Although animals display a rich variety of shapes and patterns, the genetic changes that explain how complex forms arise are still unclear. Here we take advantage of the extensive diversity of Heliconius butterflies to identify a gene that causes adaptive variation of black wing patterns within and between species. Linkage mapping in two species groups, gene-expression analysis in seven species, and pharmacological treatments all indicate that cis-regulatory evolution of the WntA ligand underpins discrete changes in color pattern features across the Heliconius genus. These results illustrate how the direct modulation of morphogen sources can generate a wide array of unique morphologies, thus providing a link between natural genetic variation, pattern formation, and adaptation.Mü llerian mimicry | Wnt pathway | Mendelian genetics | evolutionary-developmental biology

DNA 'barcoding' relies on a short fragment of mitochondrial DNA to infer identification of specimens. The method depends on genetic diversity being markedly lower within than between species. Closely related species are most likely to share genetic variation in communities where speciation rates are rapid and effective population sizes are large, such that coalescence times are long. We assessed the applicability of DNA barcoding (here the 5' half of the cytochrome c oxidase I) to a diverse community of butterflies from the upper Amazon, using a group with a well-established morphological taxonomy to serve as a reference. Only 77% of species could be accurately identified using the barcode data, a figure that dropped to 68% in species represented in the analyses by more than one geographical race and at least one congener. The use of additional mitochondrial sequence data hardly improved species identification, while a fragment of a nuclear gene resolved issues in some of the problematic species. We acknowledge the utility of barcodes when morphological characters are ambiguous or unknown, but we also recommend the addition of nuclear sequence data, and caution that species-level identification rates might be lower in the most diverse habitats of our planet.

Ecological speciation occurs when ecologically-based divergent selection causes the evolution of reproductive isolation. While there are many empirical examples of this process, there exists a poorly characterized stage during which the traits that distinguish species ecologically and reproductively segregate in a single population. Using a combination of genetic mapping, mate choice experiments, field observations, and population genetics, we studied a butterfly population with a mimetic wing color polymorphism and found that they exhibited partial color-based assortative mate preference. These traits represent the divergent, ecologically-based signal and preference components of sexual isolation that usually distinguish incipient and sibling species. The association between behavior and recognition trait in a single population may enhance the probability of speciation and provides an example for the missing link between an interbreeding population and isolated species.Research focused on a variety of biological systems has yielded compelling examples of ecological speciation (1, Table S1). Some of the specific traits that have diverged due to natural selection and cause reproductive isolation as a result include host choice in phytophagous insects such as Rhagoletis flies (2) and Timema walking-sticks (3), body size in stickleback fish (4), coloration in cichlid fish (5) and poison-dart frogs (6,7), and flowering time (8) and pollinator (9) in plants. While previous work on ecological speciation has been instrumental in characterizing the direct link between natural selection and speciation (10,11), it has largely focused on systems in which populations are highly differentiated (Table S1). However, the means by which interbreeding populations transition to assortative mating on the basis of a trait under divergent natural selection are generally unknown.Mimetic wing patterns in Heliconius butterflies provide a clear example of a trait involved in ecological speciation (12). Heliconius butterflies are chemically defended and warninglycolored. Their evolutionary history has been marked by widespread color pattern divergence among closely-related species and geographic subpopulations (13). This is combined with convergence among distantly-related species as a result of natural selection for Müllerian mimicry (13), or mimicry among mutually protected species. Furthermore, closely-related