Animal genomes possess highly conserved cis-regulatory sequences that are often 
found near genes that regulate transcription and development. Researchers have 
proposed that the strong conservation of these sequences may affect the 
evolution of the surrounding genome, both by repressing rearrangement, and 
possibly by promoting duplicate gene retention. Conflicting data, however, have 
made the validity of these propositions unclear. Here, we use a new 
computational method to identify phylogenetically conserved noncoding elements 
(PCNEs) in a manner that is not biased by rearrangement and duplication. This 
method is powerful enough to identify more than a thousand PCNEs that have been 
conserved between vertebrates and the basal chordate amphioxus. We test 42 of 
our PCNEs in transgenic zebrafish assays--including examples from vertebrates 
and amphioxus--and find that the majority are functional enhancers. We find that 
PCNEs are enriched around genes with ancient synteny conservation, and that this 
association is strongest for extragenic PCNEs, suggesting that cis-regulatory 
interdigitation plays a key role in repressing genome rearrangement. Next, we 
classify mouse and zebrafish genes according to association with PCNEs, synteny 
conservation, duplication history, and presence in bidirectional promoter pairs, 
and use these data to cluster gene functions into a series of distinct 
evolutionary patterns. These results demonstrate that subfunctionalization of 
conserved cis-regulation has not been the primary determinate of gene duplicate 
retention in vertebrates. Instead, the data support the gene balance hypothesis, 
which proposes that duplicate retention has been driven by selection against 
dosage imbalances in genes with many protein connections.