I aligned 1759 panorthologs shared between the E. coli collection and S. enterica using MAFFT (Katoh and Standley 2013), and counted the amino-acid differences between all pairs of genomes using Biopython (Cock et al. 2009). I then used this distance matrix to construct a neighbor-joining tree (Saitou and Nei 1987). I affixed the 12 nodes for the 50,000-generation LTEE clones to the node of their ancestor, REL606, in the tree; their branch lengths are the number of nonsynonymous mutations in the 1759 panorthologs in each LTEE clone. I made this figure using the ETE Toolkit for phylogenomic data (Huerta-Cepas et al. 2016).