Multi-omics analysis reveals the evolution, function, and regulatory mechanisms of SPF pheromones in Anurans

Pheromones play a pivotal role in chemical communication across various taxa, with protein-based pheromones being particularly significant in amphibian courtship and reproduction. In this study, we investigate the Emei music frog (Nidirana daunchina), which utilizes both acoustic and chemical signals for communication. Base on a de novo assembled genome of a male Emei music frog, we identify substantial expansion in four pheromone-related gene families associated with chemical communication. Notably, six members of the two-domain three-finger protein (2D-TFP) family, belonging to the sodefrin precursor-like factor (SPF) pheromone system, exhibited high and specific expression in the male post-axillary glands during the breeding season. Structural and evolutionary analyses confirm the presence of the SPF system across amphibians, classifiable into four distinct classes (two within urodeles and two within anurans). We propose a complete regulatory network governing SPF secretion via the hypothalamic-pituitary-testicular-breeding gland axis, and suggest testosterone synthesis as the pivotal pathway. Behavioral experiments further reveal a previously unknown female-attractant role of SPF in anurans. Overall, these findings not only highlight the underestimated diversity and function of pheromones in anurans, but also provide important insights into the evolution of protein-based pheromones in vertebrates.