Anaeramoebae are a divergent lineage of eukaryotes that shed light on the transition from anaerobic mitochondria to hydrogenosomes

Eukaryotes that live in low-oxygen environments often possess mitochondrion-related organelles (MROs) that are metabolically tailored to life without oxygen. Until recently, molecular investigations of the diversity of MRO functions and biogenesis were largely restricted to organelles of microbial eukaryotes (protists) with parasitic lifestyles, such as the hydrogenosomes of Trichomonas vaginalis (Parabasalia) and the mitosomes of Giardia intestinalis (Fornicata). However, in order to disentangle adaptations to parasitism from those that underpin anaerobiosis, it is critical to survey the MROs of diverse, free-living protists as well.Here we investigated the Anaeramoebae, a new phylum-level lineage of free-living marine anaerobic protists, by deeply sequencing transcriptomes of two Anaeramoeba species. Unexpectedly, the inclusion of Anaeramoeba sequences in phylogenomic analyses dramatically reordered the deep branching order of the metamonad tree, placing Anaeramoebae as a sister group to Parabasalia in a clade representing one of the two major lineages of Metamonada. Metabolic reconstructions of the Anaeramoeba MROs revealed many classical mitochondrial features previously not seen in metamonads, including a disulfide relay import system, propionate production, and amino acid metabolism. Anaerobic metabolism of these MROs is also expanded with at least two distinct hydrogen-producing pathways that use trimeric or monomeric FeFe-hydrogenases. Our findings suggest that the cenancestor of Metamonada likely had an MRO with many more classical mitochondrial features than previously anticipated and demonstrate how discoveries of novel protistan lineages of high taxonomic rank continue to transform our understanding of early eukaryote evolution.