Balancing mechanics and metabolism: Elevational variation in the microanatomy of hummingbird flight muscle.

Factors varying along elevational gradients impose strong aerodynamic and physiological constraints on powered flight, yet the internal microanatomical variation associated with elevational context in animals under such conditions remains poorly understood. In hummingbirds, sustained hovering requires extreme muscular power output, making the pectoralis muscle a key tissue for evaluating anatomical variation associated with elevational context. We tested whether pectoralis microanatomy differed among hummingbirds sampled at three elevationally distinct sites spanning a ~1500 m gradient in the Colombian Andes. Using tissue morphometry of trichrome-stained transverse sections of the pectoralis, we measured collagen-rich interstitial extracellular matrix fraction as an anatomical proxy and quantified fiber-profile cross-sectional area, packing density, and size heterogeneity. Collagen-rich ECM fraction differed among sites, with the highest values at the mid-elevation site and the lowest values at the high-elevation site. Fiber-profile cross-sectional area tended to be lower and packing density higher at the high-elevation site, although site-level contrasts showed broad uncertainty. Because species identity and site could not be fully disentangled in this limited dataset, these patterns are interpreted as site-associated anatomical variation rather than definitive evidence of elevation-driven adaptation. These results suggest that hummingbird pectoralis microanatomy differs among sampled elevational contexts in a trait-specific manner, with the strongest evidence in fiber geometry. More broadly, our findings highlight that multiple components of muscle microarchitecture, including the extracellular matrix, are consistent with diffusion-related expectations rather than direct evidence of functional performance differences.