Decoding Dual Regulatory Layers: Integrating Differential Expression and Alternative Splicing Dynamics in the Evolution of Laryngeal Echolocation Across Chiropteran Lineages

Understanding the molecular mechanisms underlying phenotypic novelties is fundamental to deciphering the evolution of biodiversity. As a pivotal driver of phenotypic divergence, gene regulation operates through multiple layers, including transcriptional dynamics and post-transcriptional modifications. Laryngeal echolocation, an evolutionary breakthrough enabling bats to occupy specialized nocturnal niches, has been instrumental in their global adaptive radiation. Here, we leverage a comparative framework of two laryngeal echolocating (Rhinolophus sinicus and Myotis pilosus) and two non-laryngeal echolocating bats (Cynopterus sphinx and Rousettus leschenaultii) to dissect the contributions of differential expression (DE) and alternative splicing (AS) in shaping this sophisticated sensory system. Integrating short-read RNA sequencing with long-read isoform-resolution data from cochlear tissues, we systematically identified differentially expressed genes (DEGs) and alternatively spliced genes (ASGs). Our multi-method validation revealed distinct regulatory signatures: Upregulated DEGs in laryngeal echolocating bats showed significant enrichment for neural function (synapse organization and neuron development), while ASGs are predominantly associated with epigenetic regulation (protein methylation, histone modification, and chromosome organization). Notably, cross-comparative analyses demonstrated a higher-than-expected overlap between DEGs and ASGs, with two key regulators (SRRM4 and MAP1B) consistently identified across all four interspecies comparisons. These conserved candidates exhibited dual regulatory modalities, suggesting their pleiotropic roles in coordinating transcriptional and post-transcriptional programs. Intriguingly, we detected varying levels of selection pressure acting on DEGs and ASGs, implying different evolutionary constraints on these regulatory layers. Overall, our findings establish that both DE and AS contribute to the molecular architecture of laryngeal echolocation, though their interplay—whether synergistic or independent—requires further mechanistic interrogation.