Genomic Evidence of Hybridization Between Two Species in <i>Nicotiana</i> sect. <i>Alatae</i>

BACKGROUND AND AIMS: Hybridization is recognised as a significant evolutionary force in plants. Advances in genomics have revealed pervasive introgression, which often challenges traditional species boundaries. This study aimed to investigate the genetic structure and hybridization patterns in an atypical population with intermediate morphology, suggestive of hybridization between Nicotiana alata and N. forgetiana, two closely related species with divergent pollination syndromes that were previously thought to maintain strong reproductive isolation. METHODS: We employed a reduced genome representation genotyping approach and population genomic analyses to assess gene flow between the two species. A total of 66 individuals were sampled from seven populations, including sympatric and allopatric sites. Our analyses focused on detecting population structure, signals of hybridization and introgression, and evolutionary relationships. KEY RESULTS: We uncovered extensive and bidirectional gene flow between these species. The sympatric population AP01 comprises two distinct genetic lineages (AP01-A and AP01-B) with different ancestry profiles and corresponding phenotypic variation. Notably, despite genomic admixture, some hybrid individuals retain canonical floral traits, raising questions about the morphological assessment of species identity in this system. Unexpectedly, an allopatric N. alata population (Ala05) also revealed signals of admixture, clustering genetically with N. forgetiana despite displaying N. alata morphology and being isolated from known contact zones. Genetic differentiation between the two N. alata populations was as high as that between the species, indicating strong genetic structure within the species. CONCLUSIONS: This study presents genomic evidence of a natural hybrid zone in diploid Nicotiana sect. Alatae. We show that gene flow between N. alata and N. forgetiana is more extensive than previously considered, and that floral morphology is an unreliable predictor of genetic ancestry. The discovery of cryptic introgression in an allopatric population suggests that N. alata may harbor unrecognized complexity, warranting broader genomic sampling across its range.