Abstract
Genetic determinants of speciation in closely related species are poorly understood. We sequenced and analyzed transcriptomes of swallowtail butterflies Heraclides cresphontes (northeastern species) and Heraclides rumiko (southwestern species), a pair of mostly allopatric sister species whose distribution ranges overlap narrowly in central Texas. We found that the two swallowtails confidently differ (F(ST) > 0.5 for both species) in about 5% of genes, similarly to the divergence in another pair of swallowtail species Pterourus glaucus (southern species) and Pterourus canadensis (northern species). The same genes tend to diverge in both species pairs, suggesting similar speciation paths in Heraclides and Pterourus. The most significant differences for both species pairs were found in the circadian clock genes that were conserved within each species and diverged strongly between species (P-value < 0.01 and F(ST) > 0.7). This divergence implied that adaptations to different climates and photoperiod at different latitudes or differences in mating behavior, including mating time and copulation duration, may be possible factors in ecological or behavioral-based speciation. Finally, we suggest several nuclear DNA regions that consistently and prominently differ between the sister swallowtail species as nuclear barcodes for swallowtail identification, with the best barcode being an exon from the protein TIMELESS.