The prey-processing behavior and jaw-adducting musculature of tetraodontiform fishes provide a novel system for studying the evolution of muscles and their function. The history of this clade has involved a pattern of repeated ‘duplications’ of jaw muscles by physical subdivision of pre-existing muscles. As a result, the number of adductor mandibulae muscles in different taxa varies from as few as two to as many as eight. We used electromyography (EMG) to quantify motor-pattern variation of adductor mandibulae muscles in four tetraodontiform species during feeding events on prey items that varied in durability and elusiveness. Statistical analyses of variation in EMG variables revealed significant differences in motor patterns between duplicated muscles derived from a common ancestral muscle in seven of nine cases examined. Overall individual EMG timing variables (e.g. relative onset or duration of bursts) were slightly less likely to diverge functionally than amplitude variables (e.g. relative intensity of bursts). Functional divergence was found in significant overall differences between muscles and twice as frequently in significant muscle-by-prey interaction terms. Such interactions represent an underappreciated way in which motor patterns can evolve and diversify. Regional variation was documented in undivided muscles in two species, indicating that it is possible for functional subdivision to precede anatomical subdivision. This study shows that phylogenetic increases in the number of tetraodontiform jaw adductor muscles have been associated with increases in the functional complexity of the jaws at the level of muscle activation patterns.

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