Phylogeny Orders HSE Evolution Figure 5. The distribution of HSEs is most variable in Hsp27. In the melanogaster subgroup, it is the only heat-shock gene with no HSE within 200 bp of the TSS. The multispecies genome alignment provides support for a rearrangement in D. ananassae and one or more deletions in the obscura group that remove intervening sequence and explain differences in distance to the TSS. We conservatively assume a single origin of proximal HSE sequence in Drosophila Hsp27 promoters, with subsequent modifications of length and distance to the TSS. A second, more distal HSE is found in all species, and appears to be orthologous in the melanogaster and obscura groups. A gain of a third distal HSE has occurred in the melanogaster subgroup. D. willistoni and D. virilis also have a third HSE, suggesting lineage specific gains. However, although the regions compared are directly adjacent to an orthologous coding region, ambiguity exists in the relationships of individual HSE in D. willistoni and the species of the subgenus Drosophila, and these HSEs are colored white. doi:10.1371/journal.pone.0010669.g005 our results, we repeated all analyses with HSEs from a single, arbitrarily chosen gene copy in each species. The results were not appreciably different than those for the more inclusive dataset. Discussion Currently studies of cis-regulatory element (CRE) evolution emphasize (a) the discovery of novel CREs through their evolutionary conservation [42,43], (b) the change in the combi- nation of heterotopic CREs in cis-regulatory modules and networks [1,44,45], (c) evolved differences in CREs in sister taxa [3,46–50], and (d) the deduction of the general properties of CRE evolution through data-mining on a multigenome-wide scale [7,51,52], among others. While these emphases are laudable, they curiously have bypassed the distribution and nature of discrete evolutionary changes in CREs on a phylogenetic scale (e.g., PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10669