Convergent evolution of broadband reflectors underlies metallic colorations in butterflies

Supplementary Files for Ren, Day et al. 2020 : Convergent evolution of broadband reflectors underlies metallic coloration in butterflies

Methods
Scanning electron microscopy (SEM)

For surface imaging of scales of distinct identities (TIF files), wing patterns of interest were excised and mounted on SEM stubs with double-sided carbon tape, and color imaged under the Keyence VHX-5000 microscope for registration of scale type (see PDF files: layered overlays of SEM and color images). Samples were sputter-coated with two 12.5 nm layers of gold for improving sample conductivity, with the second layer applied after tilting the stub by 45°. SEM images were acquired on a FEI Teneo LV SEM, using secondary electrons (SE) and an Everhart-Thornley detector (ETD) using a beam energy of 2.00 kV, beam current of 25 pA, and a 10 μs dwell time. Individual images were stitched using the Maps 3.10 software (ThermoFisher Scientific). To minimize charging for high magnification views of scale surface morphology, individual scales were collected by brushing the surface of the wing with an eyelash tool, then dusted onto an SEM stub with double-sided carbon tape. Stubs were sputter-coated with one 12.5 nm layer of gold, and imaged at 2.00 kV / 25 pA with a 10 μs dwell time. 

SEM pixel profile acquisitions and morphometric analysis

Scales were analyzed using our custom semi-automated R pipeline that derives ultrastructural parameters from large SEM images (Day et al., 2019 :  https://doi.org/10.1002/dvdy.63). Briefly, for the Vanessa cardui eyespot, a Keyence 300X micrograph and SEM image of the Cu1-Cu2 wing region were overlaid to register the color and positional information for each scale. Then, morphometric analyses were performed on the high-resolution images obtained from stitched 1500x micrographs. For each scale, one straight line segment was drawn across the full width of the scale, perpendicular to the visible ridges and another perpendicular to the visible crossribs, using FIJI. Plots of the pixel gray values was extracted using the plot profile function. The pixel intensity profiles derived from the inter-ridge and inter-crossribs segments were then analysed using our custom SEMolina semi-automated pipeline in order to measure several ultrastructural features, as described below. All the SEMolina code is available on the GitHub repository at https://github.com/Hanliconius. For the metallic scale analysis, the same process was used to derive morphometric measurements of scale widths and ridge distances on 25 scales of each color from 8 species.