Multiple Alignment Accuracy. A: Alignment accuracy varies across tools and divergences. Mean four species alignment accuracy for each tool was measured as a function of total divergence distance. B: Alignment accuracy improves with the presence of transcription factor binding sites. Mean improved alignment accuracy of enhancers over background sequences for four species alignments was measured as a function of total divergence distance. C: Dividing a fixed total divergence up with more species improves alignment accuracy. Mean Mlagan alignment accuracy for two, three and four species trees was measured as a function of total divergence distance. D: Adding in-group species to a pair of species has no effect on the alignment accuracy of the pair. Mean improved alignment accuracy of three species alignments over two species alignments, where the divergence distance between Seq1 and Seq3 in the three species alignment was the same as the divergence distances between Seq1 and Seq2 in the two species alignment, was measured as a function of divergence distance. E & F: Alignment accuracy varies across branches in a tree and is best for leaf-to-leaf alignments and worst for node-to-node alignments, with the exception of highly diverged enhancers. Mean Clustalw alignment accuracy along branches in three and four species trees subtracted from mean two species alignment accuracy, where divergence along each branch is the same as the two species divergence, was measured in background sequences (E) and enhancers (F) as a function of divergence distance.