Phylogeny Orders HSE Evolution Figure 2. Organization of identified HSEs for Hsp22 in 13 Drosophila species. Numbers indicate length of HSE in 5 bp units. Rectangles represent HSEs, whose length (in 5 bp subunits) is indicated. HSEs of the same color indicate probable homologs. Each circle, arbitrarily indicates a deduced evolutionary gain (+) or loss (2) of an HSE, and is placed arbitrarily at the center of the segment in which it occurs. Below a heat map expresses the similarity of each 5 bp subunit to the canonical sequence: G represents nGAAn-type motifs, while T represents nTTCn-type motifs. Columns correspond to the order of HSEs (from left to right) in the promoter maps. Key to heat maps: the number represents the information content of each actual subunit as a percent of the value calculated for the consensus sequence. Genome alignment indicates all proximal HSEs of 11 species occur in a conserved block near the TSS, with the exception of D. grimshawi. These HSEs are colored yellow. Point substitutions and small indels give rise to length variation in HSEs and a small gap in D. ananassae. In D. grimshawi, two copies of Hsp22 appear to be present. The copy whose coding sequence most closely aligns with those of the other 11 species lacks a conserved TSS or promoter and may not be expressed. Both a conserved TSS and promoter are present in the second copy, and included in the figure. A distal HSE is present in all species but D. grimshawi. These vary in size and distance from the TSS. However, all appear to be orthologous in whole genome alignments, as well as in local alignments with multiple algorithms, with the exception of those in D. willistoni and D. mojavensis. If correct, this implies parallel losses and re-evolutions of HSEs, which we reject by parsimony. The figure presents a simpler scenario in which only a single loss occurs. In D. ananassae, parsimony suggests that the two proximal HSEs arose from insertion into or mutation of a single ancestral HSE. doi:10.1371/journal.pone.0010669.g002 nGAAn (G-test: G = 15.19, df = 1, p,0.0001), despite an overall ratio of nTTCn-type subunits to nGAAn-type of 1.01. The orientation of an HSE appears conserved across species. This is true of most HSEs (Figures 2–9) and is especially clear in the proximal HSE of Hsp83. (c) Gapped HSEs, which are common and conserved across species. We classify HSEs as gapped if one or more internal 5 bp subunits score ,50% of the maximum possible for their motif type, and are flanked by subunits scoring .50%. Despite prior treatment in the literature as anomalies (see Introduction), gapped HSEs are commonplace in the 13 species of Drosophila. Gapped HSEs occur in every gene in our data set, in both proximal and distal HSEs, and in all species. The gapping, moreover, is conserved, appearing as light stripes in the heat maps in Figures 2– 9. In Hsp23, for example, a low-scoring 5 bp unit (in grey) persists in 2 separate HSEs in melanogaster group (Figure 3) species diverging as much as 12 MYA, as do 2 tandem 5 bp units in the second most proximal HSE. (d) Diversity in the organization of heat-shock promoters. Overall, species vary in the aggregate length of HSE sequence in PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10669