Second-chance speciation in the Clouded Apollo butterfly.

Contact zones, where genetic lineages meet, provide a unique opportunity to study speciation and reveal how reproductive barriers emerge.1,2,3 However, species ranges are dynamic, contact zones may be ephemeral,4,5 and reinforcement-selection against hybrids-is an elusive phenomenon.6,7,8 We use an integrative approach-genomic, chemical, and morphological data coupled with ecological and demographic modeling-to evaluate lineage limits, reconstruct contact history, and infer reproductive isolation strength and mechanisms in the protected Clouded Apollo butterfly (Parnassius mnemosyne) in Europe.9 The status of this taxon is highly debated: a potential cryptic species (Parnassius turatii) edging P. mnemosyne sensu stricto in the Eastern Alps has been proposed, yet evidence remains inconsistent in the absence of genomic data.10,11,12,13,14,15,16,17,18 We reveal two genetic lineages that diverged about 0.8 million years ago and experienced two successive episodes of secondary contact. During the last glaciation, the lineages met in western Europe, which resulted in admixture and the formation of hybrid Pyrenean populations before reproductive isolation had evolved. In contrast, genomic data show no detectable gene flow at their present-day contact zone in the Eastern Alps, despite morphological and ecological similarity. Evidence of mutual chemical character displacement in males at this contact suggests bidirectional reinforcement in action. These findings portray a system in which lineages that once exchanged genes now re-encounter each other and reproductive isolation is generated-a second chance for speciation to succeed-and demonstrate that the Clouded Apollo comprises two cryptic species, with implications for EU legislation implementation and IUCN assessments.9,19,20.