How barbule orientation drives iridescent coloration in Coeligena hummingbirds.

Hummingbirds produce intense iridescent signals through multilayered melanosomes arranged within feather barbules. Because barbules are typically inclined relative to the feather surface, their orientation alters the direction of peak reflectance. Yet reflectance is commonly measured under normal incidence relative to the feather plane, implicitly assuming coplanar barbules. Here, we show that not accounting for this geometry in the design of reflectance measurements may influence the interpretation of the results, depending on the reference frame adopted. By integrating experimental angular measurements with multilayer optical modeling, we predict the angle of maximum reflectance and infer barbule inclination (δ) from nanostructural morphology. Reanalysis of published spectra across multiple hummingbird species suggests that a substantial proportion of reported measurements may be more consistently interpreted under geometries influenced by barbule inclination, rather than under conventional normal-incidence conditions defined at the feather plane. These estimates depend on the assumptions of the optical model and should be interpreted within the context of standard experimental conventions. Together, our results highlight barbule inclination as a key geometric parameter linking microstructure to signal directionality and establish a framework for standardizing optical measurements of iridescent coloration.