Phylogeny Orders HSE Evolution Figure 9. Sizes and positions of identified HSEs for Hsp83 in 13 Drosophila species. Numbers indicate length of HSE in 5 bp units. Genome alignment indicates all proximal HSEs of 12 species occur in a conserved block near the TSS. These HSEs are colored yellow. Length variation in proximal HSEs is due to both point substitutions and small indels. A distal HSE in the subgenus Drosophila only is equally consistent with a single gain in this subgenus or a single loss of this HSE in Sophophora. Sequence of an outgroup (Culex pipiens) implicates the latter scenario by parsimony. doi:10.1371/journal.pone.0010669.g009 genetic walking. Drosophila is a speciose group with a detailed phylogenetic tree (http://flybase.org/static_pages/allied-data/ phylogeny/Drosophilidae-Tree/list3.html) and representative stocks (http://stockcenter.ucsd.edu) available, features lacking for many other taxa. Another limitation is that sequence variation, especially in non-transcribed sequence, often impedes the alignments on which the analysis depends, thereby reducing sample size and power [6,7]. We attempted to include members of the Hsp60 gene family in the analysis, for example, but were thwarted for lack of confidence in their orthology and alignment. In a sister study [64] of 117 primarily single-copy promoters in the 12 sequenced Drosophila genomes, despite extensive manual curation only 33% had conservation of the TSS sufficient to be alignable in all 12 species. Lack of conservation may limit the application of our approach to most genes, which are less well conserved than heat-shock genes [55,56]. As noted, phylogenetic context also discovers aspects that are unique vs. repeatable and constrained vs. variable in evolution, thereby posing functional hypotheses for follow-up. Ordinarily caveats (e.g., chance resemblances among species, mutations in trans that compensate for mutations in cis) apply to this concept [6], but are minimal in the present study due to the detailed pre- existing mechanistic understanding of regulation of the heat-shock response. The prevalence and conservation of gapped HSEs, tail- to-tail organization, and interactions between positions 1 and 5 in nGAAn-type motifs are among these discoveries. In yeast, HSE variants beginning with nGAAn or nTTCNn (head-to-head or tail-to-tail, respectively) have distinct binding properties and biological activities [26], as tail-to-tail variants can bind more HSF trimers than head-to-head variants of equal size. In D. melanogaster, all that is known is that both variants bind HSF with similar affinity in vitro [21]. Thus, either the benefit of the tail-to- tail arrangement or some other explanation (e.g., genetic linkage) is yet to be discovered for Drosophila. Gapped HSEs have been documented in a number of heat-inducible genes in yeast and are capable of binding HSF [34], but to our knowledge have not been described in Drosophila. In the yeast, Schizosaccharomyces pombe, the modulation of heat-shock expression through HSF appears to vary based on HSE architecture, suggesting that whether typical or gap-type HSEs occur in a promoter may be related to gene- or stress-specific differences in regulation [65]. Although induction of heat-shock gene expression is not identical in yeast and flies [38], the nature of the HSF-HSE interaction is well-conserved [17,19]. This fact, together with the conservation of gap-type HSEs across PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10669