Data and code from: DNA metabarcoding reveals dietary divergence among sympatric swallows and flycatchers

Aerial insectivore (AI) populations have been in steep decline in North America since the 1970s, with swallows, swifts, and nightjars declining more rapidly than flycatchers. As AI share a common diet of flying insects, reductions in insect abundance are likely one of the major factors driving population decline. Previous studies have shown major dietary differences between swallows and flycatchers; flycatchers have exhibited more diverse, generalist diets than swallows. However, no study has directly compared the diets of sympatric swallows and flycatchers using the same method of dietary analysis. To investigate these differences, we compared the diets of six AI species living in sympatry during the breeding season. We collected fecal samples from adult Riparia riparia (Bank Swallow), Hirundo rustica (Barn Swallow), Petrochelidon pyrrhonota (Cliff Swallow), Tachycineta bicolor (Tree Swallow), Empidonax alnorum (Alder Flycatcher), and E. minimus (Least Flycatcher). We used DNA metabarcoding to identify the taxonomic composition of invertebrates in the feces and compared the richness of genera by insect order, insect family, and dipteran family between all species. Through a Bray-Curtis distance-based redundancy analysis, we identified significant differences in dietary composition between bird species at all three levels; however, the greatest amount of dissimilarity is seen in the dipterans consumed. E. alnorum, E. minimus, H. rustica, and T. bicolor had broader, more generalist diets than P. pyrrhonota and R. riparia. By comparing the diets between multiple species living in sympatry, our study improves our understanding of a possible cause of disproportionate population declines observed among AI species. 

Methods
We caught adult birds of Hirundo rustica (Barn Swallow), Riparia riparia (Bank Swallow), Petrochelidon pyrrhonota (Cliff Swallow), Tachycineta bicolor (Tree Swallow), Empidonax minimus (Least Flycatcher), and E. alnorum (Alder Flycatcher) during the 2022 breeding season in New Brunswick, Canada. Birds defecated in paper bags, banded (with morphology data such as age, sex, wing chord, etc. also taken), and released. 

Fecal samples were stored at -20 °C until packaged in 1.5ml microcentrifuge tubes and sent to the Canadian Centre for DNA Barcoding (CCDB) at the University of Guelph (Guelph, ON) for DNA metabarcoding analysis. DNA fragments extracted from each sample were amplified using arthropod-specific primers described by Zeal et al. (2011), which target a 157 base-pair section of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Two polymerase chain reaction (PCR) replicates were performed for each sample. The amplified DNA was sequenced alongside negative controls via next-generation sequencing and assigned identities by comparing reads to the Barcode of Life Data System (BOLD) reference library using the basal local alignment search tool (BLAST) algorithm. Only sequences with at least a 95 % match across 100 base pairs with the reference sequence and a minimum of 100 reads were considered confident detections. We only included detections with identification to genus or species in our analyses.

Statistical analyses included calculation of frequency of occurrence (FOO) and Bray-Curtis dissimilarity matrices at the level of invertebrate order, family, and dipteran family. We then used distance-based redundancy analyses to assess the effects of consumer species, Julian date of sampling, time from civil twlight, and breeding habitat type on the dietary dissimilarity matrices.