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

## TL;DR

This paper shows how eDNA from songbird nests can reveal diet, parasites, and predators, offering new insights into biodiversity and ecological interactions.

## Contribution

A novel eDNA extraction protocol and LNA blocking oligonucleotide for improving detection of rare species in bird nests.

## Key findings

- A novel eDNA extraction protocol and LNA blocker significantly improved detection of non-sparrow species in nests.
- eDNA from 22 nests detected 126 species, primarily insects, including diet items, ectoparasites, and nest predators.
- The LNA blocker reduced sparrow reads by ~100% without affecting other passerines, increasing species richness by 31%.

## Abstract

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.

We present a novel method for extracting eDNA from whole bird nests and a LNA blocking oligonucleotide designed to reduce metabarcoding reads from nest building sparrows. Metabarcoding of 22 nests from a montane sagebrush steppe ecosystem detected 126 species, primarily insects, including putative diet items, ectoparasites, and confirmed nest predators.