Table S1: Amino acid positions and coverages of the 148 single-gene alignments used in this study 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. <i>Barthelona</i> spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one <i>Barthelona</i> 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.

本研究报道了巴氏鞭毛虫类（barthelonids）的系统发育定位，该类群为此前仅通过光学显微镜开展研究的小型厌氧鞭毛虫。我们从地理分布迥异的区域分离得到巴氏鞭毛虫属（Barthelona）物种，并建立了五株实验室培养菌株。我们以其中一株巴氏鞭毛虫菌株PAP020的转录组数据为材料，开展了大规模多基因系统发育（系统基因组学）分析。本次分析稳健地将菌株PAP020定位于梭形鞭毛虫分支（Fornicata）的基部，表明巴氏鞭毛虫类代表一个深分支的后滴虫类（Metamonad）分支。结合巴氏鞭毛虫类的厌氧/微好氧特性，以及对菌株PAP020开展的初步电子显微镜观察结果，我们推测巴氏鞭毛虫类拥有结构与功能均简化的线粒体，即线粒体相关细胞器（MROs）。我们基于菌株PAP020的转录组数据，预测了其线粒体相关细胞器中的代谢通路，并将其与梭形鞭毛虫类线粒体相关细胞器的代谢通路进行了对比。本研究提出，菌株PAP020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（substrate-level phosphorylation）的线粒体/线粒体相关细胞器酶类。与之相反，我们检测到了推定的胞质ATP生成酶——乙酰辅酶A合成酶（acetyl-CoA synthetase），这表明菌株PAP020依赖胞质中合成的ATP。我们提出，在包含巴氏鞭毛虫类与其他梭形鞭毛虫类的分支中，线粒体相关细胞器的底物水平磷酸化功能发生了两次独立的丢失事件。