1. Smithsonian Tropical Research Institute, Apartado postal 0843-03092, Panamá, República de Panamá
2. Instituto de Genética, Universidad de los Andes, Carrera 1E No 18ª–10, PO Box 4976, Santafé de Bogotá D.C., Colombia
3. Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, UK
4. *These authors contributed equally to this work

Correspondence to: Jesús Mavárez^1,4Mauricio Linares² Correspondence and requests for materials should be addressed to M.L. (Email: mlinares@uniandes.edu.co) or J.M. (Email: mavarezj@si.edu). The sequences have been deposited in GenBank under accession numbers DQ445384–DQ445414 (Distal-less) and DQ445416–DQ445457 (Invected).

Speciation is generally regarded to result from the splitting of a single lineage. An alternative is hybrid speciation, considered to be extremely rare, in which two distinct lineages contribute genes to a daughter species. Here we show that a hybrid trait in an animal species can directly cause reproductive isolation. The butterfly species Heliconius heurippa is known to have an intermediate morphology and a hybrid genome¹, and we have recreated its intermediate wing colour and pattern through laboratory crosses between H. melpomene, H. cydno and their F₁ hybrids. We then used mate preference experiments to show that the phenotype of H. heurippa reproductively isolates it from both parental species. There is strong assortative mating between all three species, and in H. heurippa the wing pattern and colour elements derived from H. melpomene and H. cydno are both critical for mate recognition by males.

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