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短膜虫类（Breviates）是一类厌氧/微需氧鞭毛虫，其细胞内具有产氢化酶的线粒体相关细胞器（mitochondrion-related organelles, MRO），且无线粒体基因组（mitochondrial genome, mtDNA）。短膜虫类与变形鞭毛虫类（Apusomonads）、后鞭毛生物（Opisthokonts，该类群包含动物与真菌）共同构成奥巴扎类群（Obazoa）。为鉴定奥巴扎类群中新型微需氧生物，我们采集了潮间带泥滩样本，采用基于流式细胞术的单细胞分选技术构建了单细胞扩增基因组（single-cell amplified genomes, SAGs）文库。通过系统发育基因组学分析，我们发现了一个与已知短膜虫类亲缘关系密切的新演化支。 令人意外的是，我们在该短膜虫类相关（breviate-related, BR）的SAGs中成功获取到了多个mtDNA，这些mtDNA编码了电子传递链复合物的绝大多数功能基因。此类BR型mtDNA还携带了rpl21、rnpB等罕见线粒体基因——这类基因在多数其他真核生物中要么发生丢失，要么通常由核基因组编码。 在对应BR SAGs的部分核基因组中，我们检测到了编码厌氧呼吸与产氢相关酶的基因（例如[FeFe]氢化酶、丙酮酸：铁氧还蛋白氧化还原酶），这表明此类生物可能为兼性厌氧菌。 此外，我们还从变形鞭毛虫类的SAGs中获得了mtDNA，与该类群已报道的mtDNA相比，此类mtDNA的基因丰度显著更高。 本次对奥巴扎类群内一新演化支的鉴定与初步表征，为深入解析短膜虫类与变形鞭毛虫类的线粒体还原进化过程提供了新的研究路径，同时凸显了无需依赖显微镜的研究方法——如荧光激活细胞分选与单细胞基因组学——在发现新演化支中的重要作用。 --- **文件夹说明：** > 注：树文件中的红色标记为弱化的旁系同源基因、污染序列或异常蛋白，在最终分析中已被移除。 ### Figure1_FigureS1_PhyloFisherFig1_Pooled_SAGs 用于生成图1系统发育树的文件： - `concat_matrix`：矩阵文件及索引 - `genes`：用于构建该矩阵的基因文件 - `treefiles`：基于PMSF C60模型生成的中间与最终树文件 ### Fig1S_NotPooled_SAGs 用于生成图1S系统发育树的文件： - `concat_matrix`：矩阵文件及索引 - `genes`：用于构建该矩阵的基因文件 - `treefiles`：基于PMSF C60模型生成的中间与最终树文件 ### Figure2_and_FigureS2_18S_tree_and_algnments 1. **Fig2**：新鉴定的BR（短膜虫类相关）与APU（变形鞭毛虫类相关）类群的18S rDNA序列，与Torruella等人2022年发表的18S rRNA基因比对序列（https://doi.org/10.1111/jeu.12956）进行了同源比对。本文件夹包含两个基于GTRFR4模型与Modelfinder（MFP）选项构建的系统发育树文件，`opimoda_wBRAP*trimal`文件用于两棵树的推断。此外还包含MAFFT比对文件、修剪后的比对文件以及所有IQ-TREE输出结果。 2. **FigS2_EPAanalysis**：用于开展EPA分析与PaPaRa比对的参考树文件（nwk格式）与比对文件（phy格式）。所有中间文件均可在`EPA_analysis`文件夹中获取；`treefiles_raxml`文件夹包含了图S2所用的系统发育树文件；似然加权比（likelihood-weight ratio, LWR）数据可在`EPA_analysis`文件夹中查询。 ### Figure3A_Williamson2025_dataset_sequences_treescleaned_alignments 用于构建系统发育矩阵的BR与APU类群SAGs的确认基因序列： - `concatenated_matrix_iqtree`：矩阵文件与IQ-TREE分析文件 - `initial_sgtree_preclean`：用于筛选对应SAGs直系同源基因的初始单基因树 - `Williamson2025_gene_name.txt`：基因文件列表及其对应基因名称 ### Figure3B_ETC_sequences_and_trees 1. **`Concatenated_IQtrees`**：用于生成图2B、2C的矩阵文件与树文件 2. **`Core_ETC_24_genes`**：用于生成单基因树的文件 3. 额外文件：同时包含图S2所需的分析文件（使用Modelfinder估算系统发育树） ### Figure4_Mitochondrial_genomes 本研究获得的14个线粒体基因组，用于生成图4。 ### Figure5_splitSDH2_alignments 来自各类真核生物的SDH2/SDHB蛋白比对序列，以及本研究获得的眼虫门（Euglenozoa）类群的分裂基因序列。 ### Figure6_mitochondria_proteins_MRO_MTS 用于在BR类群SAGs中筛选MRO与线粒体蛋白的单基因树，数据集来自Import_Chaperones、Iron_sulfur与MRO_genes。同时包含使用MitoFates、MitoProt、TargetP与TPpred3进行线粒体靶向序列分析的结果。 ### BRAPU_protMar2026 文件夹 包含3个子文件夹： 1. `predicted_euk_aa`：使用Augustus软件从原始组装序列中预测得到的蛋白氨基酸序列（原文笔误为preducted_euk_aa） 2. `proteome_for_phylofisher`：用于使用PhyloFisher构建系统发育树的蛋白质组文件 3. `raw_assemblies`：原始核苷酸组装序列