Co-option of a tendon-associated osteogenic program drives the evolutionary plasticity of intermuscular bones in teleosts.

Intermuscular bones (IBs) are critical yet poorly understood evolutionary specializations. These bones exhibit considerable morphological variability, proposed as adaptations to teleost diversification; however, the explicit adaptive and evolutionary mechanisms remain unresolved. By integrating comparative morphological analysis of 360 teleost species and eco-functional performance assays, we demonstrate that IB complexity is associated with ecological niche expansion and enhanced burst swimming capacity, underscoring IBs as versatile adaptations. Single-cell transcriptomic profiling across four ray-finned fish species reveals that the teleost-specific emergence of IBs originated from a tendon-associated ossification pathway. This evolutionary pathway is mechanistically linked to the co-option of existing cell types and gene networks, including the osteogenic program initiated by runx2b and the critical tendon microenvironment maintained by mkxa/b. Furthermore, we provide evidence of repeated IB loss events associated with alterations or losses of these key osteogenic and tendon niche-regulating genes, highlighting the variability of this adaptive trajectory. Our findings reveal a distinct tendon-associated osteogenic system in teleosts, providing a mechanistic framework for IB evolution during teleost adaptive radiation.