Metabarcoding reveals seasonal shifts in the Allegheny woodrat's generalist diet

The Allegheny woodrat (Neotoma magister) population has been declining for over a century, with reduced food availability being a leading hypothesis. Allegheny woodrats consume nuts, fungi, and vegetation, but no study has used a molecular tool, such as metabarcoding, to describe diet more accurately. Furthermore, few studies address seasonal diet changes in Allegheny woodrats. To address this gap, we performed a year-long DNA metabarcoding study from 2022 to 2023 by collecting fresh fecal (n = 180) and latrine (n = 240) samples from 2 populations located in Pennsylvania and New Jersey, USA. We used chloroplast trnL and internal transcribed spacer 1 (ITS1) markers to identify plants and fungi in the woodrat diet. We amplified and sequenced samples, then identified them to species using OBItools software and databases. We identified 123 families, 173 genera, and 156 species of plants and fungi in the Allegheny woodrat diet from the ITS and trnL dataset containing 19,208,635 reads. The summer season had higher diversity and richness than winter and hard mast items were not detected year-round. Fungi and invasive plant species were consumed more frequently than anticipated in each season. Fresh fecal samples detected more dietary items than latrine fecal samples, including more rare items. Findings will inform conservation plans and natural habitat enhancement actions, such as what to provide in cultivated food plots and diets for captive-bred individuals.