Phylogeny Orders HSE Evolution Figure 3. Sizes and positions of identified HSEs for Hsp23 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. Parsimony suggests that two proximal HSEs are ancestral, and their variation in relative size and spacing arose from point substitutions and small indels. The absence of one member of the cluster in only one of the 12 species (D. willistoni) is therefore considered a loss. A single gain of two distal HSEs in a common ancestor of the melanogaster-obscura groups, a gain of a third distal HSE n D. ananassae alone, and a gain of a single distal HSE in D. mojavensis are more parsimonious than alternative scenarios. doi:10.1371/journal.pone.0010669.g003 their heat-shock promoter regions (G-test: G = 22.810, df = 11, p,0.019). Few if any heat-shock promoters are identical in the number, arrangement, and size of their HSEs (except that all include at least one HSE), suggesting that organizations sufficient for heat-shock responsiveness are multiple. Although the distal HSEs are more variable than the proximal HSEs, even the latter are not uniform. For example, whereas most promoters in the present study included a proximal (or solo) HSE within 67 bp of the TSS, the proximal HSE is 94–137 bp from the TSS in the Hsp23 promoters of all species and .270 bp from the TSS in the Hsp27 promoters of all melanogaster subgroup species, anomalies evidently conserved for .40 MY and .10 MY, respectively. In other cases, HSEs come and go during evolution, merge and diverge, move proximally or distally, and expand and contract – but with resultant patterns conserved for millions of years. Evolution of HSE features in heat-shock promoters We cannot unambiguously decipher the relationships of the distal HSEs in Hsp27 promoters of the subgenus Drosophila (Figure 5) species and D. willistoni, and so exclude them from the following analysis. While the presence or absence of the remaining HSEs varies, this variation is always consistent with the phylogeny. That is, in no case does a feature disappear in a clade and reappear in a descendant clade. This consistency, combined with the principle of parsimony, allows the unambiguous assignment of evolutionary gains or losses of HSEs to specific periods in cladogenesis (Figures 2–9): (a) Multiple origins of unique HSEs. In Hsp23, three such origins of distal HSEs occurred independently after D. mojavensis, D. willistoni, and D. ananassae each diverged from the other species. In Hsp26, two such origins of distal HSEs occurred independently after D. willistoni, and D. virilis each diverged from the other species. A distal HSE evolved in DnaJ-1 of D. grimshawi after it diverged from the other species. No such events occurred in the other genes examined. (b) Unique losses of HSEs. A distal HSE disappeared in Hsp22 of D. grimshawi after it diverged from the other species, and is thus absent in this species only. A distal HSE disappeared in the Hsp83 of a common ancestor of all Sophophora (Figure 9) species examined, and is thus absent in all. This event cannot unambiguously be designated as a loss or a gain from the data for the 13 species, but the Hsp83 sequence of an outgroup (Culex) indicates the HSE was lost in Sophophora rather than gained in a common ancestor of the non-Sophophora species. (c) Origins of HSEs with subsequent retention in all descendant species. In Hsp22, HSE features evolved prior to the divergence of the 13 species, although the distal HSE disappeared after D. grimshawi diverged. [An alternative explanation, that the state in D. grimshawi was ancestral and a distal HSE of similar size and position originated independently first in a common ancestor of D. virilis and D. mojavensis and second in a common ancestor of all other species, can be rejected as less parsimonious.] In Hsp23 and in Hsp27, a distal HSE evolved uniquely in a common ancestor of the Sophophora species (but after D. willistoni diverged for Hsp23), appearing in all descendant species. In Hsp26, a pair of distal HSEs evolved uniquely in a common PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10669