Supplemental data from: Genomic signatures of speciation in butterflies

Studies of life rely on classifying organisms into species. Contrary to a frequent belief, simple and quantitative standards for species delineation are lacking, and debates about species delimitation create obstacles for conservation biology, agriculture, legislation, and education. To tackle this key biological question, we have chosen butterflies as model organisms. We sequenced and analyzed transcriptomes of 186 butterfly specimens representing pairs of close but clearly distinct species, conspecific populations, and taxa that are debated among experts. We find that species are robustly separated from conspecific populations by the combination of two measures computed on Z-linked genes: the fixation index that detects hiatus between species, and the extent of gene flow that quantifies reproductive isolation. These criteria suggest that all 9 butterfly pairs that caused experts' disagreement are distinct species, not populations or subspecies. When applied to Homo, our criteria agree..., Sample Collection We assembled 25 pairs of butterfly counterparts from the eastern and western sides of the Central Texas suture zone. For each pair, we gathered 2 - 42 specimens on each side. Our analysis focused on protein-coding sequences. For 21 of the 25 pairs, we obtained their sequences using RNA-seq. For the remaining pairs in the Calephelis, Calycopis, Phoebis, and Pterourus genera, we did not have specimens preserved for RNA-seq. Instead, we relied on previously published genomic DNA data. These genomic DNA data were obtained via Illumina sequencing of paired-end libraries, targeting 10-fold coverage for each library. We obtained protein-coding sequences for these four genera by mapping the genomic reads to their annotated reference genomes and extracting the coding regions. Library Preparation and Sequencing Specimens for RNA-seq libraries were euthanized upon capture by thorax pinching, and their bodies (excluding wings and genitalia) were preserved in RNAlater solution. Tot..., , # Butterfly RNA Analysis Pipeline This repository contains a comprehensive analysis pipeline for studying butterfly speciation and divergence using RNA-seq data. The analysis is organized into 6 main stages, each focusing on different aspects of genomic and transcriptomic analysis. ## Overview This pipeline analyzes nuclear and mitochondrial DNA sequences from multiple butterfly species to study: * Species delineation using fixation indices * Divergence hotspots identification * Bayesian phylogenetics and phylogeography (BPP) * Hybrid zone analysis ## Directory Structure ### `previous/` Contains older versions of the analysis results as compressed tar.gz files. These serve as backups of previous iterations of each analysis stage. **Files:** * `reference_genomes.tar.gz` - Previous version of reference genome data * `stage1_integrate_nuclear_data.tar.gz` - Previous nuclear data integration results * `stage2_integrate_mito_data.tar.gz` - Previous mitochondrial data integration res..., ,