Evolutionary innovation relies partially on changes in gene regulation. While a 
growing body of evidence demonstrates that such innovation is generated by 
functional changes or translocation of regulatory elements via mobile genetic 
elements, the de novo generation of enhancers from non-regulatory/non-mobile 
sequences has, to our knowledge, not previously been demonstrated. Here we show 
evidence for the de novo genesis of enhancers in vertebrates. For this, we took 
advantage of the massive gene loss following the last whole genome duplication 
in teleosts to systematically identify regions that have lost their coding 
capacity but retain sequence conservation with mammals. We found that these 
regions show enhancer activity while the orthologous coding regions have no 
regulatory activity. These results demonstrate that these enhancers have been de 
novo generated in fish. By revealing that minor changes in non-regulatory 
sequences are sufficient to generate new enhancers, our study highlights an 
important playground for creating new regulatory variability and evolutionary 
innovation.