ABSTRACT Endomyxans are a poorly sampled and incompletely resolved aggregate of Rhizarian lineages that fall outside Filosa and Retaria. Among them, “Novel Clade 12” (NC12; Bass et al. 2009) is an environmental clade comprised primarily of sequences derived from anoxic sediments, hitherto lacking a morphologically-characterised representative. We have cultivated a marine anaerobic eukaryotroph, SSF, that we identify as the first representative of NC12. SSF is a teardrop-shaped cell with two unequal flagella emerging a third of the way down the cell behind a distinctive row of refractile globules. The posterior end of the cell is filled with food vacuoles. There is a surface thickening discernible in light microscopy. We also describe another distinct anaerobe eukaryotrophic lineage, also cultivated from marine sediment: PG. It consists of large pyriform cells with a substantial trailing “tail” and two unequal flagella, the posterior exceptionally long. In small subunit ribosomal RNA gene phylogenies, it falls outside the characterised clades and forms a distinct novel rhizarian lineage in its own right. Together, SSF and PG represent two additional independent adaptations to anoxic conditions within Rhizaria.
The complexity of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase) has increased considerably relative to the homologous complex in bacteria. Comparative analyses of CI composition in animals, fungi and land plants/green algae suggest that novel components of mitochondrial CI include a set of 18 proteins common to all eukaryotes and a variable number of lineage-specific subunits. In plants and green algae, several purportedly plant-specific proteins homologous to gamma-type carbonic anhydrases (gammaCA) have been identified as components of CI. However, relatively little is known about CI composition in the unicellular protists, the characterizations of which are essential to our understanding of CI evolution.We have performed a tandem mass spectrometric characterization of CI from the amoeboid protozoon Acanthamoeba castellanii. Among the proteins identified were two gammaCA homologs, AcCa1 and AcCa2, demonstrating that gammaCA proteins are not specific to plants/green algae. In fact, through bioinformatics searches we detected gammaCA homologs in diverse protist lineages, and several of these homologs are predicted to possess N-terminal mitochondrial targeting peptides.The detection of gammaCAs in CI of Acanthamoeba, considered to be a closer relative of animals and fungi than plants, suggests that gammaCA proteins may have been an ancestral feature of mitochondrial CI, rather than a novel, plant-specific addition. This assertion is supported by the presence of genes encoding gammaCAs in the nuclear genomes of a wide variety of eukaryotes. Together, these findings emphasize the importance of a phylogenetically broad characterization of CI for elucidating CI evolution in eukaryotes.
