The colouration of the common butterflies Aglais urticae (Small Tortoiseshell), Aglais io (Peacock) and Vanessa atalanta (Red Admiral), belonging to the butterfly subfamily Nymphalinae, is due to the species-specific patterning of differently coloured scales on their wings. We investigated the scales’ structural and pigmentary properties by applying scanning electron microscopy, (micro)spectrophotometry, and imaging scatterometry. The anatomy of the wing scales appears to be basically identical, with an approximately flat lower lamina connected by trabeculae to a highly structured upper lamina, which consists of an array of longitudinal, parallel ridges and transversal crossribs. Isolated scales observed at the abwing (upper) side are blue, yellow, orange, red, brown, or black, depending on their pigmentation. The yellow, orange and red scales contain various amounts of 3-OH-kynurenine and ommochrome pigment, black scales contain a high density of melanin, and blue scales have a minor amount of melanin pigment. Observing the scales from their adwing (lower) side always revealed a structural colour, which is blue in the case of blue, red and black scales, but orange for orange scales. The structural colours are created by the lower lamina, which acts as an optical thin-film. Its reflectance spectrum, crucially determined by the lamina thickness, appears to be well-tuned to the scales’ pigmentary spectrum. The colours observed locally on the wing are also due to the degree of scale stacking. Thin films, tuned pigments and combinations of stacked scales together determine the wing colouration of nymphaline butterflies.

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