Although the ultrastructural characteristics listed above are expected to be present in most, if not all, members of the Symbiontida (the ultrastructural and molecular phylogeny of another lineage in this clade BV-6 molecular weight will be published shortly; Breglia, Yubuki, Hoppenrath and Leander, in preparation), this remains to be demonstrated with improved knowledge of euglenozoan diversity from both ultrastructural and molecular phylogenetic perspectives. Phylogenetic (apomorphy-based) diagnosis Euglenozoa Cavalier-Smith 1981 Symbiontida taxon nov. Yubuki, Edgcomb, Bernhard & Leander, 2009 Apomorphy Rod-shaped epibiotic bacteria above superficial layer

of mitochondrion-derived organelles with reduced or absent cristae, homologous to the organization in Calkinsia aureus, the type species (Figures 2, 4). Extended diagnosis of the type species Calkinsia aureus find more Lackey, 1960, emend., Yubuki, Edgcomb, Bernhard &

Leander, 2009 Paraxonemal rods present in flagella; kinetoplast DNA and pellicle strips absent; long complex transitional zone between the basal bodies and the axonemes. Rod-shaped epibiotic bacteria on perforated orange extracellular matrix. Cell with a large nucleus on the anterior ventral side and a battery of tubular extrusomes linked to an extrusomal pocket located adjacent to the nucleus. Feeding apparatus supported by both fibrous structures and microtubules that are derived from ventral root (VR). Small subunit ribosomal RNA gene sequence (EU753419) distinguishes Calkinsia aureus from all other symbiontid Inhibitor Library cell assay species. Conclusion Molecular phylogenies inferred from SSU rDNA demonstrate that C. aureus is closely related to several marine environmental sequences collected from low-oxygen environments, forming a novel subgroup within the Euglenozoa, referred to here as the “”Symbiontida”". Improved understanding of these flagellates is necessary for Calpain further demonstrating the cellular identity of the Symbiontida and for reconstructing the evolutionary radiation

of the euglenozoan lineage. In this study, we characterized the detailed ultrastructure of C. aureus and demonstrated all of the euglenozoan synapomorphies (e.g. flagellar apparatus) and several cellular innovations associated with symbiotic interactions with epibiotic bacteria (e.g., complex extracellular matrix). We also demonstrated novel ultrastructural systems found in this species, such as the extrusomal pocket. Environmental sequencing surveys from different low-oxygen environments around the world suggest that many symbiontid lineages have yet to be discovered and characterized. Continued exploration into the overall diversity of this group should contribute significantly to our understanding of eukaryotic evolution, especially in low-oxygen environments.