Chemical signatures in the preen oil of pied flycatchers: testing reproducibility and exploring ontogeny

Preen oil – the secretion from the uropygial gland of birds – may have diverse functions in avian reproduction: protection against eggshell bacteria, olfactory crypsis against nest predators and olfactory mate choice. To investigate such functions, we should first characterise variation in preen oil composition, but also confirm that previously described patterns are robust. Replication studies are crucial to test the reproducibility of previous findings, but are rarely undertaken in chemical ecology. Here, we conducted an almost exact replication of a previous study on the chemical composition of preen oil in a wild passerine bird, the pied flycatcher Ficedula hypoleuca . We aimed to estimate the reproducibility of the previous results using larger sample sizes and following a pre‐registered analysis. In addition, we explored the ontogeny of preen oil composition by comparing nestling and adult preen oil. In line with previous findings, preen oil composition was similar between breeding partners and not repeatable within individual females across breeding stages. Female preen oil changed across breeding stages more clearly than in the original study (higher richness, diversity and volatility during incubation than nestling‐rearing), further refuting a role of preen oil in olfactory crypsis in this species. Unlike the original study, we found no difference in chemical profiles between sexes (nestling‐rearing), casting doubt on the proposed role of preen oil as a sex semiochemical in this species. Nestling preen oil differed from adults, was more similar to adult males than to adult females, but was not more similar to parents than to non‐parents. We found family chemical signatures, which, along with the breeding pair signature, suggests an influence of the nest environment on preen oil composition. Our study highlights the importance of replication and provides novel insights into the function and development of preen oil.