Ultrastructure and molecular phylogeny of Calkinsia aureus: cellular identity of a novel clade of deep-sea euglenozoans with epibiotic bacteria

Background: The Euglenozoa is a large group of eukaryotic flagellates with diverse modes of nutrition. The group consists of three main subclades - euglenids, kinetoplastids and diplonemids - that have been confirmed with both molecular phylogenetic analyses and a combination of shared ultrastructural characteristics. Several poorly understood lineages of putative euglenozoans live in anoxic environments, such as Calkinsia aureus, and have yet to be characterized at the molecular and ultrastructural levels. Improved understanding of these lineages is expected to shed considerable light onto the ultrastructure of prokaryote-eukaryote symbioses and the associated cellular innovations found within the Euglenozoa and beyond. Results: We collected Calkinsia aureus from core samples taken from the low-oxygen seafloor of the Santa Barbara Basin (580 - 592 m depth), California. These biflagellates were distinctively orange in color and covered with a dense array of elongated epibiotic bacteria. Serial TEM sections through individually prepared cells demonstrated that C. aureus shares derived ultrastructural features with other members of the Euglenozoa (e.g. the same paraxonemal rods, microtubular root system and extrusomes). However, C. aureus also possessed several novel ultrastructural systems, such as modified mitochondria (i.e. hydrogenosome-like), an "extrusomal pocket", a highly organized extracellular matrix beneath epibiotic bacteria and a complex flagellar transition zone. Molecular phylogenies inferred from SSU rDNA sequences demonstrated that C. aureus grouped strongly within the Euglenozoa and with several environmental sequences taken from low-oxygen sediments in various locations around the world. Conclusion: Calkinsia aureus possesses all of the synapomorphies for the Euglenozoa, but lacks traits that are specific to any of the three previously recognized euglenozoan subgroups. Molecular phylogenetic analyses of C. aureus demonstrate that this lineage is a member of a novel euglenozoan subclade consisting of uncharacterized cells living in low-oxygen environments. Our ultrastructural description of C. aureus establishes the cellular identity of a fourth group of euglenozoans, referred to as the "Symbiontida".Botany, Department ofScience, Faculty ofZoology, Department ofReviewedFacult