Transcriptome analysis of anuran breeding glands reveals a surprisingly high expression and diversity of NNMT-like genes

In many amphibians, males have sexually dimorphic breeding glands, which can produce proteinaceous or volatile pheromones, used for intraspecific communication. In this study we analyse two types of glands in the Mexican treefrog Ptychohyla macrotympanum (Hylidae)-large ventrolateral glands and small nuptial pads on their fingers-using histology, whole-transcriptome sequencing, and phylogenetic analyses. We found strong differences in glandular tissue composition and gene expression patterns between the two breeding gland types. In both glands we only found low expression of protein pheromone candidates. Instead, in the ventrolateral glands, gene expression was strikingly dominated by nicotinamide N-methyltransferase (NNMT)-like genes. The diversity of these genes was remarkably high, with at least 68 distinct NNMT-like genes. Our comparative phylogenetic analysis of the diversity of NNMT-like genes across vertebrates indicates that the extreme diversity of this gene is largely a frog-specific phenomenon and can be traced to large numbers of relatively recent gene duplications occurring independently in many lineages. The strong dominance and astonishing diversity of NNMT-like genes found in anurans in general, and in their sexually dimorphic breeding glands specifically, suggests an important function of NNMT-like proteins for anuran reproduction, possibly being related to volatile pheromone production.