Evaluation of the Role of Functional Constraints on the Integrity of an Ultraconserved Region in the Genus Drosophila Carlos Dı´az-Castillo1*, Xiao-Qin Xia2, Jose´ M. Ranz1* 1 Department of Ecology and Evolutionary Biology, University of California Irvine, Irvine, California, United States of America, 2 Institute of Hydrobiology, Chinese Academy of Science, Wuhan, China Abstract Why gene order is conserved over long evolutionary timespans remains elusive. A common interpretation is that gene order conservation might reflect the existence of functional constraints that are important for organismal performance. Alteration of the integrity of genomic regions, and therefore of those constraints, would result in detrimental effects. This notion seems especially plausible in those genomes that can easily accommodate gene reshuffling via chromosomal inversions since genomic regions free of constraints are likely to have been disrupted in one or more lineages. Nevertheless, no empirical test has been performed to this notion. Here, we disrupt one of the largest conserved genomic regions of the Drosophila genome by chromosome engineering and examine the phenotypic consequences derived from such disruption. The targeted region exhibits multiple patterns of functional enrichment suggestive of the presence of constraints. The carriers of the disrupted collinear block show no defects in their viability, fertility, and parameters of general homeostasis, although their odorant perception is altered. This change in odorant perception does not correlate with modifications of the level of expression and sex bias of the genes within the genomic region disrupted. Our results indicate that even in highly rearranged genomes, like those of Diptera, unusually high levels of gene order conservation cannot be systematically attributed to functional constraints, which raises the possibility that other mechanisms can be in place and therefore the underpinnings of the maintenance of gene organization might be more diverse than previously thought. Citation: D´ıaz-Castillo C, Xia X-Q, Ranz JM (2012) Evaluation of the Role of Functional Constraints on the Integrity of an Ultraconserved Region in the Genus Drosophila. PLoS Genet 8(2): e1002475. doi:10.1371/journal.pgen.1002475 Editor: Mohamed A. F. Noor, Duke University, United States of America Received July 29, 2011; Accepted November 29, 2011; Published February 2, 2012 Copyright: 2012 D´ıaz-Castillo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by start-up funds and a grant from NSF (DEB-0949365) to JMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: crlsdiazcastillo@gmail.com (CD-C); jranz@uci.edu (JMR) Introduction Collinearity conservation, i.e. conservation of local gene order, across distantly related phyla is often viewed as the result of functional constraints that prevent the occurrence of breaks of chromosomal rearrangements during evolution. The nature of these constraints is still poorly understood. They may merely reflect the presence of yet-to-be annotated protein and nonprotein- coding genes in intergenic regions [1–3]. A second type of constraints may be linked to the existence of regulatory domains, i.e. genomic regions associated with complex regulatory inputs. These regulatory domains can adopt at least two forms. One common form of complex regulatory inputs corresponds to genes that are coordinately expressed or repressed [4–7]. Alternatively, regulatory constraints can adopt the form of long-range regulatory interactions, which often involve the interdigitation of cis- regulatory sequences with genes that are not their targets [8– 10]. These regulatory domains are enriched for noncoding DNA (highly conserved noncoding elements or HCNEs) with putative regulatory potential [11–16]. These HCNEs tend to be found in the vicinity of protein-coding genes that participate in key processes during development, such as regulation of gene expression and signal transduction [10,12,17]. Disruption of genomic regions under constraints can be accompanied by alteration of gene activity, as illustrated by chromosomal rearrangements that modify gene expression as a result of the separation of a gene from its regulatory sequences [18–20]. These alterations in gene activity may have a detrimental effect, which would lead to conservation of gene organization [19,21]. Highly rearranged genomes, such as those of the Diptera, are especially suitable for analyzing the presence of regulatory-based constraints that preserve collinearity since regions free of them are likely to have been disrupted in one or more lineages. Gene order comparisons have helped to delineate collinear blocks [22] across nine Drosophila species that represent ,380 million years (myr) of total divergence time [23,24]. A minimum of ,6,100 chromo- somal breakpoints have been estimated to have occurred in 2,688 intergenic regions [22] scattered across the main chromosomal elements (the so-called Muller’s elements A–E) that constitute the Drosophila genome [25,26]. The analysis of the expected patterns of evolution of gene organization under different evolutionary scenarios indicate that fragile regions, i.e. those that accumulate most chromosomal breaks during evolution [27], are the main factor that explains the patterns of gene arrangement across Drosophila species [22]. Constraints nevertheless may be responsible for ,15% of gene order conservation and their presence is positively correlated with the size of the collinear block [22]. The top 1% largest collinear blocks, or ultraconserved regions [22], are PLoS Genetics | www.plosgenetics.org 1 February 2012 | Volume 8 | Issue 2 | e1002475