Figure 4. Two-way hierarchical clustering of the average levels of expression estimated for the 1% transcripts exhibiting the lowest P values across the six strains under study according to one-way ANOVA. (A) Males; (B) females. For both sexes, the patterns of expression among red-eyed strains, i.e. those with (INV1, INV2) or without (REC) the disrupted ultraconserved region CG15121–CG16894, are more similar to each other than either of them is to any of the white-eyed strains (SIM1, REV1, REV2), all of them carrying the ultraconserved region in intact condition. Thirty-six one-color hybrid- izations were performed involving three biological replicates for each sex-by-strain combination. Red, high expression; green, low expression. doi:10.1371/journal.pgen.1002475.g004 in gene expression showed that a few of them were related to genes in the ultraconserved region. However, these alterations in mRNA levels are in fact the result of pigmentation differences, given that these alterations were found invariably between red-eyed and white-eyed strains, regardless of whether the former carry the disrupted (INV1 and INV2) or the intact form (REC) of the ultraconserved region (Figures S14 and S15). Searches for biologically coherent patterns among differentially expressed genes indicated that, for example for females, statistically significant enrichment was found for functional classes related to perception of visual stimuli (Table S18). Further, patterns of sex bias in gene expression were not affected either (Figure S16; Dataset S3). Hence, no discernible effect on the levels of mRNA of genes both inside and outside of the ultraconserved region CG15121– CG16894 was detected as a result of its disruption. Discussion The interplay among organization, function, and evolution of eukaryotic chromosomes is still poorly understood. Collinearity conservation is an important genomic feature that can conflate all three aspects, especially in genomes characterized by their ease in accommodating structural variation. Up to date, no empirical evaluation has been performed on the effects of disrupting the integrity of one of the largest genomic regions whose overall gene organization has been preserved over a large time scale. Importantly, this conservation entails species with very different behaviors and ecologies and therefore subject to very different selective pressures [46,47]. Our results show an absence of detrimental effects on the carriers of the disrupted ultraconserved Collinearity Conservation in Eukaryotes region for a variety of traits associated with viability and fertility. Although the absence of detrimental phenotypic effects could result from a limited ability to detect differences as statistically significant, the comparison of actual and estimated ideal samples sizes shows that this potential limitation could be the explanation in only a few cases (Table S19). Therefore, at least in Diptera genomes, our results show that an unusually high degree of collinearity conservation coupled with enrichment for functional coherent patterns is not necessarily associated with severe detrimental effects upon perturbation [20,48]. The ultraconserved region CG15121–CG16894 harbors at least three sets of protein-coding genes that may be associated with regulatory-based constraints. The genes fall into the following broad categories: detection of chemical stimuli; sperm manufac- turing and performance; and developmental processes. The only phenotype detected in association with our disruption was the more attenuated odorant response to attractant volatile com- pounds. Although variation in olfactory response occurs both within and between species [49,50], it has been shown that the altered function of Obp genes, including some in the region studied, can affect fitness components in D. melanogaster [51]. Transcrip- tome characterization of adult flies did not uncover any obvious consequences in the activity of the Obp genes due to our disruption as measured by mRNA abundance, and magnitude and direction of sex bias. This lack of evidence for misregulation cannot rule out though that some other expression attributes, such as the spatial distribution of transcripts, had not been altered since they would have gone unnoticed by our approach. The evolutionary relevance of the maintenance of the clustering of Obp genes throughout the genus Drosophila is reinforced by the presence of an orthologous arrangement in different mosquito species to an extent not explained by chance [37]. In fact, the maintenance of the arrangement that includes a core cluster of Obp genes and the gene Toll-7 for ,970 myr was unexpected due to the extent of rearrangement undergone by the genomes of the Diptera involved [22,33,34] (Figure S2B). Regardless, the conservation of the cluster of Obp genes and Toll-7 would not explain the collinearity conservation observed elsewhere in the ultraconserved region under study. In relation to the two other biological signatures suggestive of constraints, we detected no evidence that our perturbation resulted in a detectable phenotype. The genes included in the two intertwined gene expression neighborhoods of the ultraconserved region CG15121–CG16894 do not show evidence of altered mRNA levels or malfunction that could result in impaired male fertility. Whether the constituent genes are under a tight coordinated regulation [52] that could affect the long-term stability of the region does not seem to be upheld by our results. This conclusion would be reinforced by the genus-wide lability shown by clusters of male-biased genes in expression [22]. The absence of misregulation, as measured by mRNA abundance, is consistent with the results obtained when D. melanogaster male- specific gene neighborhoods spanning over hundred of kb were disrupted [28]. Further, protein-coding genes presumably responsive to long- range regulation mediated by HCNEs during development are found scattered across the ultraconserved region CG15121– CG16894 at both sides of the disruption. The lack of a detrimental phenotype could reflect that the region is a composite of several autonomous genomic regulatory domains, each of them under the control of a particular HCNE peak. Under this scenario, our disruption would have separated different genomic regula- tory domains without affecting any long-range interaction be- tween HCNEs and their targets. Nevertheless, marked autonomy PLoS Genetics | www.plosgenetics.org 6 February 2012 | Volume 8 | Issue 2 | e1002475