Supplement- raw data and R scripts.zip from Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using <i>in situ</i> hybridization chain reaction (HCR). We then experimentally demonstrated a novel function for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a postulated Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

解析新物种中全新适应性性状的遗传基础，是生物学领域的核心科学问题之一。本研究以巴哈马圣萨尔瓦多岛特有的、发生适应辐射的营养特化鳉类为研究对象，揭示了galr2在脊椎动物颅面发育中的全新功能。我们通过原位杂交链反应（in situ hybridization chain reaction, HCR）证实，食鳞鳉的galr2基因上游存在一处推定的性别决定区Y（Sry）转录因子结合位点的缺失；同时发现不同鳉类物种的梅克尔软骨中，galr2的表达存在显著的空间差异。随后，我们通过向胚胎施加Galr2抑制剂，实验验证了Galr2在颅面发育中的全新功能。在两种营养特化种中，Galr2抑制均会缩短梅克尔软骨的长度并提高软骨细胞密度，但在泛食性遗传背景的个体中未观察到此现象。基于食鳞鳉中推定的Sry结合位点缺失导致galr2表达下调这一现象，我们提出了食鳞鳉颌骨延长的潜在调控机制。食鳞鳉的梅克尔软骨中Galr2受体数量更少，这可能通过限制发育过程中推定的Galr2激动剂与受体结合的机会，最终导致成体个体颌骨变长。本研究结果表明，将非模式生物系统中候选适应性单核苷酸多态性（SNPs）与高度分化的表型关联起来，以解析脊椎动物全新基因功能的研究方法，其应用价值正日益凸显。