Molecular phylogenetic analysis of the heterotrophic Chrysophyte genus Paraphysomonas (Chrysophyceae), and the design of rRNA-targeted oligonucleotide probes for two species

Nanoflagellate protists are algae and protozoa (2-20 mu m in size) that play important ecological roles in freshwater and marine microbial communities as primary producers and as consumers of prokaryotic and eukaryotic prey. There is little biogeographical information for most of these minute protists despite their significant role in aquatic food webs. In addition, the evolutionary relationships among some of these species and their affinities to other protistan taxa are unclear, These circumstances are largely a consequence of the fact that small protists possess few readily apparent morphological features on which to base taxonomic and phylogenetic schemes and with which to identify them in natural assemblages. As an alternative approach for addressing these issues, we sequenced the small-subunit ribosomal RNA genes of four species of the colorless chrysophyte genus Paraphysomonas. A phylogenetic analysis based on that sequence information was performed, and oligonucleotide probes for two commonly occurring species of Paraphysomonas were designed and tested. Phylogenetic analyses of these four species confirmed the affinity of the genus Paraphysomonas with other chrysophyte species. High sequence similarity among three of the species (P. imperforata Lucas, P. bandaiensis Takahashi, and P. foraminifera Lucas) supported a previous phylogenetic grouping of these species based on the morphology of the scales produced by these species, In particular, sequence similarity between P, imperforata and P. foraminifera indicated that this speciation was a recent evolutionary event. However, a fourth species (P, vestita (Stokes) de Saedeleer) possessing similar scale morphology to P. bandaiensis, P, imperforata, and P. foraminifera showed considerable sequence dissimilarity in comparison to these latter three species. Oligonucleotide probes were successfully designed for the species P, imperforata and P, bandaiensis and applied together with a recently developed quantitative in situ hybridization procedure. The development of species-specific oligonucleotide probes for these nanoflagellate species and their application for counting nanoflagellates in natural water samples provide tools for studying these ecologically important species.