Genomics and ecological modelling clarify species integrity in a confusing group of butterflies.

Recent advances in both genomics and ecological modelling present new, multidisciplinary opportunities for resolving species boundaries and understanding the mechanisms that maintain their integrity in regions of contact. Here, we use a combination of high-throughput DNA sequencing and ecological niche modelling to resolve species boundaries and niche divergence within the Speyeria atlantis-hesperis (Lepidoptera: Nymphalidae) complex, a confusing group of North American butterflies. This complex is notorious for its muddled species delimitations, morphological ambiguity, and extensive mitonuclear discordance. Our admixture and multispecies coalescent-based analyses of single nucleotide polymorphisms identified substantial divergences between S. atlantis and S. hesperis in areas of contact, as well as between distinct northern and southern lineages within S. hesperis. Our results also provide evidence of past introgression relating to another species, S. zerene, which previous work has shown to be more distantly related to the S. atlantis-hesperis complex. We then used ecological models to predict habitat suitability for each of the three recovered genomic lineages in the S. atlantis-hesperis complex and assessed their pairwise niche divergence. These analyses resolved that these three lineages are significantly diverged in their respective niches and are not separated by discontinuities in suitable habitat that might present barriers to gene flow. We therefore infer that ecologically-mediated selection resulting in disparate habitat associations is a principal mechanism reinforcing their genomic integrity. Overall, our results unambiguously support significant evolutionary and ecological divergence between the northern and southern lineages of S. hesperis, sufficient to recognize the southern evolutionary lineage as a distinct species, called S. nausicaa based on taxonomic priority.