Behavioral smearing and physiological secretions drive divergent microbiome assembly during breeding in the crested...

Host-microbiota interactions represent a key axis in animal adaptation, especially in species displaying pronounced seasonal variation in behavior and physiology. In avian species, behavioral processes associated with reproduction may influence symbiotic microbial communities, yet the underlying mechanisms remain poorly resolved. The endangered crested ibis ( Nipponia nippon) exhibits a distinctive seasonal transition in plumage coloration, shifting from white in the non-breeding period to gray during breeding, a change linked to smearing behavior and deposition of black secretions from the neck region. In the present study, 16S rRNA sequencing across three body sites was performed to profile body surface microbiomes during breeding (gray-feather) and non-breeding (white-feather) stages. Breeding individuals exhibited lower microbial diversity, consistent with an influence of black neck secretions on microbiome structure. Microbial communities were differentiated more strongly by season than by body site, and microbial similarity among body sites increased during breeding, supporting redistribution of microbes through smearing behavior. Community assembly also showed clear season- and site-specific variation. Neck feathers exhibited a 36.5% better fit to the neutral model, indicating a stronger contribution of stochastic assembly, likely associated with microbial dispersal during smearing of black secretions. In contrast, neck skin showed a 36.3% lower neutrality and 11.87% more host-selected variants, indicating stronger deterministic selection associated with breeding-related secretions. These findings support a dual regulatory framework during breeding, in which behavioral smearing promotes microbial dispersal while physiological secretion strengthens host filtering. Such coordinated regulation likely drives seasonal microbiome variation and contributes to seasonal adaptation. Overall, this work provides novel insight into the integration of behavior and physiology in shaping host-microbiota interactions during critical life stages and establishes a microbiome-based perspective for crested ibis conservation.