Abstract Fish skulls are often highly kinetic, with multiple linkage and lever systems powered by a diverse suite of muscles. Comparative analysis of the evolution of soft-tissue structures in the fish skull is often limited under traditional approaches, while new imaging techniques like diceCT (diffusible iodine-based contrast-enhanced computed tomography) allow for high-resolution imaging of muscles in situ. Darters (Percidae: Etheostomatinae) are a diminutive and species-rich clade of lotic freshwater fishes, which show diverse head shapes believed to be associated with different foraging strategies. We used diceCT to sample all major cranial adductors and abductors responsible for movement of the jaw, hyoid, operculum, and suspensorium from 29 species. We applied comparative phylogenetic approaches to analyse the evolutionary trends in muscle size across the clade. We found two major patterns: (i) darter cranial muscles show fundamental trade-offs relating to investment in musculature, as well as buccal expansion vs. biting attributes; early divergence in muscle size appears to be associated with shifts in habitat use and foraging; (ii) darter adductor mandibulae show high variation in architecture (fibre orientation, divisions). This study highlights how new imaging techniques can provide novel insights into the anatomy of even well-sampled/represented clades.
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