Phylogeny Orders HSE Evolution Figure 4. Sizes and positions of identified HSEs for Hsp26 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. By parsimony, an upstream distal HSE cluster arose in a common ancestor of the melanogaster subgroup, and a single distal HSE arose independently in D. willistoni and D. virilis. The distal element in D. willistoni appears due to a local duplication of sequence proximal to the TSS. Duplication of the Hsp26 coding sequence appears to have occurred in D. mojavensis with both copies having a similar proximal promoter architecture. doi:10.1371/journal.pone.0010669.g004 ancestor of the melanogaster subgroup species, appearing in all descendant species. A distal HSE similarly evolved in Hsp27. In DnaJ-1 a distal HSE evolved in a common ancestor of D. persimilis and D. pseduoobscura, and a pair of distal HSEs arose separately in a common ancestor of the melanogaster group (Figure 6) species, in each case appearing in all descendant species. (d) HSEs of Hsp70 are anomalous in their conservatism (Figure 8). The only routinely multi-copy gene surveyed includes four HSEs of similar size and location in the vast majority of its proximal promoter regions (except in a single copy each in D. santomea and D. willistoni). The data support 14 discrete evolutionary events (gains or losses) distributed among 24 branches in the phylogeny, with 0, 1, or 2 events per branch (Figure 10). Lengths of these three classes of branches average 5.2, 19.5, and 29.7 MY respectively; this variation is significant (Kruskal-Wallis test, 2 df, p = 0.0012). HSEs have thus neither appeared nor disappeared in the individual species of the melanogaster subgroup or the two species of the obscura group (Figures 2–9) presumably because the time since their divergence has been insufficient. HSE sequence variation and subunit site composition bias The sequences of HSEs seem far more variable than their number, size, and position. With Hsp70 HSEs excluded, overall HSEs vary among species (Tamura-Nei gamma distance: mean = 0.410, SE = 0.089; transversions only) as much as do the third positions of codons within the coding sequence of the genes in which they occur. (synonymous: mean = 0.413, S.E = 0.095; HSE: mean = 0.410, SE = 0.089; transversions only). By contrast, certain positions within HSEs are highly constrained. We analyzed 872 nGAAn-type 5 bp subunits and 880 nTTCn-type 5 bp subunits. While our findings (Figures 11 and 12) largely conform to previously reported patterns, some departures are evident. For example, whereas Position 1 in nTTCn has absolutely no nucleotide preference as previously reported, in the corresponding position in nGAAn (position 5) A accounts for 42.7% of the bases. Also, in position 5 in nTTCn only 40.5% of the motifs are T, less than previously reported. Furthermore, even between otherwise poorly conserved sites in the 5 bp motifs, certain combinations of nucleotides were more prevalent than expected by chance for nGAAn-type motifs (Figure 12). For example, As in both position 1 and position 5 (denoted A1A5) were excessively more frequent than expected from their joint probability, as was A3A4. By contrast, A1T5 and C1A5 were excessively infrequent. Excesses and deficiencies in nTTCn-type motifs were smaller. The foregoing analysis of 5 bp subunits included those from all HSEs in promoters we examined, including those in all copies of paralogous genes. To assess whether this universal inclusion affects PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10669