Pinguicula (Lentibulariaceae, Lamiales) is a monophyletic genus consisting of ca. 80–100 carnivorous species distributed across subarctic, temperate, and tropical regions. To date, phylogenetic relationships within the genus remain insufficiently resolved, and with the exception of P. balcanica, no samples from Türkiye have been included in such studies. Moreover, Türkiye’s role in the evolutionary history of the genus has been entirely overlooked. Neither the recently described P. habilii, endemic to Muğla in southwestern Anatolia, nor other taxonomically problematic taxa (i.e., P. crystallina, P. megaspilaea, and P. hirtiflora) from Anatolia, Cyprus, and Greece have been comprehensively included in phylogenetic studies. Although these taxa have occasionally appeared in earlier works, they have never been analyzed within a focused and integrative phylogenetic framework. Furthermore, key questions regarding the timing and geographic origin of the Türkiye and Cypriot lineages, the historical dispersal routes leading to their present-day distributions, and the historical biogeographic processes that have shaped these patterns have remained unexplored. To address these gaps and to establish a comprehensive phylogenetic framework for the genus, we conducted a molecular phylogenetic study of Pinguicula. This study incorporated newly generated DNA sequences together with publicly available sequences retrieved from GenBank, including nuclear DNA (ITS region) and plastid DNA regions (trnH-psbA and rpL32-trnL). Our Maximum Likelihood (ML) and Bayesian Inference (BI) analyses revealed that, although the genus is monophyletic, the Pinguicula crystallina/hirtiflora complex is still undergoing speciation, likely due to its relatively recent origin. Within this complex, only P. megaspilaea was recovered as strongly monophyletic, whereas its phylogenetic placement differed substantially between the nuclear and plastid datasets. Similarly, P. balcanica was recovered as two well-supported clades in the nuclear phylogeny, but as a single clade in the rpL32-trnL plastid phylogeny, representing an additional case of cytonuclear discordance within the genus. Taken together, these results indicate a complex and dynamic evolutionary history within Pinguicula, in which reticulate evolution and geographic isolation appear to have played significant roles. On the other hand, our molecular dating and ancestral biogeographic analyses indicated that the genus Pinguicula most likely originated in Europe approximately 24.69 million years ago, that the Pinguicula crystallina/hirtiflora complex evolved in Greece around 6.46 million years ago, subsequently expanded into Anatolia, and later dispersed from Anatolia to Cyprus.
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