Phylogeny Orders HSE Evolution Figure 7. Sizes and positions of identified HSEs for Hsp68 in 13 Drosophila species. Numbers indicate length of HSE in 5 bp units. Multispecies genome alignment juxtaposes Hsp68 and Hsp70 copies in some species. Hence, all coding regions called as Hsp68 in this study were confirmed through amino-acid states at diagnostic sites following Kellet and McKechnie (2005), and local alignments were used to assess conservation in some species. No recognizable copy of Hsp68 was found in D. willistoni. All proximal HSEs occur in a conserved block near the TSS. Upstream regions are divergent, and no distal HSEs are recognized outside the melanogaster and obscura groups. High levels of sequence divergence in the regions including and flanking the distal HSEs ambiguate the orthology of the HSEs between the melanogaster and obscura groups. Parsimony implicates a single gain of a distal HSE rather than two independent gains. Finally, the distal HSE could have arisen in the common ancestor of the melanogaster and obscura groups, or it could have arisen in the common ancestor of all species and been lost in the common ancestor of D. mojavensis, D. virilis, and D. grimshawi. Parsimony supports the former scenario. doi:10.1371/journal.pone.0010669.g007 of evolutionary events, the gain or loss of entire HSEs, changes in size and location of HSEs can similarly be dated. The dating can unambiguously exclude evolutionary explanations. We ourselves have posited that the heat-shock expression system in Drosophila melanogaster (and perhaps also D. simulans and D. yakuba) is an adaptation to the unique thermal environment that this species encounters. While D. melanogaster’s thermal niche may be unique, the organization of its HSEs is both unremarkable and preceded the evolution of the species. The same is true of the desert species, D. mojavensis, and D. yakuba vs. its less-thermotolerant sister species, D. santomea [40]. With respect to a linkage between regulatory evolution and speciation, none is evident in the heat-shock promoters under study. Finally, the gain or loss of HSEs is evident only for long time periods, as none is detectable in species diverging during the last 10 MY (Figures 2–9). Nonetheless, our analysis has several significant limitations. Obviously it is only as good as the phylogeny on which it rests. Although the relationships of the 13 species examined in the present study are well established [39,63], the 12 whose genomes have been sequenced were chosen to achieve objectives [39] other than the elucidation of HSE evolution. Hence, they are less than ideal for present purposes. For example, inclusion of D. montium, whose subgroup diverged from the ananassae subgroup, would have enabled localization of the evolution of an HSE in Hsp23 of D. ananassae to before or after the divergence. In particular, the time segments pertinent to the evolution of the Drosophila subgenus species, obscura subgroup species, and D. willistoni are too long to time evolutionary events precisely. This limitation can be addressed by selective resequencing of species whose divergence times progressively subdivide lengthy time segments; i.e., phylo- PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10669