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. <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.

本研究报道了巴泰勒虫类（barthelonids）的系统发育位置：该类群为此前仅通过光学显微镜（light microscopy）开展研究的小型厌氧鞭毛虫。我们从不同地理区域分离得到巴泰勒纳虫属（*Barthelona*）物种，并构建了5株实验室菌株。以其中一株巴泰勒纳虫菌株PAP020的转录组数据（transcriptomic data）为基础，开展了大规模多基因系统基因组学（phylogenomic）分析。分析结果稳健地将菌株PAP020定位至纺锤虫类分支（Fornicata clade）的基部，表明巴泰勒虫类属于深度分支的后滴类（Metamonad）分支。结合巴泰勒虫类的厌氧/微好氧属性，以及针对菌株PAP020开展的初步电子显微镜（electron microscopy）观察结果，我们推测巴泰勒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）。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内的代谢通路，并将其与纺锤虫类的线粒体相关细胞器代谢通路进行了比较。本研究提出：由于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，菌株PAP020无法在线粒体相关细胞器中生成ATP。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依赖胞质中生成的ATP。我们提出：在包含巴泰勒虫类与其他纺锤虫类的分支中，线粒体相关细胞器曾两次独立丢失底物水平磷酸化通路。