Aerobic sister lineage of Breviates has gene-rich mitochondrial genomes

Breviates are anaerobic/micro-aerophilic flagellates that harbor hydrogen-producing MROs and lack a mitochondrial genome (mtDNA). Breviates alongside apusomonads and opisthokonts (the group that includes animals and fungi) 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 found a new lineage closely related to known breviates based on phylogenomics. Surprisingly, we recovered several mtDNAs in the breviate-related (BR) SAGs, which encode most of the genes involved in electron transport chain complexes. These BR mtDNAs harbour rare mitochondrial genes such as rpl21 and rnpB that are lost or usually encoded in nuclear genomes in most other eukaryotes. In some of the nuclear genomes of the corresponding 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 SAGs of apusomonads that are gene-rich compared to known mtDNAs from this group. The identification and initial characterization of a new clade within Obazoa provides avenues to better understand the process of mitochondrial reductive evolution in breviates and apusomonads, and highlights the important role of microscope-independent approaches, such as fluorescence-activated cell sorting and single cell genomics, in discovering new lineages.Folder descriptions:NOTE**: red labels in the treefiles are dimmed paralogs, contaminants, or different proteins and were removed in the final analyses.Figure1_FigureS1_PhyloFisherFig1_Pooled_SAGs: files used to produce Fig 1 tree.concat_matrix: matrix file and indicesgenes: gene files used to construct the matrixtreefiles: intermediate and final tree files under the model PMSF C60Fig1S_NotPooled_SAGs: files used to produce Fig 1S treeconcat_matrix: matrix file and indicesgenes: gene files used to construct the matrixtreefiles: intermediate and final tree files under the model PMSF C60Figure2_and_FigureS2_18S_tree_and_algnmentsFig2: 18S rDNA sequences of newly identified BR and APU aligned with the 18S rRNA gene alignments obtained from Torruella et al. 2022 (https://doi.org/10.1111/jeu.12956). This folder contains two treefiles inferred using GTRFR4 model and Modelfinder (MFP) option. "opimoda_wBRAP*trimal" file was used to infer both trees. Also contains mafft aligned file, trimmed file, and all IQ-TREE outputs.FigS2_EPAanalysis: reference treefile (nwk) and alignment file (phy) used to conduct EPA analysis and PaPaRa alignment. All the intermediate files can be found in the folder "EPA_analysis". "treefiles_raxml" folder contains the treefile used in Fig S2. The liklihood-weight ratio (LWR) data can be found in "EPA_analysis".Figure3A_Williamson2025_dataset_sequences_treescleaned_alignments: confirmed genes from BR and APU SAGs used in the matrixconcatenated_matrix_iqtree: matrix and iqtree filesinitial_sgtree_preclean: initial single-gene trees to screen orthologs for the corresponding SAGsWilliamson2025_gene_name.txt: list of gene file and its corresponding gene namesFigure3B_ETC_sequences_and_treesConcatenated_IQtrees: matrix and treefiles used for Fig 2BCore_ETC_24_genes: files used to generate single-gene treesAdditional files: also contain files for Fig S2 (used modelfinder to estimate the tree)Figure4_Mitochondrial_genomes14 Mitochondrial genomes from this study. Used to generate Fig 4.Figure5_splitSDH2_alignmentsAlignments of SDH2/SDHB from eukaryotes and split genes from euglenozoans and from this studyFigure6_mitochondria_proteins_MRO_MTSsingle-gene trees used to find MRO and mitochondrial proteins in the BR SAGs from Import_Chaperones, Iron_sulfur, and MRO_genes.also contain results for searching mitochondrial targeting sequence analyses, using MitoFates, MitoProt, targetP, and TPpred3.BRAPU_protMar2026 Folderthree sub-folders: 1) preducted_euk_aa, contains amino acid sequences of predicted proteins from raw asseblies using augustus; 2) proteome_for_phylofisher, contains proteome used to construct trees using PhyloFisher; 3) raw_assemblies, contains nucleotide assemblies