Phylogeny Orders HSE Evolution Figure 6. Sizes and positions of identified HSEs for DNAJ-1 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, with the exception of D. willistoni. However, local alignments upstream of the DNAJ-1 ortholog in this species indicate conservation of this HSE. Conserved proximal HSEs are colored yellow. Minor length variation in proximal HSEs are presumably due to small indels and point substitutions. Although divergence in the D. ananassae promoter region ambiguates the orthology of its distal HSE, its location, size, and parsimony suggest a single origin of a distal HSEs in a common ancestor of the melanogaster group. Distal HSEs arose independently in the obscura group and in D. grimshawi. doi:10.1371/journal.pone.0010669.g006 Figures 2–9). [By contrast, comparable investigations of coding sequence (e.g., [53]) and phenotype (e.g., [54], Figure 1 in [50]) are numerous.] Thus, the present study provides a unique glimpse at the evolution of cis-regulatory elements (CREs) and the promoters that include them. As foreseen (see Introduction), the combination of a large pre-existing literature on the focal CRE’s structure and function, a well-established and detailed phylogeny, and aligned orthologous promoters of the included clades underlie this analysis, and ought to permit similar analyses wherever this combination is available elsewhere. Indeed, [6] have similarly described gains and losses of bicoid and hunchback binding sites, and [3] changes in the structure and function of an enhancer element, in a subset of the 13 species investigated in the present study. HSEs are always present in the genes we have studied, and presumably the promoters that include them uniformly can respond to activated HSF. By inference, the Drosophila heat-shock promoters studied never completely gain or lose HSF responsive- ness, but evolve the number of HSFs that can bind, binding affinity, and/or the locations of the HSEs, which putatively affects their ability of HSF-HSE complexes to interact with the transcriptional apparatus. Three studies examining the gain and loss of other CREs in Drosophila implicate more rapid turnover [6,7,52]; each reports, for example, turnover in the melanogaster subgroup species, within which we detect none. By contrast, while HSEs appear or disappear (but never entirely) in evolution, they more frequently expand or contract, merge or separate, and/or migrate towards or away from the TSS. A possible explanation is that the genes hosting CREs in the work of [6,7,52] are evolving more rapidly than heat-shock genes. The prior studies examine CREs involved in early development, whose evolution is responsible for the divergent phenotypes of the species. By contrast, the heat-shock response and its major genes are notoriously highly conserved [55,56] and considerably predate complex eukaryotes [57–59]. An approximate counterpart con- cerns duplicate genes, wherein one member of a duplicate pair can evolve rapidly after duplication. In genes free to evolve in this manner, especially recent duplicates [60], both gene expression and cis-regulatory sequence evolve more rapidly than in non- duplicate genes whose evolution is constrained [61]. [Hsp70, the one multi-copy gene in the present study, is the only gene with almost no turnover of HSEs; its evolution, however, is constrained by gene conversion, at least among transcribed sequence [62]]. HSEs occur in numerous genes other than heat-shock genes; whether HSEs turn over more rapidly in less-conserved non-heat- shock genes might confirm or reject this explanation. The phylogenetic context bounds the dates of each evolutionary event. While we have limited our dating to the most conspicuous PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10669