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.

引言：关于社会行为演化的核心问题之一是，当行为依赖于共享的神经、分子与激素机制时，如何产生广泛的行为多样性。对近缘类群进行比较研究，可为揭示共享机制中哪些组分最具演化可塑性提供关键洞见。
方法：我们针对两种非互斥假说展开区分，以此阐释保守神经机制如何演化以产生社会行为差异——即神经元数量或活性的改变。我们在两种新近分化的三棘刺鱼（Gasterosteus aculeatus）生态型中检验上述假说：普通生态型存在育幼行为，而白色生态型不具备育幼行为。我们采用以pS6（转录活性神经元标记物）为标记的双标记荧光免疫组织化学技术，量化两种影响脊椎动物育幼行为的视前区（preoptic area）神经肽能细胞类型的数量与活性：甘丙肽（galanin）与催产素（oxytocin）。
结果：两种生态型的视前区整体活性、甘丙肽与催产素神经元的数量均无显著差异，但二者的甘丙肽和催产素神经元活性存在显著区别。普通生态型中，这些神经元的活性随生殖阶段发生变化，而白色生态型则无此变化规律。当普通生态型的雄性个体开始育幼时，其神经元活性在交配后达到峰值，这提示从求偶过渡至育幼行为需要这些特定视前神经元的活性发生改变。
结论：综上，本研究表明，快速的行为演化是通过特定视前区神经肽能神经元的活性（而非数量）改变实现的。