Cetaceans have evolved unique limb structures, such as flippers, due to genetic changes during their transition to aquatic life. However, the full understanding of the genetic and evolutionary mechanisms behind these changes is still developing. By examining 25 limb-related protein-coding genes across various mammalian species, we compared genetic changes between aquatic mammals, like whales, and other mammals with unique limb structures such as bats, rodents, and elephants. Our findings revealed significant modifications in limb-related genes, including variations in the Hox, GDF5, and EVX genes. Notably, a relaxed selection in several key genes was observed, suggesting a lifting of developmental constraints, which might have facilitated the emergence of morphological innovations in cetacean limb morphology. We also uncovered non-synonymous changes, insertions, and deletions in these genes, particularly in the polyalanine tracts of HOXD13, which are distinctive to cetaceans or convergent with other aquatic mammals. These genetic variations correlated with the diverse and specialized limb structures observed in cetaceans, indicating a complex interplay of relaxed selection and specific mutations in mammalian limb evolution.

Annotated orthologous transcripts were retrieved from the phylogenomic dataset by McGowen et al. and from Genbank.  We also used the OrthoMaM v12 database to check orthologs where possible. The coding sequences for the bowhead whale (Balaena mysticetus) came from the public platform The Bowhead Whale Genome Resource. For 28 genomes fully or partially lacking annotation, the gene sequences were identified by reciprocal BLAST+ searches using annotated orthologues of the related mammals as queries against reference genome assemblies. Multiple alignments for limb-related genes and partial HOXD13 sequences were built using translated protein sequences with MAFFT 7.