Laryngeal Transcriptomic Insights into Echolocation Call Frequency Divergence in Closely Related Rhinolophus Species

Acoustic divergence is widely recognized as a key driver of speciation and niche differentiation in vocal animals. In echolocating horseshoe bats (Rhinolophus), the larynx is specialized for producing high-duty-cycle signals used in foraging, navigation, and species recognition. While the ecological role of echolocation is established, the molecular mechanisms regulating laryngeal frequency remain unclear. We compared the laryngeal transcriptomes of three closely related, sympatric Rhinolophus species with distinct resting frequencies (RFs): R. episcopus (~46 kHz), R. siamensis (~66 kHz), and R. osgoodi (~85 kHz). This comparison identified 511 differentially expressed genes. High-frequency species upregulated genes involved in cytoskeletal dynamics and muscle contraction, such as cell adhesion molecules and motor proteins, while low-frequency species upregulated genes related to cellular homeostasis and metabolic maintenance. Weighted gene co-expression network analysis revealed two RF-correlated modules: a high-frequency module enriched in aerobic respiration and carbon metabolism and a low-frequency module enriched in lipid metabolism. Protein-protein interaction analysis identified ACTC1, vital for muscle contraction, as a hub gene. Evolutionary analysis showed that ACTC1 is highly conserved, with no significant positive selection, indicating that transcriptional regulation, rather than coding-sequence divergence, is the primary driver of the observed functional differences. These findings suggest that RF variation likely results from transcriptional remodeling in laryngeal superfast muscles. This study provides the first transcriptomic evidence linking laryngeal gene expression with acoustic divergence and offers new insights into the genetic basis of bat echolocation.