2.2. Psittacofulvin colour gamut

The 26 NMNH parrot feathers each featured a region of barbs made up of predominantly one colour (e.g. magenta; appendix A). The coloured regions of these parrot feathers were analysed on site in the collections of the Division of Birds, NMNH. Three spectra were collected from nearby positions on each feather using a Raman microscope (Nomadic, BaySpec, San Jose CA, USA) that had a 1064 nm excitation Nd:YAG laser with a 512 pixel InGaAs array detector for a maximum spectral range of 277–1886 cm⁻¹ (electronic supplementary material, tables S1 and S2). A 40× microscope objective (EPlan, Nikon Instruments, Tokyo, Japan) with a 25 µm confocal pinhole produced a minimum spot size diameter of 2 µm. A Raman spectrum from a white wing feather of a red-footed booby was collected as a β-keratin spectral standard (electronic supplementary material, table S1). Triplicate Raman spectra from each feather were preprocessed by first reducing the spectral range to 900–1800 cm⁻¹, followed by Savitzky–Golay filtering (sgolay::signal 0.7–6) [28] and baseline correction (baseline::baseline 1.2-1) [29] in R v. 3.4.1 [30]. Triplicate preprocessed spectra were then intensity normalized (i.e. intensities scaled between 0 and 1) and averaged (electronic supplementary material, table S2). Peak parameters were calculated using Spectragryph 1.2 [31].

Raman instruments that produced higher resolution spectra over larger wavenumber ranges were used to provide a second spectral dataset from five of the 26 NMNH parrot feathers (electronic supplementary material, table S3). This second spectral dataset was produced to support peak assignments made for the 26 feather dataset, and was based on feathers that could be transported out of the Division of Birds to two additional Raman instruments. Spectra were collected with a Nicolet Almega XR spectrometer (Thermo Electron Corporation, Madison, WI, USA) using 780 nm excitation to produce spectra across 100–3500 cm⁻¹ at 3 cm⁻¹ resolution, and an NXR FT-Raman module coupled to a 6700 FTIR spectrometer (Thermo Electron Corporation) which used 1064 nm excitation to produce spectra across 100–3700 cm⁻¹ at 4 cm⁻¹ resolution.