DNA metabarcoding reveals rangewide variation in aquatic diet of a riparian avian insectivore, the Prothonotary warbler

Riparian avian insectivores not only depend on terrestrial insect prey but also benefit from the inclusion of aquatic prey during critical life history periods. Diets identified herein show that Prothonotary Warbler (Protonotaria citrea) nestlings were provisioned with aquatic prey throughout the breeding season across their range, but with variation in prey frequency of occurrence and taxonomy. Anthropogenic activity and climate change may impact the trophic link especially between aquatic and riparian habitats by altering the presence, abundance, and timing of prey availability. Thus, we used DNA metabarcoding of fecal samples to quantify the frequency of occurrence of nestling diet items at nine sites across their breeding range that differed in expected aquatic prey consumption. We analyzed spatial and temporal differences in the occurrence and multivariate diet assemblages of each prey source. Lepidoptera was the predominant terrestrial prey occurring in diets across space and time..., During the 2018 and 2019 breeding seasons, we sampled 9 Prothonotary Warbler populations at sites monitored annually throughout their breeding range. Local nest monitors collected fecal sacs from nestlings aged 6-11 days during routine nest visits. Nestlings readily defecate when handled during banding, enabling collection of fresh fecal samples. We stored fecal sacs in glass vials with 96% ethanol at room temperature to preserve DNA until extraction. We extracted DNA from samples using the Qiagen Fast DNA Mini Stool kit, modifying the manufacturer’s instructions to increase yield of degraded DNA (Trevelline et al. 2016). A two-stage targeted amplicon sequencing protocol was used to amplify prey DNA from our DNA extractions (Naqib et al. 2018). The first PCR stage amplified sequences with the primers LCO1490 and CO1-CFMRa (hereafter, ANML; Jusino et al. 2019). The second PCR stage added unique 10-bp barcodes to each sample, allowing samples to be pooled during sequencing while retaining..., Most data processing and analyses were performed in R (4.0.3; R Core Team 2020). R scripts are available from the corresponding author upon reasonable request.
Biopython was used to perform the NCBI BLAST and sequences identification consensus. Python scripts are available from the corresponding author upon reasonable request.