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

Abstract 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 species 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. Diversity of these genes was remarkably high, with at least 68 distinct NNMT-like genes. Our phylogenetic comparative 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.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 species 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. Diversity of these genes was remarkably high, with at least 68 distinct NNMT-like genes. Our phylogenetic comparative 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. Supplementary datasets accompanying the paper: - final RNAseq assemblies of the ventrolateral glands and the nuptial pads of Ptychohyla macrotympanum - fasta-file of all Ptychohyla-NNMT-like genes found in this study

摘要 在诸多两栖动物中，雄性个体拥有性二态繁殖腺（sexually dimorphic breeding glands），此类腺体可产生蛋白质类或挥发性信息素，用于种内通讯。本研究针对墨西哥雨蛙科（Hylidae）褶蛙属物种大鼓膜褶蛙（Ptychohyla macrotympanum）的两类腺体——大型腹外侧腺以及指部小型婚垫——采用组织学、全转录组测序及系统发育分析开展研究。结果显示，两类繁殖腺的腺体组织组成与基因表达模式存在显著差异。两类腺体中蛋白质类信息素候选物的表达量均较低。与之相反，腹外侧腺的基因表达显著以烟酰胺N-甲基转移酶（nicotinamide N-methyltransferase, NNMT）样基因为主导。此类基因的多样性极高，至少存在68种不同的NNMT样基因。本研究针对脊椎动物NNMT样基因多样性开展的系统发育比较分析表明，该基因的极端多样性主要为蛙类特有现象，其起源可追溯至多个支系中独立发生的大量较新基因重复事件。总体而言，无尾目动物（anurans），尤其是其性二态繁殖腺中所发现的NNMT样基因的强主导性与惊人多样性，暗示NNMT样蛋白在无尾目动物繁殖中发挥重要功能，可能与挥发性信息素的产生相关。本文配套补充数据集：
- 大鼓膜褶蛙腹外侧腺与指部婚垫的最终RNAseq组装结果
- 本研究中发现的所有褶蛙属NNMT样基因的FASTA格式文件