Table S2: Putative MRO proteins of Barthelona sp. strain PAP020 and their predicted subcellular localizations. from Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP

We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching Metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected the putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.本研究报道了巴瑟隆虫类（barthelonids）的系统发育位置——这类小型厌氧鞭毛虫此前仅通过光学显微镜开展过相关研究。我们从不同地理区域分离得到巴瑟隆虫属（Barthelona spp.）菌株，并构建了5株实验室培养株系。以其中一株巴瑟隆虫（PAP020）的转录组数据为基础，我们开展了大规模多基因系统发育（系统基因组学）分析。分析结果稳健地将PAP020菌株定位在Fornicata演化支的基部，表明巴瑟隆虫类属于一个深度分支的后滴虫（Metamonad）演化支。结合巴瑟隆虫类的厌氧/微好氧特性，以及对PAP020菌株的初步电子显微镜观察结果，我们推测巴瑟隆虫类拥有功能与结构均简化的线粒体，即线粒体相关细胞器（mitochondrion-related organelles, MROs）。基于该菌株的转录组数据，我们对其线粒体相关细胞器中的代谢通路进行了预测，并与Fornicata类群的线粒体相关细胞器代谢通路进行了比对。本研究发现PAP020菌株无法在线粒体相关细胞器中合成ATP，因为未检测到参与底物水平磷酸化的线粒体/线粒体相关细胞器酶类。反之，我们检测到了假定的胞质ATP生成酶——乙酰辅酶A合成酶（acetyl-CoA synthetase），这表明PAP020菌株依赖胞质中生成的ATP。我们提出，在包含巴瑟隆虫类与其他Fornicata类群的演化支中，曾发生过两次独立的线粒体相关细胞器底物水平磷酸化通路丢失事件。