Developmental regulation of conserved non-coding element evolution provides insights into limb loss in squamates.

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Limb loss shows recurrent phenotypic evolution across squamate lineages. Here, based on three de novo-assembled genomes of limbless lizards from different lineages, we showed that divergence of conserved non-coding elements (CNEs) played an important role in limb development. These CNEs were associated with genes required for limb initiation and outgrowth, and with regulatory signals in the early stage of limb development. Importantly, we identified the extensive existence of insertions and deletions (InDels) in the CNEs, with the numbers ranging from 111 to 756. Most of these CNEs with InDels were lineage-specific in the limbless squamates. Nearby genes of these InDel CNEs were important to early limb formation, such as Tbx4, Fgf10, and Gli3. Based on functional experiments, we found that nucleotide mutations and InDels both affected the regulatory function of the CNEs. Our study provides molecular evidence underlying limb loss in squamate reptiles from a developmental perspective and sheds light on the importance of regulatory element InDels in phenotypic evolution.