Elevational Adaptation in the Moth Thitarodes xiaojinensis: HSP60A-Mediated Thermal Tolerance Evolution Under Gene Flow

Climate warming intensifies thermal selection on high-elevation species, yet the molecular mechanisms enabling rapid adaptation under ongoing gene flow remain poorly understood. We examined differentiation between High-elevation (High) and Low-elevation (Low) ecotype samples of Thitarodes xiaojinensis, an endemic ghost moth of the Qinghai-Tibet Plateau, using SLAF-seq, RNA-seq profiling and survival experiments. Phylogenetic analyses confirm the two ecotype samples are conspecific, and they differ in forewing phenotype. Population structure at K = 2 uncovers elevation-correlated clustering despite weak genome-wide structure (optimal K = 1). Genome-wide F _ST scans (Weir & Cockerham) identify HSP60A as a prominent outlier locus (F _ST = 0.391, top 1.4% of genome-wide windows). Targeted PCR amplification and sequencing confirmed a nonsense mutation (Glu378*) in the HSP60A coding sequence. This derived allele was carried by 80% (4/5) of Low individuals and by none of 5 High individuals (0 of 10 chromosome copies, one-sided 95% upper bound on allele frequency ≈ 26%), suggesting that it is associated with incipient ecological divergence between the two ecotypes. Co-expression network analysis grouped the chaperone genes into two systems with divergent thermal responses. The small HSPs were induced 2.3-fold from 8°C to 16°C, whereas the classical chaperones were not induced, and HSP60A was consistently placed within the latter (median biweight midcorrelation +0.56 with classical chaperones versus -0.27 with small HSPs, p = 3.5 × 10^-9). Across the three temperatures the Low ecotype expressed HSP60A at lower levels than the High ecotype, significant under log₂(TPM + 1) (p = 0.020) but not under the other two scales, and survived better at 16°C (p = 0.0342). These results link a coding variant at HSP60A, its lower transcript level in the Low ecotype and the better survival of that ecotype at 16°C, and identify HSP60A as a candidate locus for ecotype-informed conservation of the Ophiocordyceps sinensis host insect.