Phylogenomic test of mitochondrial clues to archaic ancestors in a group of hybridizing swallowtail butterflies

Genomics has revolutionized our understanding of hybridization and introgression, but most of the early evidence for these processes came from studies of mitochondrial introgression. To expand these evolutionary insights from mitochondrial patterns, we evaluate phylogenetic discordance across the nuclear genomes of a hybridizing system, the Papilio machaon group of swallowtail butterflies. This species group contains three hybrid lineages (P. brevicauda, P. joanae, and P. m. kahli) that are geographically disjunct across North America and have complete fixation of a mitochondrial lineage that is otherwise primarily found in P. m. hudsonianus, a boreal subspecies of the Holarctic P. machaon. Genome-wide nuclear markers place the three hybrid lineages as a monophyletic group that is sister to P. polyxenes/P. zelicaon rather than P. machaon, although ancient hybridization between a subspecies of P. machaon and the ancestor of these three lineages is also shown by their greater nuclear affinity to P. m. hudsonianus than to other subspecies of P. machaon. Individuals from contemporary hybrid swarms in Alberta, where mitochondrial DNA fixation has not occurred, were more intermediate between their respective parent species, demonstrating diversity in mito-nuclear discordance following hybrid interactions. Our new phylogenetic findings for the P. machaon species group also include: subspecific paraphyly within P. machaon itself across its Holarctic distribution; paraphyly of P. zelicaon relative to P. polyxenes; and more divergent placement of a Mediterranean species, P. hospiton. These results provide the first comprehensive genomic evaluation of relationships within this species group and provide insight into the evolutionary dynamics of hybridization and mitochondrial introgression.