ABSTRACT
BACKGROUND AND AIMS: Species delimitation can be challenging when analysing recently diverged species, especially those taxonomically synonymised due to morphological similarities. We aimed to untangle the relationships between two grassland species, Petunia guarapuavensis and Petunia scheideana, exploring the dynamics of fast divergence and addressing their species delimitation. METHODS: We used a low-coverage genome sequencing and population genomic approach to distinguish species and populations between P. guarapuavensis and P. scheideana. Our analysis focused on detecting structuration, hybridisation/introgression, and phylogenetic patterns. We employed demographic models to support species delimitation while exploring potential phylogeographic barriers influencing gene flow. KEY RESULTS: Our findings indicated differentiation between the two species and revealed another lineage, which was phylogenetically distinct from the others and had no evidence of gene flow with them. The presence of a river acted as a phylogeographic barrier, limiting gene flow and allowing for structuration between closely related lineages. The optimal species delimitation scenario involved secondary contact between well-established lineages. CONCLUSIONS: The rapid divergence observed in these Petunia species explains the lack of significant morphological differences, as floral diagnostic traits in species sharing the pollinators tend to evolve more slowly. This study highlights the complexity of species delimitation in recently diverged groups and emphasises the importance of genomic approaches in understanding evolutionary relationships and speciation dynamics.