Large herbivores may affect ecosystem processes and states, but such effects can be difficult to quantify, especially within multispecies assemblages. To better understand such processes and improve our predictive ability of systems undergoing change, herbivore diets can be studied using controlled feeding trials (or cafeteria tests). With some wildlife, such as large herbivores, it is impractical to empirically verify these findings, because it requires visually observing animals in forested environments, which can disturb them from their natural behaviors. Yet, in field-based cafeteria trials it is nearly impossible to differentiate selection between herbivore species that forage on similar plants and make very similar bite marks. However, during browsing ungulates leave saliva residue which includes some buccal cells and DNA that can be extracted for species identification. Here we used a newly developed eDNA-based method (biteDNA) to test the browsing preferences of four sympatric ungulate species in the wild. Overall, food preferences varied between species, but all species strongly preferred deciduous over coniferous species. Our method allows the study of plant-animal interactions in multispecies assemblages at very fine detail.

Many processes in wild populations are difficult to study. Genetic data, often non-invasively collected, may provide a solution to these difficulties and are increasingly used to study behavioral, demographic, ecological, and evolutionary processes. Moreover, the improved sensitivity of genetic methods now allows analyses of trace amounts of DNA left by animals in their environment (e.g., saliva, urine, epithelial cells). Environmental DNA (eDNA) thus offers new opportunities to study a range of historic and contemporary questions. Here, we present a species and sex diagnostic kit for studying browsing in a multispecies temperate ungulate assemblage. Using mitochondrial sequences deposited in Genbank, we developed four single nucleotide polymorphisms (SNPs) for identifying four temperate ungulate species. We also sequenced portions of the Amelogenin gene on the X- and Y-chromosomes and developed six SNPs (three on the X-chromosome and three on the Y-chromosome) for sex determination. We tested the SNP assays on high and low quality/quantity DNA samples.