Genomic Analysis and Population Divergence Driven by Geographic Isolation in Neotetracus sinensis

Mountain systems are natural laboratories for evolution, where rugged topography and ecological heterogeneity restrict gene flow. In the Hengduan Mountains, Neotetracus sinensis (N. sinensis), the sole species of its genus, occupies cool montane forests and has limited dispersal. Here we assemble a high-quality reference genome and resequenced individuals from two isolated ranges in Yunnan, specifically Gaoligong Mountains (GLG) and Wuliang Mountains (WL), to test how geographic isolation shapes genomic variation. Genome-wide SNPs reveal clear population structure, with PCA and phylogeny concordantly resolving two distinct clades. Relatedness analysis shows no close-kin duplicates, and ROH are uniformly short, indicating outbred genomes. Demographic reconstructions show a shared trajectory marked by expansion ~1.0 Mya and a prolonged decline through the Middle-Late Pleistocene. Despite a lower long-term Nₑ, WL population displays contemporary nucleotide diversity (π), consistent with very recent gene flow or rapid post-bottleneck recovery. To robustly assess genomic differentiation and mitigate biases from the small WL sample size, we employed a strict two-tiered filtering approach. The intersection of the top 1% relative (F _st) and absolute (D _xy) divergence windows identified 5 core genomic islands, notably highlighting the DNA repair gene MGMT as a key candidate for environmental adaptation. Concurrently, functional enrichment of the broader top 1% F _st regions indicated an overrepresentation of pathways associated with synaptic and neuromodulatory signaling, cytoskeleton-linked intracellular transport, and circadian regulation. These results provide a genomic framework for understanding how geographic isolation and montane environments shape divergence in N. sinensis.