When COI barcodes deceive: complete genomes reveal introgression in hairstreaks

Two species of hairstreak butterflies from the genusCalycopisare known in the United States:C. cecropsandC. isobeon. Analysis of mitochondrial COI barcodes ofCalycopisrevealedcecrops-like specimens from the eastern US with atypical barcodes that were 2.6% different from either USA species, but similar to Central AmericanCalycopisspecies. To address the possibility that the specimens with atypical barcodes represent an undescribed cryptic species, we sequenced complete genomes of 27Calycopisspecimens of four species:C. cecrops,C. isobeon,C. quintanaandC. bactra. Some of these specimens were collected up to 60 years ago and preserved dry in museum collections, but nonetheless produced genomes as complete as fresh samples. Phylogenetic trees reconstructed using the whole mitochondrial and nuclear genomes were incongruent. While USACalycopiswith atypical barcodes grouped with Central American speciesC. quintanaby mitochondria, nuclear genome trees placed them within typical USAC. cecropsin agreement with morphology, suggesting mitochondrial introgression. Nuclear genomes also show introgression, especially betweenC. cecropsandC. isobeon. About 2.3% of eachC. cecropsgenome has probably (p-value < 0.01, FDR < 0.1) introgressed fromC. isobeonand about 3.4% of eachC. isobeongenome may have come fromC. cecrops.The introgressed regions are enriched in genes encoding transmembrane proteins, mitochondria-targeting proteins and components of the larval cuticle. This study provides the first example of mitochondrial introgression in Lepidoptera supported by complete genome sequencing. Our results caution about relying solely on COI barcodes and mitochondrial DNA for species identification or discovery.