Quantifying Iridescent Feather Nanostructures

To understand the morphological (nanostructural) traits underlying divergence in iridescent coloration, we used a transmission electron microscope (TEM; see below) to image cross-sections of three to five iridescent feather barbules from three body regions (crown, gorget and tail) per single individual of each parental species and the hybrid, following the protocol developed by Shawkey et al. [29 (link)]. Briefly, we dissected iridescent feather barbs, dehydrated them in ethanol, infiltrated the feathers with Embed 812 resin, and cured them at 60°F. We then sectioned the polymerized blocks into approximately 90 nm sections and imaged them on a Philips CM200 TEM at the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign. On each TEM image, we measured several traits known to be involved in iridescent colour production [17 (link)]: (i) melanosome platelet thickness (pt), (ii) amount of keratin between melanosomes (ker), (iii) air space diameter (air), (iv) number of melanosome layers, (v) thickness of top surficial melanosomes (pt_top), and (vi) keratin cortex thickness (cortex). For air space diameter, we took measurements both parallel (air_par) and perpendicular to the feather barbule surface (air_perp). This was done to determine whether deformation had occurred during sectioning (e.g. the resin often ‘pulls away’ from the outer barbule surface resulting in perpendicular deformation). We made the assumption that air spaces should be roughly isometric in cross-section, thus we took the average of perpendicular and parallel measurements. In total, we took 2421 individual measurements from 33 TEM images (9–13 images per taxon).