Geographic isolation and reduced population sizes can lead to local extinction, low efficacy of selection, and decreased speciation. However, population differentiation is an essential step of biological diversification. In allopatric speciation, geographically isolated populations differentiate and persist until the evolution of reproductive isolation and ecological divergence complete the speciation process. Pitcairnia flammea allow us to study the evolutionary consequences of habitat fragmentation on naturally disjoint rock outcrop species from the Brazilian Atlantic Rainforest (BAF). Our main results showed low to moderate genetic diversity within populations, and deep population structuring caused by limited gene flow, low connectivity, genetic drift and inbreeding of long-term isolation and persistence of rock outcrops populations throughout Quaternary climatic oscillations. Bayesian phylogenetic and model-based clustering analyses found no clear northern and southern phylogeographic structure commonly reported for many BAF organisms. Although, we found two main lineages diverging by approximately 2 Mya during the early Pleistocene, species delimitation analysis assigned most of the populations as independent evolving entities, suggesting an important role of disjoint rock outcrops in promoting high endemism in this rich biome. Lastly, we detected limited gene flow in sympatric populations although some hybridization and introgression were observed, suggesting a continuous of speciation processes in this species complex. Our data not only inform us about the extensive differentiation and limited gene flow found among Pitcairnia flammea species complex, they also contain information about the mechanisms that shape the genetic architecture of small and fragmented populations of isolated rock outcrop of recently radiated plants.

Two plastid DNA regions (rpl32-trnL and rps16-trnK) were amplified and sequenced for a subset of 176 individual from the populations sampled, using the primers described by Shaw et al. (2007). All polymerase chain reaction (PCR) amplifications were carried out in a total volume of 20 μL containing 10 ng DNA template, 1× GoTaq buffer, 2.5 mMMgCl2, 0.25 mMdNTP mix, 5 pmol forward and reverse primers and 0.5 U GoTaq DNA polymerase (Promega) and run using the following parameters: initial denaturation at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min, 54°C (rpl32-trnL) or 58°C (rps16-trnK) for 1 min, and 72°C for 1 min, and a final extension of 10 min at 72°C. PCR products were sent for both forward and reverse sequencing to Macrogen (Seul, Korea).