The timing of early cellular evolution, from the divergence of Archaea and Bacteria to the origin of eukaryotes, is poorly constrained. The ATP synthase complex is thought to have originated prior to the Last Universal Common Ancestor (LUCA) and analyses of ATP synthase genes, together with ribosomes, have played a key role in inferring and rooting the tree of life. We reconstruct the evolutionary history of ATP synthases using an expanded taxon sampling set and develop a phylogenetic cross-bracing approach, constraining equivalent speciation nodes to be contemporaneous, based on the phylogenetic imprint of endosymbioses and ancient gene duplications. This approach results in a highly resolved, dated species tree and establishes an absolute timeline for ATP synthase evolution. Our analyses show that the divergence of ATP synthase into F- and A/V-type lineages was a very early event in cellular evolution dating back to more than 4 Ga, potentially predating the diversification of Archaea and Bacteria. Our cross-braced, dated tree of life also provides insight into more recent evolutionary transitions including eukaryogenesis, showing that the eukaryotic nuclear and mitochondrial lineages diverged from their closest archaeal (2.67-2.19 Ga) and bacterial (2.58-2.12 Ga) relatives at approximately the same time, with a slightly longer nuclear stem-lineage.

早期细胞进化的时间，从古细菌和细菌的分化到真核生物的起源，很少受到限制。 ATP 合酶复合体被认为起源于最后的通用共同祖先 (LUCA) 之前，对 ATP 合酶基因和核糖体的分析在生命之树的推断和扎根方面发挥了关键作用。我们使用扩展的分类单元采样集重建了 ATP 合酶的进化历史，并开发了一种系统发育交叉支撑方法，基于内共生和古代基因重复的系统发育印记，将等效的物种形成节点限制为同时发生。这种方法产生了高度解析的、有日期的物种树，并建立了 ATP 合酶进化的绝对时间表。我们的分析表明，ATP 合酶分化为 F 型和 A/V 型谱系是细胞进化中非常早期的事件，可以追溯到 4 Ga 以上，可能早于古细菌和细菌的多样化。我们的交叉支撑、过时的生命树还提供了对包括真核发生在内的更近期的进化转变的洞察，表明真核细胞核和线粒体谱系与其最接近的古菌（2.67-2.19 Ga）和细菌（2.58-2.12 Ga）亲属的分化程度约为同时，具有稍长的核干谱系。