Using cytochrome b to identify nests and museum specimens of cryptic songbirds

Understanding demography of vertebrate populations requires quantifying reproductive success. For taxa that are difficult to distinguish, estimates of reproductive success can be biased if species are misidentified or if breeding concludes before identification is confirmed. We surveyed desert grasslands where three species of cryptic sparrows breed: Peucaea botterii, P. cassinii, and Aimophila ruficeps (Emberizidae). Nests, eggs, and nestlings of these species are similar, making it difficult to differentiate nests without observing adults, which can be challenging. We collected seven types of material from nests for DNA analysis: maternal cells from exterior surfaces of unhatched eggs, epithelial cells from the oropharyngeal cavity of nestlings, eggshells, feathers, feather sheaths, feces, and fecal sacs. From these materials, we amplified and sequenced a fragment of the diagnostic locus, mitochondrial cytochrome b (cyt b), and analyzed the data in a phylogenetic framework to classify nests to species. We validated our classification by sequencing the same locus from feathers of museum specimens. Overall, 72% of samples from nests yielded high-quality sequences. We identified to species 44 of 51 nests and identified museum specimens with archival ages of up to 47 years. Our study extends previous research by demonstrating the efficacy of standard kits, inexpensive reagents, low DNA concentrations, and diverse materials in classifying nests of grassland sparrows. Compared to more invasive methods of acquiring DNA, the approaches we describe are less likely to affect nesting behavior and bias estimates of nesting success of birds, issues especially important for species of conservation concern.