Asynchronous life cycles contribute to reproductive isolation between two Alpine butterflies

Geographic isolation often leads to the emergence of distinct genetic lineages that are at least partially reproductively isolated. Zones of secondary contact between such lineages are natural experiments that allow investigation of how reproductive isolation evolves and co-existence is maintained. While temporal isolation through allochrony has been suggested to promote reproductive isolation in sympatry, its potential for isolation upon secondary contact is far less understood. Sampling two contact zones of a pair of mainly allopatric Alpine butterflies over several years and taking advantage of museum samples, we show that the contact zones have remained geographically stable over several decades. Furthermore, they seem to be maintained by the asynchronous life cycles of the two butterflies, with one reaching adulthood primarily in even and the other primarily in odd years. Genomic inferences document that allochrony is leaky and that gene flow from allopatric sites scales with the degree of geographic isolation. Overall, we show that allochrony has the potential to contribute to the maintenance of secondary contact zones of lineages that diverged in allopatry. Many species have persisted in warmer geographic refugia during the last glaciation period. Since then, they have recolonized formerly glaciated areas, often coming into contact with close relatives from other refugia. Coexistence between these lineages is possible if there are enough isolating barriers that prevent gene flow between them. However, closely related taxa often lack strong barriers and form contact zones instead, where they hybridize to some degree. The factors that maintain such contact zones over time differ among species and are often unknown. Here, we studied two contact zones between a pair of closely related Alpine butterfly lineages. Over the 4 years that we sampled, we found that one lineage occurred predominantly in even and the other one predominantly in odd years, suggesting that their biennial life cycle enables them to spatially coexist in contact zones, with limited interbreeding. Using museum specimens, we confirmed that temporal isolation has been at play for at least several decades, and based on observation record data, we showed that biannual life cycles broadly differed between the two lineages across Switzerland. However, temporal isolation is leaky and hybridization occurs, especially when the contact zone is more connected to other populations. Taken together, our study highlights how asynchronous biennial life cycles, which are common in Alpine butterfly species, have the potential to contribute as a barrier to gene flow and may promote coexistence.