PREMISE OF THE STUDY: Orchids in the genus Ophrys represent extraordinary cases of tight coevolution between plants and their pollinators, and as a result, they present opportunities for studying hybridization, or a lack thereof, during speciation. However, few studies assess the real effect of hybridization in diversification. The three most representative species of section Pseudophrys in the western Iberian Peninsula—O. dyris, O. fusca, and O. lutea—were chosen to study evolutionary relationships and examine speciation.

METHODS: Using eight specific nuclear microsatellite loci, 357 individuals from 28 locations were studied; 142 of these samples were also studied with four plastid microsatellite loci. Data were analyzed using Bayesian cluster analysis, a median-joint network, and multivariate analysis.

KEY RESULTS: Many O. dyris and O. fusca specimens had three or four alleles and were therefore treated as tetraploid. Ophrys dyris is poorly genetically separated from O. fusca, and pure populations are rare. Ophrys fusca and O. lutea are distinct, but hybrids/introgressed individuals were detected in most of the populations and supported by plastid haplotypes. Ophrys fusca is subdivided into three well-delimited genetic lineages with a strict geographic correspondence confirmed by plastid haplotypes.

CONCLUSIONS: Because postzygotic barriers are weak, leakage in this highly specialized orchid-pollinator system contributes to hybridization and introgression. These leakages may have occurred during periods of past climate change, promoting homogenization and the potential for generations of new biodiversity via production of novel genotypes/phenotypes interacting with pollinators.