Sex identification in embryos and adults of Darwin’s finches

Abstract Darwin’s finches, endemic to the Galapagos and Cocos islands, are an iconic example of adaptive radiation and evolution under natural selection. Comparative genetic studies using embryos of Darwin’s finches have shed light on the possible evolutionary processes underlying the speciation of this clade. Molecular identification of the sex of embryonic samples is important for such studies, where this information often cannot be inferred otherwise. We tested a fast and simple chicken embryo protocol for extraction of genomic DNA on Darwin’s finch embryos. In addition, we suggest modifications to two of the previously reported PCR primer sets for CHD1 , a gene used for sexing in adult passerine birds. The sex of all 29 tested embryos of six species of Darwin’s finches was determined successfully by PCR, using both primer sets. Hatchlings/nestlings and fledglings are also impossible to distinguish visually. This includes juveniles of sexually dimorphic species which are yet to moult in adult-like plumage and beak colouration. Furthermore, four species of Darwin’s finches are monomorphic, males and females looking alike. Therefore, sex assessment in the field can be a source of error, especially with respect to juveniles and mature monomorphic birds outside of the mating season. We caught 567 juveniles and adults belonging to six species of Darwin’s finches and only 44% had unambiguous sex-specific morphology. We sexed 363 birds by PCR, including individuals sexed based on marginal sex specific morphological traits (N=278) and birds which were impossible to classify in the field (N=39). For birds with marginal sex specific traits, PCR results revealed a 13% sexing error rate. This demonstrates that PCR based sexing can improve field studies on Darwin’s finches, especially when individuals with unclear sex-related morphology are involved. The protocols used here provide an easy and reliable way to sex Darwin’s finches throughout ontogeny, from embryos to adults.