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Background  Parabasalia are single-celled eukaryotes (protists) that are mainly comprised of endosymbionts of termites and wood roaches, intestinal commensals, human or veterinary parasites, and free-living species. Phylogenetic comparisons of parabasalids are typically based upon morphological characters and 18S ribosomal RNA gene sequence data (rDNA), while biochemical or molecular studies of parabasalids are limited to a few axenically cultivable parasites. These previous analyses and other studies based on PCR amplification of duplicated protein-coding genes are unable to fully resolve the evolutionary relationships of parabasalids. As a result, genetic studies of Parabasalia lag behind other organisms.    Principal Findings  Comparing parabasalid EF1α, α-tubulin, enolase and MDH protein-coding genes with information from the  Trichomonas vaginalis  genome reveals difficulty in resolving the history of species or isolates apart from duplicated genes. A conserved single-copy gene encodes the largest subunit of RNA polymerase II (Rpb1) in  T. vaginalis  and other eukaryotes. Here we directly sequenced  Rpb1  degenerate PCR products from 10 parabasalid genera, including several  T. vaginalis  isolates and avian isolates, and compared these data by phylogenetic analyses.  Rpb1  genes from parabasalids, diplomonads,  Parabodo ,  Diplonema  and  Percolomonas  were all intronless, unlike intron-rich homologs in  Naegleria ,  Jakoba  and  Malawimonas .    Conclusions/Significance  The phylogeny of Rpb1 from parasitic and free-living parabasalids, and conserved Rpb1 insertions, support Trichomonadea, Tritrichomonadea, and Hypotrichomonadea as monophyletic groups. These results are consistent with prior analyses of rDNA and GAPDH sequences and ultrastructural data. The Rpb1 phylogenetic tree also resolves species- and isolate-level relationships. These findings, together with the relative ease of Rpb1 isolation, make it an attractive tool for evaluating more extensive relationships within Parabasalia.

Phylogeny of Parasitic Parabasalia and Free-Living Relatives Inferred from Conventional Markers vs. Rpb1, a Single-Copy Gene