Aerobic sister lineage of breviates has gene-rich mitochondrial genomes

The tree of eukaryotes has gone through numerous overhauls in recent decades, leading to a growing consensus on major supergroups. This stabilization has revealed that anaerobic eukaryotic lineages evolved convergently and that the variety of non-canonical mitochondria in these anaerobes evolved in parallel. These non-canonical mitochondria, known as mitochondrion-related organelles (MROs), are found in organisms that thrive in oxygen-limited environments and have highly reduced or no mitochondrial genomes (mtDNA). Breviates are anaerobic/microaerophilic flagellates that harbor hydrogen-producing MROs and lack mtDNA. Breviates, alongside apusomonads and opisthokonts, form a lineage called Obazoa. To identify novel microaerophiles within Obazoa, we sampled an intertidal mudflat and used flow cytometry-based single-cell sorting to generate single-cell amplified genomes (SAGs). We identified a new lineage closely related to known breviates using phylogenomics. Surprisingly, we recovered several mtDNAs in the breviate-related (BR) SAGs, with genes encoding most of the electron-transport chain complexes. These BR mtDNAs harbor rare mitochondrial genes such as rpl21 and rnpB that are lost or usually encoded in nuclear genomes in most eukaryotes. In several nuclear genomes of the BR SAGs, we detected genes encoding enzymes for anaerobic respiration and hydrogen production (e.g., [FeFe]-hydrogenase and pyruvate:ferredoxin oxidoreductase), suggesting that these organisms could be facultative anaerobes. We additionally recovered mtDNAs from apusomonad SAGs that are gene-rich compared with other known apusomonad mtDNAs. The identification and initial characterization of a new clade within Obazoa provide avenues to better understand the process of mitochondrial reductive evolution and highlight the important role of microscope-independent approaches in discovering new lineages.