Resolving the origins of membrane phospholipid biosynthesis genes using outgroup-free rooting

One of the key differences between Bacteria and Archaea are their canonical membrane phospholipids, which are synthesized by distinct biosynthetic pathways with non-homologous enzymes. This \"lipid divide\" has important implications for the early evolution of cells and the type of membrane phospholipids present in the last universal common ancestor (LUCA). One of the main challenges in studies of membrane evolution is that the key biosynthetic genes are ancient and their evolutionary histories are poorly resolved. This poses major challenges for traditional rooting methods because the only available outgroups are distantly related. Here, we address this issue by using the best available substitution models for single gene trees, by expanding our analyses to the diversity of uncultivated prokaryotes recently revealed by environmental genomics, and by using two complementary approaches to rooting that do not depend on outgroups. Consistent with some previous analyses, our rooted gene trees support extensive inter-domain horizontal transfer of membrane phospholipid biosynthetic genes, primarily from Archaea to Bacteria. They also suggest that the capacity to make archaeal-type membrane phospholipids was already present in LUCA.