of the amplified DNA than would be expected based on pigment ratios determined by HPLC. While these data should be interpreted with caution due to the biased nature of PCR (for a discussion see below), the same group also demonstrated the presence of several novel prymensiophyte lineages in their 18S rDNA clone libraries, which had no equivalent among cultured species. Using a similar PCR based approach in order to amplify and clone picoplankton rbcL gene sequences the diversity of picophytoplankton in the oligotrophic Gulf of Mexico has also been studied (Pichard et al. 1997b, Paul et al. 2000b). In the first study only five unique rbcL gene-sequences were recovered. These sequences however demonstrated the presence of algae spanning the diversity of almost the entire form I clade. In the later study the presence of a diverse array of eukaryotic algae in a low salinity coastal plume was detected. Eukaryotic rbcL DNA sequences were related to prasinophytes, prymnesiophytes, diatoms and pelagophytes and shared between 85 and 99% similarity with cultured representatives in GenBank. In addition this study illustrated how unknown levels of genetic diversity within the picoplankton may not only exist in the form of divergent and novel lineages, but also in the form of small micro-diverse clades of closely related sequences. Several other studies have since also addressed the diversity of picoeukaryotes in the environment using 18S PCR based clone libraries (Diez et al. 2001, Moon-van der Staay et al. 2001, Vaulot et al. 2001). These studies further support the notion that the oceanic picoplankton (both heterotrophic as well as autotrophic) is genetically very diverse and yet contains a considerable number of undescribed lineages. In order to identify and characterize some of the classified and non-classified species in the environment FISH (Fluorescent In-Situ Hybridization) and DGGE (Denaturant Gradient Gel Electrophoresis) in particular has been useful. Using FISH several novel groups have been observed both in the Pacific Ocean and in coastal waters indicating their ubiquity (Vaulot et al. 2001). Two basal stramenopile lineages have been studied in field samples and enrichments in the northwestern Mediterranean Sea using FISH (Massana et al. 2002). Cells were 2-3µm in diameter and bacteriovorus. One particular lineage may have accounted for as much as 46% of the heterotrophic flagellates suggesting the tremendous importance of this unclassified stramenopile as a bacterial grazer.