Recently diverged species may hybridize in their contact zones if complete reproductive isolation has not yet emerged. Petunia inflata and P. interior are closely related species with a narrow geographic distribution in Argentina and southern Brazil. They share morphological features, genetic markers, pollinators, and occupy a transitional area between the Pampa grasslands and the Brazilian Atlantic forest. Here, we used genomic data to verify species boundaries, identify putative hybrids, and shed light on their speciation process. We characterized 59 individuals from allopatric and contact zones using genotyping-by-sequencing technology, resulting in a final dataset with 21,759 neutral single nucleotide polymorphisms used to perform structure, demographic, and hybridization analyses. These species belong to distinct evolutionary lineages that hybridized after secondary contact. A combination of geographic distance, elevation, and climate explains the genetic divergence between species. Niche overlap analysis revealed that even though these species have overlapping distributions and similar habitat preferences, they are more distinct than expected by chance, rejecting the niche conservatism hypothesis. Demographic analysis suggested that the species diverged during the Last Glacial Maximum but came into secondary contact during the mid-Holocene. The secondary contact led to limited gene flow between species and bidirectional introgression. The distribution expansion of these species ancestor to a transitional zone between biomes, associated with elevational ranges and habitat fragmentation, promoted speciation and niche differentiation.
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