Ostreococcus spp. are common worldwide oceanic picoeukaryotic pelagic algae. The complete genomes of three strains from different ecological niches revealed them to represent biologically distinct species despite their identical cellular morphologies (cryptic species). Their tiny genomes (13 Mb), with approximately 20 chromosomes, are colinear and densely packed with coding sequences, but no sexual life cycle has been described. Seventeen new strains of one of these species, Ostreococcus tauri, were isolated from 98 seawater samplings from the NW Mediterranean by filtering, culturing, cloning, and plating for single colonies and identification by sequencing their ribosomal 18S gene. In order to find the genetic markers for detection of polymorphisms and sexual recombination, we used an in silico approach to screen available genomic data. Intergenic regions of DNA likely to evolve neutrally were analyzed following polymerase chain reaction amplification of sequences using flanking primers from adjacent conserved coding sequences that were present as syntenic pairs in two different species of Ostreococcus. Analyses of such DNA regions from eight marker loci on two chromosomes from each strain revealed that the isolated O. tauri clones were haploid and that the overall level of polymorphism was approximately 0.01. Four different genetic tests for recombination showed that sexual exchanges must be inferred to account for the between-locus and between-chromosome marker combinations observed. However, our data suggest that sexual encounters are infrequent because we estimate the frequency of meioses/mitoses among the sampled strains to be 10(-6). Ostreococcus tauri and related species encode and express core genes for mitosis and meiosis, but their mechanisms of cell division and recombination, nevertheless, remain enigmatic because a classical eukaryotic spindle with 40 canonical microtubules would be much too large for the available approximately 0.9-microm(3) cellular volume.