Lineage-specific expansion and positive selection of NPC2 genes in the crab spider Ebrechtella tricuspidata and other arachnids

Niemann-Pick type C2 (NPC2) proteins have been proposed as soluble carriers for semiochemicals in arthropods, serving as putative functional substitutes for the canonical odorant-binding proteins (OBPs) largely absent in arachnids. However, the evolutionary mechanisms driving NPC2 diversification in spiders—particularly in ambush predators relying heavily on chemical cues—remain unclear. We identified a minimum expressed repertoire of 13 high-confidence NPC2 genes in the crab spider Ebrechtella tricuspidata via comprehensive transcriptomic profiling. Comparative phylogenomics across 21 arthropod species revealed that while the generalist herbivore mite Tetranychus urticae underwent a significant repertoire expansion (+ 9 genes, P < 0.001), the E. tricuspidata lineage exhibited a modest numerical gain (+ 3 genes). Despite this, transcriptomic data revealed a striking functional dichotomy within the spider repertoire. The basal singleton EtriNPC2-2 was highly enriched in the abdomen, suggesting a conserved role in visceral lipid transport. In contrast, the derived Etri-B subclade (EtriNPC2-1, -3) and the singleton EtriNPC2-4 were predominantly expressed in chemosensory appendages (palps and legs). This spatial expression shift coincided with strong signatures of episodic positive selection acting on the Etri-B lineage. Structural modeling identified a key adaptive substitution (L33→Q31) at the pocket entrance of EtriNPC2-3, serving as a prime candidate for future functional validation regarding ligand access in the sensillar lymph. Our findings suggest a clear pattern of neofunctionalization, where gene duplication enabled spider NPC2s to diverge from an ancestral visceral function toward a specialized sensory role. This linkage between spatial expression shifts and adaptive sequence evolution provides essential molecular evidence for how ambush spiders evolved a complex chemosensory system in the absence of insect-type OBPs.