DOI: 10.5061/dryad.rfj6q57n6
title: Data from: <em>Araucan</em> regulates butterfly wing iridescence by coordinating scale structure and pigmentation

description: Butterfly wings exhibit a remarkable diversity of structural iridescent
 colors, yet the genetic regulation of iridescence remains poorly
 understood. Here, we show that the Iroquois-complex transcription factor
 gene araucan plays a role in modulating wing scale iridescence in the
 common buckeye butterfly, Junonia coenia. Using CRISPR-Cas9 knockouts, we
 demonstrate that loss of araucan function causes dorsal wing scales to
 shift from golden-brown to blue iridescence, and eyespot center scales to
 shift from saturated purple-violet to dull grey-brown. These phenotypes
 are associated with changes in thickness of the lower lamina in scale
 cells, a structural feature known to influence thin-film interference,
 along with reduction of pigmentation in ground scales. Together, these
 results identify araucan as a single transcription factor that determines
 coloration by simultaneously regulating both photonic architecture and
 light-absorbing pigmentation, and show that these effects can be regulated
 in pattern-specific manner. Our findings provide a framework for linking
 gene regulation to the spatially coordinated evolution of structural and
 pigmentary components of color.
description: # Data from: *Araucan* regulates butterfly wing iridescence by
 coordinating scale structure and pigmentation Dataset DOI:
 [10.5061/dryad.rfj6q57n6](https://doi.org/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 --- ##### 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`). --- ##### 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 ``` --- #### 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)**. --- ##### 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. --- ##### 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)**. --- ##### 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.