Interactions between diet and gut microbiota are associated with metabolic adaptations during autumn migration preparation in sympatric migratory waterbirds

To investigate food resource utilization and gut microbiota adaptation in sympatric migratory birds during autumn migration preparation at high altitudes (> 4,500 m), we studied black-necked cranes, ruddy shelducks, and bar-headed geese in the Yellow River Source Area of Sanjiangyuan National Park. Fecal DNA metabarcoding, 16 S rRNA sequencing, Spearman correlation, and PICRUSt2-based prediction of MetaCyc/KEGG pathways were used to characterize dietary composition, gut microbial community structure, and predicted microbial functional potential. Dietary composition differed among species: black-necked cranes specialized on the tipulid Tipula subcunctans and the plant Argentina anserina ; ruddy shelducks relied primarily on Stuckenia pectinata and diverse animal foods; and bar-headed geese exhibited intermediate dietary diversity, with Puccinellia distans and Stuckenia pectinata as the main plant foods, together with a variety of animal foods. Gut microbiota was dominated by Firmicutes, with Ligilactobacillus as the most abundant genus in all three species; its high abundance in black-necked cranes was consistent with their starch-rich diet. Significant diet-microbiota correlations were detected in black-necked cranes and ruddy shelducks but not in bar-headed geese. Predicted functional profiles differed among species: black-necked cranes showed enrichment in both degradation and biosynthesis pathways, ruddy shelducks predominantly in biosynthesis pathways, and bar-headed geese in pathways related to energy metabolism, electron transport, and cofactor biosynthesis. These functional profiles represent computationally predicted microbial functional potential rather than directly measured metabolic activities. These findings reveal interspecific differences in dietary composition, gut microbial community structure, and predicted functional potential during autumn migration preparation, and provide exploratory insights into potential microbial contributions to migratory energetics. Validation through metagenomics, metabolomics, and host physiological measurements is needed.