Euglenozoa is a major phylum of excavate protozoa (comprising euglenoids, kinetoplastids, and diplonemids) with highly unusual nuclear, mitochondrial, and chloroplast genomes. To improve understanding of euglenozoan evolution, we sequenced nuclear small-subunit rRNA genes from 34 bodonids (Bodo, Neobodo, Parabodo, Dimastigella-like, Rhynchobodo, Rhynchomonas, and unidentified strains), nine diplonemids (Diplonema, Rhynchopus), and a euglenoid (Entosiphon). Phylogenetic analysis reveals that diplonemids and bodonids are more diverse than previously recognised, but does not clearly establish the branching order of kinetoplastids, euglenoids, and diplonemids. Rhynchopus is holophyletic; parasitic species arose from within free-living species. Kinetoplastea (bodonids and trypanosomatids) are robustly holophyletic and comprise a major clade including all trypanosomatids and most bodonids (‘core bodonids’) and a very divergent minor one including Ichthyobodo. The root of the major kinetoplastid clade is probably between trypanosomatids and core bodonids. Core bodonids have three distinct subclades. Clade 1 has two distinct Rhynchobodo-like lineages; a lineage comprising Dimastigella and Rhynchomonas; and another including Cruzella and Neobodo. Clade 2 comprises Cryptobia/ Trypanoplasma, Procryptobia, and Parabodo. Clade 3 is an extensive Bodo saltans species complex. Neobodo designis is a vast genetically divergent species complex with mutually exclusive marine and freshwater subclades. Our analysis supports three phagotrophic euglenoid orders: Petalomonadida (holophyletic), Ploeotiida (probably holophyletic), Peranemida (paraphyletic).
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