In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation.

Functional validation of candidate genes involved in adaptation and speciation remains challenging. Here, we exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification, we quantified cell-typespecific gene expression in the histological context of growing feather follicles. Expression of Tyrosinase Related Protein 1 (TYRP1), Solute Carrier Family 45 member 2 (SLC45A2) and Hematopoietic Prostaglandin D Synthase (HPGDS) was melanocyte-limited and significantly reduced in follicles from hooded crow, explaining the substantially lower eumelanin content in grey versus black feathers. The central upstream Melanocyte Inducing Transcription Factor (MITF) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations.