Abstract
BACKGROUND: Sexual dimorphism in skeletal muscle is a well-recognized biological phenomenon of adaptive evolution, yet its developmental genetic basis remains poorly understood. In many anurans, males develop hypertrophied forelimb muscles to facilitate amplexus (mating embrace) during breeding season, offering an ideal model to dissect the ontogeny of sex-specific muscle growth. The aim of this study was to determine the developmental onset of sexual dimorphism in the flexor carpi radialis (FCR) muscle and to characterize the transcriptional landscape driving this phenotypic divergence. METHODS: We first established the developmental timeline of sexual dimorphism in the FCRmuscle of Bufo gargarizans through morphological analysis. Based on the identified onset time, we performed comparative RNA sequencing at three critical post-metamorphic stages: pre-dimorphism (PD; 2 months post-metamorphosis), onset of dimorphism (OD; 4 months post-metamorphosis), and adult (AD; 24 months post-metamorphosis), to capture transcriptional dynamics associated with phenotype emergence. RESULTS: The results showed that males exhibited significantly greater FCR mass than females beginning at the OD stage and this difference persisted into adulthood. Transcriptomic profiling revealed a marked increase in sex‑biased differentially expressed genes (DEGs) at OD, coinciding with the emergence of morphological divergence. Time‑series analysis identified two major expression trajectories: 415 genes showed a sharp increase in male-biased expression from PD to OD, followed by a decline in AD, whereas 177 genes increased continuously from PD through AD. Integrated analysis highlighted eight candidate genes, comprising seven collagen isoforms (COL1A1, COL2A1, COL4A1, COL6A1, COL6A2, COL6A3, COL6A6) and an integrin β subunit (ITGB6), which showed coordinated male‑biased expression at OD stage. Protein-protein interaction network predicted ITGB6 as a critical hub linking extracellular matrix (ECM) remodeling to intracellular focal adhesion and FAK signaling pathways. CONCLUSIONS: These findings support a developmental model in which collagen-integrin-mediated ECM remodeling at a critical post-metamorphic stage, well before sexual maturation. This process establishes and stabilizes a sexually dimorphic muscle phenotype optimized for the mechanical demands of amplexus. HIGHLIGHTS: Male-biased hypertrophy of the flexor carpi radialis (FCR) muscle in male B. gargarizans emerges within a specific post-metamorphic pre-maturation window, accompanied by a surge in male-biased transcription. A sex-biased co-expressed gene set comprising seven collagen isoforms and the integrin subunit ITGB6 shows coordinated upregulation specifically at the onset of sexually dimorphic muscle development. Network analysis positions ITGB6 as a mechanotransduction hub that links extracellular collagen matrix remodeling (ECM) to intracellular focal adhesion and FAK signaling pathways, providing a molecular basis for sex-specific muscle growth. In many animals, males and females differ in their physical traits known as sexual dimorphism. In frogs and toads, for example, males often have stronger forelimb muscles that help them clasp females during mating. However, it was unclear when these muscle differences first appear during development and what genetic instructions make them grow differently. We investigated these questions in Asiatic toads (B. gargarizans). Our study clarifies that males begin to exhibit significant hypertrophy of the flexor carpi radialis muscle at four months after metamorphosis, well before sexual maturation. We found that this growth is accompanied by an increase in the activity of specific genes. We identified eight candidate genes involved in extracellular matrix remodeling-a process that reorganizes the muscle microenvironment and influences its architecture. Among these, a protein called ITGB6 acts as a molecular bridge, connecting the extracellular matrix to internal signaling pathways within muscle cells that help regulate muscle growth. Our study highlights that sex-biased transcriptional changes during early development coincide with lasting differences between males and females, providing insight into the potential biological basis of sexually dimorphic traits in vertebrates.