Dataset DOI: 10.5061/dryad.rfj6q57n6

Description of the data and file structure

Microspectrophotometry – reflectance and absorbance spectra

This dataset contains micro-spectrophotometry reflectance and absorbance spectra of butterfly wing scales, including:

• Non-eyespot regions – cover scales
• Eyespot regions – both cover and ground scales

All raw data files are compressed into microspectrophotometry_data.zip.

Ultrastructure measurements – FIB-SEM

Ultrastructural features of wing scales were quantified from cross-sections obtained by Focused Ion Beam milling followed by scanning electron microscopy (FIB-SEM). We measured:

• Distance between adjacent ridges (dR–R)
• Distance between adjacent crossribs (dCR–CR)
• Thickness of the lower lamina (LL)

All measurements and the corresponding raw FIB-SEM images are contained in ultrastructure_FIBSEM.zip.

Files and variables

Microspectrophotometry – reflectance and absorbance spectra

Folder structure

Inside microspectrophotometry_data.zip:

• reflectance/absorbance raw/
  Raw spectra exported directly from the microspectrophotometer software.
• reflectance/absorbance for plots/
  Processed spectra used to generate the plots in the manuscript (e.g. averaged or cleaned data).

Within the eyespot datasets under reflectance/absorbance raw/, raw data are further organized into:

• absorbance raw data/ – absorbance spectra for eyespot cover and ground scales
• reflectance raw data/ – reflectance spectra for eyespot cover and ground scales

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File naming convention

All spectral files follow this general pattern:

[GENOTYPE]_[SCALE-TYPE]_[MEASUREMENT]_[MAGNIFICATION]_scale[NN]_[RR].txt

where:

• GENOTYPE
  • WT or control = wild-type individuals
  • araucan or araucan_ectopic_blue = araucan CRISPR-Cas9 mosaic knockouts (mKO)
• SCALE-TYPE
  • cover = cover scales
  • ground = ground scales
• MEASUREMENT
  • abs = absorbance spectrum
  • ref = reflectance spectrum
• MAGNIFICATION
  Objective magnification used for data collection, e.g.:
  • 20x = 20× objective
  • 100x = 100× objective
• scale[NN]
  Identifier for the individual scale measured.
  • For absorbance, scale01–scale05 correspond to five independently sampled scales.
  • For reflectance, scale01–scale03 correspond to a different set of three independently sampled scales.
• [RR]
  Two-digit replicate number (01, 02, 03, …) indicating repeated measurements from the same scale under identical settings.

Example:

• araucan_cover_abs_20x_scale01_01.txt
  Araucan mosaic knockout, cover scale, absorbance spectrum, 20× objective, scale 01, replicate 01.
• WT_cover_abs_20x_scale05_03.txt
  Wild-type, cover scale, absorbance spectrum, 20× objective, scale 05, replicate 03.

For each genotype (WT, araucan), there are typically 5 scales (scale01–scale05) for absorbance measurements, with multiple replicate spectra per scale (e.g. _01–_05).

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Layout of the .txt spectra files

Each .txt file is a plain-text, tab-delimited table with two columns:

1. Wavelength_nm – wavelength of incident light in nanometers (nm)
2. Value – absorbance (unitless) for abs files, or reflectance (unitless) for ref files

Example:

Wavelength_nm    Value
300              0.0123
301              0.0131
...
800              0.0456

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Ultrastructure measurements – FIB-SEM

Ultrastructural features of wing scales were quantified from cross-sections obtained by Focused Ion Beam milling followed by scanning electron microscopy (FIB-SEM). We measured:

• Distance between adjacent ridges (dR–R)
• Distance between adjacent crossribs (dCR–CR)
• Thickness of the lower lamina (LL)

All measurements and the corresponding raw FIB-SEM images are contained in ultrastructure_FIBSEM.zip.\

Eyespot ultrastructure measurements (eyespot region)

For eyespot scales, measurements are provided in an Excel file named eyespot_ultrastructure_measurement.xlsx. This file contains six sheets:

• dRR_cover
• dRR_ground
• dCRCR_cover
• dCRCR_ground
• LL_cover
• LL_ground

where:

• dRR = ridge–ridge distance
• dCRCR = crossrib–crossrib distance
• LL = lower lamina thickness
• cover / ground = scale type in the eyespot region (cover scales vs. ground scales)

Each sheet includes:

• A condition column indicating genotype:
  • WT = wild-type scales
  • mutant (e.g. araucan / araucan_ectopic_blue) = araucan CRISPR-Cas9 knockout scales
• A scale identifier (scale number) indicating which individual scale was measured
• One or more replicate measurements per scale

All distances and thickness values are reported in nanometers (nm).

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Raw FIB-SEM image files – eyespot region

Raw FIB-SEM images for eyespot scales are organized into:

• FIBSEM-eyespot_cover_scales_images/
  Raw images of eyespot cover scales, named:

  GENOTYPE_eyespot_scaleNN_RR.tiff

  where:

  • GENOTYPE = mutant (araucan knockout) or WT (wild-type control)
  • NN = individual scale measured (e.g. 01, 02, …)
  • RR = region on that scale from which the measurement was taken

• FIBSEM-eyespot_ground_scales_images/
  Raw images of eyespot ground scales, named:

  GENOTYPE_ground_scaleNN_RR.tiff

  where:

  • GENOTYPE = mutant (araucan knockout) or WT (wild-type control)
  • NN = individual scale measured
  • RR = region on that scale used to obtain the corresponding measurement

These TIFF files provide the raw image context for the ultrastructure measurements reported in the eyespot Excel file.

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Non-eyespot ultrastructure measurements (non-eyespot region)

There is an additional folder called non eyespot, which contains an Excel file named araucan_non_eyespots_measurements.xlsx with measurements from non-eyespot cover scales. This file has three sheets:

• LL – lower lamina thickness
• dRR – ridge–ridge distance
• dCRCR – crossrib–crossrib distance

Within this Excel file:

• Mutant_blue refers to araucan knockout (ectopic blue) scales.
• WT_brown refers to wild-type brown scales.
• All scale numbers correspond to individual scales that were measured.
• All distance and thickness values are reported in nanometers (nm).

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Raw FIB-SEM image files – non-eyespot region

The subfolder FIBSEM-noneyespot_cover_scales_images/ contains the raw images used to obtain the non-eyespot measurements. Files are in TIFF format and named as:

araucan_GENOTYPE_scaleNN_RR.tiff

where:

• GENOTYPE =
  • blue_EA for ectopic blue araucan mutant scales
  • ctrl_brown for wild-type brown scales
• NN = scale number (from 1–5), corresponding to individual scales in the Excel file
• RR = region on the scale from which measurements were taken (from 1–3, and in some cases 4)

These TIFF images correspond directly to the non-eyespot ultrastructure measurements in the associated Excel sheets.