Multiple ways to become red: Pigment identification in red feathers using spectrometry

Red hues are a challenge in studies on the evolution of bird coloration, as multiple pigments such as carotenoids, pheomelanin, psittacofulvins, porphyrins, turacin, haemoglobin and even exogenous iron-oxides, may confer red colors. Determining the pigment type is paramount and here we investigate the differences in spectrum reflectance for six pigments resulting in red colorations in feathers of different species, with a focus on discriminating among melanins and carotenoids. Pigment chemical identification was obtained from the literature or using High Performance Liquid Chromatography (HPLC) in our laboratory. We have also derived discriminant formulas for identification of the major known types of pigments based on parameters of the reflectance curves obtained with a portable spectrometer. Our results indicate that the reflectance patterns of coloration perceived as red patches widely differ. The distinction between carotenoid- and melanin-based reflectance curves is relatively straightforward: sigmoid versus straight slope. The spectral reflectance curves of feathers containing red psittacofulvins are sigmoid, whereas iron oxide and porphyrin-containing feathers recall pheomelanins in rendering a straight slope. In the case of turacin-based coloration, the spectral shape is unique. For the pigments with enough number of species sampled (i.e., carotenoids, melanins and psittacofulvins) the differences in reflectance shape are important enough to allow separation of carotenoid and melanin derived colorations based on reflectance curves alone.