Key molecular cladistic characters that help root the tree of life. Green bars mark major evolutionary innovations. Those explained in detail in previous publications [1, 24, 26] are labelled in blue. Those introduced for the first time or discussed in more detail in the present paper are in red. The three most fundamental changes in cell structure (the origin of unibacteria by loss of the negibacterial outer membrane [1, 5]; the neomuran revolution involving novel chromatin and glycoprotein secretion and much coadaptive macromolecular evolution [1, 5, 29, 62]; and the origin of the eukaryote cell [5, 27, 62]) are marked by thicker bars. So also are the three major transitions, whose key importance and decisiveness for rooting the tree of life are explained here for the first time: the origins of the proteasome, of flagella, and of Omp85 for insertion of OM β-barrel proteins. The three major kinds of cell from the viewpoint of their having fundamentally distinct membrane topology (eukaryotes, unibacteria, negibacteria) [5, 29, 56, 62] are shown by thumbnail sketches (isoprenoid ether lipids in red, outer membranes in blue). Thumbnail sketches also illustrate the inferred times of origin of two key cylindrical macromolecular assemblies (the OM β-barrel protein Omp85 and HslVU/proteasome ATP-dependent regulated proteases) and the two-step increased complexity of the latter. Negibacterial taxa are shown in black, Posibacteria in orange, and neomuran taxa in brown. Gracilicutes comprise four negibacterial phyla with either a very thin peptidoglycan layer or no peptidoglycan at all in their cell envelope: Proteobacteria, Planctobacteria, Spirochaetae, Sphingobacteria (Table 1 explains the formal bacterial taxon names used here for precision and brevity). Evidence for the relatively late dating of the neomuran revolution was explained in detail previously . Note that although Chlorobacteria and Endobacteria are shown as holophyletic, either or both might actually be paraphyletic; I suspect that Endobacteria may be paraphyletic as the most divergent actinobacterium has endospores, but think that Chlorobacteria are probably not. Conversely, it is uncertain whether actinobacteria are paraphyletic as shown or paraphyletic; see text – further work is needed to decide. For simplicity, five additional polarizations within Gracilicutes that are also discussed are not shown; see the more comprehensive Fig. 7 for them and additional characters mapped onto the tree. Note that the ~2.8 Gy date for the origin of cyanobacteria is based solely on hopanoid biomarkers; since no earlier organic deposits have been found that are sufficiently well preserved and with enough extractable hydrocarbons for such biomarker analysis, this is a minimum date (though its validity also depends on the assumption that such hydrocarbons have not migrated vertically in the rocks since being formed, which is hard to test).