Phylogeny Orders HSE Evolution Figure 12. Results of two-way chi-square tests. Subunits were first separated, and tested independently. On the vertical axis, the four largest letters represent the first four positions in the consensus sequence, and the horizontal represent positions 2 through 5. Small letters indicate the pair of bases are tested. Results are summarized as the number of excesses of deficiencies found in each test. Key to heat map: numbers indicated deviation from expected number of observations. doi:10.1371/journal.pone.0010669.g012 Drosophila species, argues for constraint on these motifs, and a possible functional role in Drosophila. Finally, the canonical sequence of HSE 5 bp subunits has been inferred largely from a single species, D. melanogaster, with inherent ascertainment bias. A phylogenetic and multispecies perspective can test the robustness of a canon based on a single species. Our analysis is consistent with the previously-reported conservation of sites 1 and 2 in the AGAAn motif and site 4 in the nTTCn motif [10,23], sites intimately involved in the interaction between HSE and the HSF DNA-binding domain [23]. Additionally, the correlations between PLoS ONE | www.plosone.org 12 May 2010 | Volume 5 | Issue 5 | e10669