DNA metabarcoding is a molecular technique frequently used to characterize diet composition of insectivorous birds. However, results are sensitive to methodological decisions made during sample processing, with primer selection being one of the most critical. The most frequently used DNA metabarcoding primer set for avian insectivores is ZBJ. However, recent studies have found that ZBJ produces significant biases in prey classification that likely influence our understanding of foraging ecology. A new primer set, ANML, has shown promise for characterizing insectivorous bat diets with fewer taxonomic biases than ZBJ, but ANML has not yet been used to study insectivorous birds. Here, we evaluate the ANML primer set for use in metabarcoding of avian insectivore diets through comparison with the more commonly used ZBJ primer set. Fecal samples were collected from both adult and nestling Purple Martins (Progne subis subis) at 2 sites in the USA and 1 site in Canada to maximize variation in diet composition and to determine if primer selection impacts our understanding of diet variation among sites. In total, we detected 71 arthropod prey species, 39 families, and 10 orders. Of these, 40 species were uniquely detected by ANML, whereas only 11 were uniquely detected by ZBJ. We were able to classify 54.8% of exact sequence variants from ANML libraries to species compared to 33.3% from ZBJ libraries. We found that ANML outperformed ZBJ for PCR efficacy, taxonomic coverage, and specificity of classification, but that using both primer sets together produced the most comprehensive characterizations of diet composition. Significant variation in both alpha- and beta-diversity between sites was found using each primer set separately and in combination. To our knowledge, this is the first published metabarcoding study using ANML primers to describe avian diet, and also the first to directly compare results returned by ANML and ZBJ primer sets.
DNA metabarcoding is a popular technique for characterizing diet composition by sequencing DNA of prey items found in fecal samples.
Molecular primer sets used to detect and identify prey DNA differ in efficacy, resulting in different conclusions about diet composition depending on the primer set selected.
We tested whether the recently developed ANML primer set may improve our ability to comprehensively describe diet composition for insectivorous Purple Martins as compared to the commonly used ZBJ primer set.
ANML has been shown to outperform ZBJ for characterizing diet in insectivorous bats, but ANML has not yet been tested against ZBJ for characterizing diets of insectivorous birds.
We found that the new ANML primer set detected and identified more insect prey than the ZBJ primer set, but that using both together provided the most comprehensive approach.
This study is the first to test ANML primers with bird diet samples and also to compare results between the use of ANML and ZBJ.