Song distinguishability predicts reproductive isolation between subspecies of the dark-eyed junco

The dark-eyed junco (Junco hyemalis) has experienced rapid phenotypic diversification within the last 18,000 years, resulting in several subspecies that reside in partially overlapping regions across North America. These subspecies have distinct plumage and morphology. If members of a subspecies disproportionately mate with one another, we would expect genetic differences to accumulate between the subspecies. In parallel, their learned songs could also accumulate changes. If song is used by individuals to recognize members of their own subspecies during mate selection, which would prevent the production of less fit hybrid offspring between subspecies, then song differences might co-localize with subspecies boundaries. Here, we quantify 10 song features to explore subspecies-level song variation using song recordings from community-science databases. We build a machine learning classifier to measure how accurately the subspecies songs can be distinguished from one another. Here, we show that songs of dark-eyed junco subspecies exhibit significant song-feature differences. However, these differences do not necessarily lead to distinguishability between subspecies. Notably, we find that subspecies pairs with adjacent ranges that do not hybridize have much more distinguishable songs, and also more evidence for genetic differentiation, than pairs that are known to hybridize. Thus, song distinguishability appears to have predictive power about which subspecies will hybridize, suggesting that song might play a role in reinforcing certain subspecies boundaries more than others. Finally, we analyze subspecies-level song differences alongside available genetic data and geographic coordinates to characterize the current evolutionary landscape of the dark-eyed junco subspecies complex. We observe geographic signal in the song and genetic data, indicating that individuals who share a range are more likely to share song characteristics and be genetically similar. This study illuminates the existence of subspecies-level song differences in the dark-eyed junco and provides further clarity on the role learned song plays in reinforcing reproductive boundaries between dark-eyed junco subspecies.