Male Cope’s gray treefrogs (Hyla chrysoscelis) in amplexus have elevated and correlated steroid hormones compared to solitary males

•Male gray treefrogs were sampled in the field at natural breeding aggregations.•Males were either in amplexus with a female mate or solo and vocalizing.•Corticosterone, testosterone and estradiol were measured in plasma.•Amplexed males had higher concentrations of all three steroids.•Steroid concentrations were correlated in amplexed males only. Gonadal steroid hormones are typically elevated during the breeding season—a finding known as an associated reproductive pattern. Though less studied, there is also evidence, in both sexes, for elevated adrenal/interrenal steroids, including acute elevations on the day of mating. I investigated gonadal and interrenal steroids in wild male Cope’s gray treefrogs at breeding aggregations. I collected blood from males found in amplexus with female mates (amplexed males) and males sampled at the same time and location that were actively advertising vocally and without a mate (solo males). Concentrations of plasma corticosterone, testosterone, and 17β-estradiol (CORT, T and E2, respectively) were validated and measured. These two categories of males differed in three ways: (1) amplexed males exhibited significantly elevated concentrations of all three steroids compared to solo males (CORT: +347 %; T: +60 %; and E2: +43 %); (2) these hormone profiles alone accurately predicted male mating category with ca. 83 % accuracy using a discriminant function analysis; (3) amplexed males exhibited significant between-hormone correlations (T and E2 were positively correlated and CORT and E2 were negatively correlated) whereas no correlations were found in solo males; (4) amplexed males showed a negative correlation with CORT concentration and the time of night, whereas no such pattern was present in solo males. These findings suggest an acute and strong coactivation of the interrenal and gonadal axes that could drive phenotypic integration during this fitness-determining moment. I discuss these findings and suggest experiments to determine causation, including the role of motor behavior driving endocrine states and the role of female selection on endocrine profiles.