DOI: 10.5061/dryad.2fqz612mc
title: Data from: Convergent evolution of broadband reflectors underlies metallic colorations in butterflies

description: Supplementary Files for Ren, Day et al. 2020 : Convergent
 evolution of broadband reflectors underlies metallic
 coloration in butterflies
description: <strong>Scanning electron microscopy
 (SEM)</strong><br> For surface imaging of scales of distinct
 identities (<strong>TIF files</strong>), 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 <strong>PDF files</strong>:
 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. 
 <strong>SEM pixel profile acquisitions and morphometric
 analysis</strong><br> 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 <em>Vanessa cardui </em>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 <em>FIJI</em>. 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 <em>SEMolina</em> semi-automated pipeline in order to measure several ultrastructural features, as described below. All the <em>SEMolina</em> 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.
description: TIF files: high-resolution SEM images<br> PDF files:
 color/SEM overlays<br> JPG files are provided as
 thumbnails<br> <br> <strong>Species codes and pattern
 location</strong><br> Af = <em>Anteros
 formosus,</em> ventral hindwing<br> Av = <em>Agraulis
 vanillae,</em> ventral hindwing<br> Ce = <em>Colias
 eurytheme,</em> ventral hindwing (D1 discal spot), male
 specimen<br> Ci = <em>Cigaritis lohita,</em> ventral
 hindwing<br> Ec = <em>Epargyreus clarus,</em> ventral
 hindwing<br> Ha =<em> Hypochrysops apelles,</em> ventral
 hindwing<br> Sc = <em>Speyeria cybele,</em> ventral
 hindwing<br> Vc = <em>Vanessa cardui,</em> ventral
 hindwing (Cu1-Cu2 eyespot used in Day et al., 2019 :  https://doi.org/10.1002/dvdy.63)<br> Zc = <em>Zerene cesonia,</em> ventral hindwing (D1 discal spot), male specimen