Additionally, we ran PhyloScan with some of its features disabled. In three pairs of runs, one for each motif strength, as above, we ran PhyloScan on the four clades of sequence data, but by disabling its Neuwald-Green calculation (see Methods) we did not permit PhyloScan to statistically incorporate any sites other than the best found binding site in each intergenic region. In another three pairs of runs we ran PhyloScan, permitting it to consider multiple sites within an intergenic region, but by disabling its Bailey-Gribskov calculation (see Methods) PhyloScan could not consider more than one clade, and we gave it only the sequence data from the Enterobacteriales clade. Finally, we ran MONKEY (which incorporates neither the Neuwald-Green nor the Bailey-Gribskov calculation) on the Enterobacteriales clade sequence data, in a final three pairs of runs.