The study investigates the gain, loss, and modification of gene regulatory elements (CREs) during vertebrate evolution, which are believed to underlie significant phenotypic changes. The authors identified genome-wide sets of conserved non-exonic elements (CNEEs) in five vertebrates, including humans, and inferred the evolutionary branches on which these CNEEs were under selective constraint. This analysis revealed three distinct periods of regulatory innovation: 1. **Early Vertebrate Evolution**: Regulatory gains were primarily near transcription factors and developmental genes. 2. **From 300 Mya to 100 Mya**: Innovations shifted towards extra-cellular signaling genes, followed by post-translational protein modifiers. 3. **Placental Mammals**: Regulatory innovations near transcription factors and developmental genes declined, while those near post-translational protein modifiers and intra-cellular signaling genes increased. The study used multiple alignments of vertebrate genomes and a phylogenetic hidden Markov model to identify and date CNEEs. The results were robust across different reference species and methods, indicating that these trends are not artifacts of alignment inaccuracies or methodological choices. The findings highlight the importance of CREs in evolutionary adaptation and provide insights into the functional categories of genes that have undergone regulatory innovations.The study investigates the gain, loss, and modification of gene regulatory elements (CREs) during vertebrate evolution, which are believed to underlie significant phenotypic changes. The authors identified genome-wide sets of conserved non-exonic elements (CNEEs) in five vertebrates, including humans, and inferred the evolutionary branches on which these CNEEs were under selective constraint. This analysis revealed three distinct periods of regulatory innovation: 1. **Early Vertebrate Evolution**: Regulatory gains were primarily near transcription factors and developmental genes. 2. **From 300 Mya to 100 Mya**: Innovations shifted towards extra-cellular signaling genes, followed by post-translational protein modifiers. 3. **Placental Mammals**: Regulatory innovations near transcription factors and developmental genes declined, while those near post-translational protein modifiers and intra-cellular signaling genes increased. The study used multiple alignments of vertebrate genomes and a phylogenetic hidden Markov model to identify and date CNEEs. The results were robust across different reference species and methods, indicating that these trends are not artifacts of alignment inaccuracies or methodological choices. The findings highlight the importance of CREs in evolutionary adaptation and provide insights into the functional categories of genes that have undergone regulatory innovations.