New insights into the phylogeny of eukaryotes based on ciliate Hsp70 sequences.

The current framework of the eukaryotic phylogeny is based on the analysis of a comprehensive set of sequences of the small subunit ribosomal RNA. However, phylogenies based on protein-encoding genes are not completely congruent with this picture. Since congruence between different markers is the best tool to determine evolutionary history, we focused on Hsp70 (heat-shock protein of 70 kDa), a chaperone protein which is highly conserved and is a potentially reliable phylogenetic marker. We used a PCR-based approach to sequence Hsp70s in two distinct classes of Ciliates. Seven Hsp70s were identified from Paramecium tetraurelia (Oligohymenophora) and six Hsp70s from Euplotes aediculatus (Hypotricha), encompassing orthologous genes for all major Hsp70 classes of Eukaryotes, i.e., those localized in cytosol, in endoplasmic reticulum, and in mitochondria. Three independent phylogenies of eukaryotes, based on each set of orthologous genes, have been constructed using different tree reconstruction methods. A significant advantage of Hsp70s is the existence of outgroups close to Eukaryotes for these major classes, reducing the long-branch attraction artifact due to the outgroup. The monophyly of Ciliates is supported by good bootstrap proportions in the phylogenetic reconstructions, and this phylum is generally a sister-group of Sporozoa, forming the expected Alveolates clade. The Hsp70 seems to be a suitable phylogenetic marker since it recovers all the monophyletic groups, undoubtedly defined by morphological criteria. The Hsp70 trees are, however, notably different from the rRNA ones and do not show two aspects of the classical topology, i.e., the successive emergence of deeply branching groups and the vast assembly of the major eukaryotic groups, emerging at the tip of the tree, i.e., the "terminal crown". More precisely, the Hsp70 trees do not resolve the relationships between the major groups of Eukaryotes with confidence, in keeping with the hypothesis that all these groups emerged in a great radiation that occurred at the origin of all the extant Eukaryotes.