Genomic Signatures of Independently Evolved Walking-Associated Locomotion in Fishes

Walking-associated locomotion has evolved independently in several fish lineages through distinct anatomical and biomechanical mechanisms. The spiny red gurnard (Chelidonichthys spinosus) provides an experimentally tractable model for dissecting the genetic basis of walking-associated locomotor specialization. We assembled a high-quality chromosome-level genome of the spiny red gurnard and performed a comparative genomic analysis across five walking-associated fish lineages. An integrated protein–protein interaction (PPI) network revealed high clustering coefficients and significant enrichment in fin development and locomotion pathways, indicating candidate functional similarity of lineage-specific genes at the protein interaction level. Shared positively selected genes (PSGs), including jph2, ccdc80, lrrc7, and ap3b2, were identified as candidate genes potentially associated with neuromuscular function. Specifically, in the spiny red gurnard, cdh23, showed lineage-specific positive-selection signals and amino acid changes, suggesting a potential contribution to mechanosensory adaptation. Furthermore, we identified changed conserved noncoding elements (CNEs) in each lineage, with some showing convergent changes near wnt3 and cyp26a1 in specific species pairs. A species-specific CNE deletion in the tax1bp1b intron was tested in HEK293T cells using sequences from zebrafish and spiny red gurnard; the two sequences showed different heterologous reporter activities. Overall, our analyses provide exploratory evidence for a dual pattern of genomic variation potentially underlying walking locomotor evolution: functional association at the protein network level coupled with predominantly lineage-specific yet occasionally convergent regulatory changes. This work also offers insights into environmental adaptation in fishes and a valuable genomic resource for studying convergent evolution.