Field observations can accurately detect interspecific brood parasitism in Aythya valisineria (Canvasback) nests

Abstract Validating the accuracy of protocols and field observations is important to maintain consistent data collection and inference amidst changing observers or study sites. To aid inferences on interspecific brood parasitism, we validated field observations of egg identification to the species level against mitochondrial DNA sequencing for nests where interspecific brood parasitism was suspected. Field observations of parasitic Aythya americana (redhead), and host Aythya valisineria (canvasback) eggs proved highly accurate (Aame_ID = 0.99 ± 0.05; Aval_ID = 0.99 ± 0.04) even among observers with different levels of experience (Obs1yr = 0.99 ± 0.03; Obs3yr = 0.97 ± 0.02). Hatched eggshell membranes contained the best tissue samples for successful sequencing compared to eggshell membranes earlier in development (Prseq_succ = 0.84 ± 0.08 for 0–9 days incubation, 0.63 ± 0.12 for 10–24 days incubation, and 0.88 ± 0.04 for hatched membranes), while DNA concentration following extraction was not an important predictor of sequencing success. Despite obtaining sufficient DNA concentrations, we observed 15% failure rate among samples, with 69% and 31% of the 29 failed samples being due to either a lack of quality DNA or non-target sequencing, respectively, suggesting failure was likely due to bacterial growth from exposure after eggs were abandoned or preyed upon. Our investigation showcases the utility of non-invasive molecular techniques to validate field protocols and rejects egg identification as a major source of uncertainty when trying to account for interspecific brood parasitism in parasitized A. valisineria nests.