An evolutionary shift to prioritizing mating over care is associated with consistently high androgen levels in male threespine stickleback.

Steroid hormones play a crucial role in regulating social behaviors in vertebrates, but the mechanisms by which hormones mediate the evolution of these traits remain an open question. Here, we use liquid chromatography-mass spectrometry (LC-MS/MS) to quantify a panel of steroids in breeding males of two recently diverged populations of threespine stickleback. The common ecotype provides paternal care, whereas the white ecotype has evolutionarily lost care. Hormone levels were quantified in both ecotypes at three reproductive stages: (1) after completing a nest, (2) soon after mating, when commons begin providing care and whites remove their embryos from the nest, and (3) four days after mating, when commons are performing parental care and are no longer courting females while whites are not providing care and are courting females. Androgens declined in commons when males began providing care but remained elevated in whites across stages, possibly to maintain the production of "spiggin," the androgen-dependent glue males use to construct nests. Progestogen levels were consistently low in whites and were lowest in commons after mating, suggesting an antagonistic relationship between progestogens and sexual behavior. Both ecotypes showed elevated glucocorticoid levels after mating, suggesting that the stress axis may not have diverged between ecotypes. Altogether, these results provide evidence that the ecotypes regulate steroid levels differently, supporting the ways in which they balance mating and parental effort. Our data suggest a variety of mechanisms by which steroid signaling and regulation can change during the early stages of evolutionary divergence between behaviorally distinct populations.