Pangenome and transcriptomics reveal the genomic basis associated with interspecific variation of sexually dimorphic nuptial spines in moustache toads.

Sexual dimorphism is a widespread phenotypic innovation that plays a crucial role in species diversification, yet its genetic origins and evolutionary trajectories remain poorly understood in vertebrates. Leptobrachium provides a tractable system in which male moustache toads develop maxillary nuptial spines used in male-male combat, whereas closely related pseudo-moustache toads lack this trait. To investigate the evolutionary genomics of these sexually dimorphic nuptial spines, we construct a pangenome from chromosome-level assemblies of six Leptobrachium species and integrate trans-species transcriptomic analyses. We find that keratin gene expansion predated the origin of nuptial spines, potentially providing genetic material for keratinized skin innovation. Recently duplicated hair keratin genes acquired putative Hoxc13 binding sites, and promoter reporter assays confirm that Hoxc13 can activate these genes in both moustache toads and pseudo-moustache toads. Differential hoxc13 expression may therefore be more important for affecting whether duplicated hair keratin genes are recruited to develop nuptial spines. Transcriptomic comparisons further reveal maxillary skin-specific expression of genes involved in keratinocyte proliferation and differentiation, as well as melanogenesis during the formation of nuptial spines. In Leptobrachium tengchongense, which has undergone secondary loss of nuptial spines, igfbp2, a conserved gene highly expressed in keratinized skin appendages across amphibians, shows positive selection and represents a candidate associated with this evolutionary transition. Our study provides amphibian pangenome resources and highlights how gene duplication coupled with regulatory recruitment may shape the evolution of novel sexually dimorphic traits in vertebrates.