Supplementary Material for: An evolutionary loss of parental care in stickleback is associated with differences in the activity, but not the number, of neuropeptidergic neurons in the preoptic area

Introduction: A central question about the evolution of social behavior is how extensive diversity can arise when behaviors depend on shared neural, molecular, and hormonal mechanisms. Comparing close relatives can offer insights into which components of shared mechanisms are most evolvable. Methods: We discriminate between two nonexclusive hypotheses by which conserved neural mechanisms might evolve to generate differences in social behavior: changes in the number or activity of neurons. We test these hypotheses in two recently diverged ecotypes of threespine stickleback (Gasterosteus aculeatus); the common ecotype provides parental care, while the white ecotype does not. We used double-label fluorescent immunohistochemistry with pS6, a marker of transcriptionally active neurons, to quantify the number and activity of two preoptic neuropeptidergic cell types that affect parental care across vertebrates: galanin and oxytocin. Results: Ecotypes did not differ in the overall activity of the preoptic area or the number of galanin and oxytocin neurons but did differ in the activity of galanin and oxytocin neurons. The activity of these neurons changed across reproductive stages in the common but not the white ecotype. Activity peaked after mating in commons when males began to care for their offspring, suggesting that changes in the activity of these specific preoptic neurons are required to transition from courtship to parenting. Conclusion: Overall, our study suggests that rapid behavioral evolution occurred via changes in the activity but not the number of specific preoptic neuropeptidergic neurons.