Diet and Prey Selection of Migratory Shorebirds on Virginia Mudflats Revealed by Fecal DNA Metabarcoding

Intertidal mudflats are globally important foraging habitats, yet their role in supporting migratory shorebirds at mid‐Atlantic staging sites remains poorly understood. We compared shorebird habitat use across mudflat and barrier island sand and peat substrates in the Virginia barrier island and seaside lagoon system during spring migration (14 May–2 June 2023–2024) and used fecal DNA metabarcoding with concurrent prey availability data to evaluate diet composition and prey selection of Calidris alpina (dunlin; n = 21), Calidris canutus rufa (red knot; n = 18), and Calidris pusilla (semipalmated sandpiper; n = 21) on mudflats. Shorebird abundance, species richness, and invertebrate density were highest on peat banks while invertebrate community composition differed among substrates. Focal species exhibited distinct habitat associations with C. alpina most abundant on peat and mudflats, C. c. rufa on sand and peat, and C. pusilla on mudflats. Diets also differed, with C. alpina and C. pusilla consuming mixed invertebrate assemblages comprised mainly of crustaceans and worms while C. c. rufa fed mainly on bivalves. Biofilm, a diatom‐rich microbial assemblage, was detected in all species, revealing a previously undocumented trophic pathway at mid‐Atlantic staging sites. Selectivity analyses indicated bivalve selection by C. alpina and C. c. rufa , while C. pusilla showed no evidence of prey selection, suggesting species‐specific foraging strategies. These findings underscore the importance of preserving habitat heterogeneity and connectivity in coastal staging sites to support diverse shorebird assemblages under ongoing environmental change.