Metabarcoding Close to Home: Songbird Nests as eDNA Aggregators for Trophic Ecology and Biodiversity Studies

Environmental DNA (eDNA) sampling to monitor wildlife presence has mostly focused on water but increasingly includes soil, air, and creative biotic substrates like flowers and spiderwebs. Biotic substrates are unique in that they also provide insight into ecological interactions. Here we explore the ability of eDNA from songbird nests to reveal avian trophic ecology, such as nestling diet and nest predator identity, in addition to local insect biodiversity. Twenty-two nests comprising five New World sparrow species and two nonsparrow passerines were collected in a montane sagebrush steppe ecosystem shortly after confirmed nest predation events. A novel protocol was used to extract eDNA from whole nests, and each nest was sequenced twice-with and without a blocking oligonucleotide. The blocker was designed with alternating locked nucleic acids to specifically inhibit sparrow amplification and improve detection of rare species. A total of 126 species were detected, and the blocker proved highly effective, reducing sparrow reads ~100% with no discernable coblocking of nonsparrow passerines. Species richness in sparrow nests increased by 31% with the blocker when using a minimum read threshold of 10 copies. Most detected species were insects, including likely prey items and ectoparasites of nestling birds. Predators were detected in 36% of nests. We discuss the merits of this rich and unique data source and considerations for future implementation.