Skin colour in salamanders is modulated by both epitranscriptomic methylation and gene expression

Animal colouration is a key trait in organismal biology, being involved in natural and sexual selection, competition, and communication. Amphibians use their highly diverse colouration in many ecological interactions, but the molecular bases of their colour variation are less well understood than in other vertebrate systems. While the genetic, structural, and cellular bases of pigmentation are increasingly understood in a range of models, potential epigenetic or epitranscriptomic effects are almost completely unexplored. The fire salamander (Salamandra salamandra) has striking colour patterns and polymorphisms, but the extremely large genome size of salamanders makes traditional genetic analyses infeasible. To discover loci and molecular mechanisms underlying colour differences in salamanders, we used long-read direct RNA sequencing to test the roles of RNA methylation, gene expression and their relationship on intra- and inter-individual colour variation in black, yellow and brown skinned salamanders. We found 129 differentially expressed and 281 differentially methylated genes across all pairwise comparisons. Many of the genes involved are related to pigmentation, with several directly associated with melanin production, such as Melan-A (MLANA), Premelanosome protein (PMEL), Tyrosinase (TYR) and Tyrosinase-related protein 1 (TYRP1). We found both a positive overall correlation and a significant overlap in transcripts that are differentially methylated and expressed. These findings suggest multiple molecular mechanisms, including gene expression and RNA methylation, contribute to amphibian colour diversity. RNA modifications as a promising area for understanding morphological variation in non-model animals and their impact on their ecology.