(A) Expanded set genes that reject domain monophyly (p<0.05, approximately unbiased [AU] test, with Bonferroni correction [see main text]) support significantly shorter Archaea-Bacteria (AB) branch lengths when constrained to follow a domain monophyletic tree (p=3.653 × 10–6, Wilcoxon rank-sum test). None of the marker genes from several other published analyses significantly reject domain monophyly (Bonferroni-corrected p<0.05, AU test) for all genes tested, consistent with vertical inheritance from the LUCA (last universal common ancestor) to the last common ancestors of Archaea and Bacteria, respectively. (B) Two measures of evolutionary proximity (Zhu et al., 2019), AB branch length and relative AB distance, are positively correlated (R = 0.7426499, p< 2.2 × 10–16). We considered two complementary proxies of marker gene verticality: ∆LL (C: against AB branch length; D: against relative AB length), which reflects the degree to which marker genes reject domain monophyly (C: p=0.009013 and R = –0.2317894; D: p=0.0001051 and R = –0.2213292); and the between-domain split score (E: against AB branch length; F: against relative AB length), which quantifies the extent to which marker genes recover monophyletic Archaea and Bacteria; a higher split score (see Materials and methods) indicates the splitting of domains into multiple gene tree clades due to gene transfer, reciprocal sorting out of paralogs, or lack of phylogenetic resolution (E: p=0.0005304 and R = –0.3043537; F: p=2.572 × 10–6 and R = –0.2667739). We also considered a split score based on within-domain relationships (G); between- and within-domain split scores are positively correlated: R = 0.836679, p<2.2 × 10–16, Pearson’s correlation, indicating that markers that recover Archaea and Bacteria as monophyletic also tend to recover established within-domain relationships. (H) Inferred AB length decreases as marker genes of lower verticality (larger ∆LL) are added to the concatenate. Marker genes were sorted by ∆LL, the difference in log-likelihood between the maximum likelihood gene family tree under a free topology search and the log-likelihood of the best tree constrained to obey domain monophyly. Note that 79/381 expanded set markers had zero or one archaea in the 1000-species subsample and so could not be included in these analyses; of the remaining 302 markers, 176 have AB branch lengths very close to 0 in the constraint tree (as seen in panel A). In these plots, we removed all markers with an AB branch length of <0.00001; see Figure 1—figure supplements 1–13 for all plots. Nonlinear trendlines were estimated using LOESS regression.