Riparian habitats are characterized by substantial flows of emergent aquatic insects that cross the stream-forest interface and provide an important source of prey for insectivorous birds. The increased availability of prey arising from aquatic subsidies attracts high densities of Neotropical migratory songbirds that are thought to exploit emergent aquatic insects as a nestling food resource; however, the prey preferences and diets of birds in these communities are only broadly understood. In this study, we utilized DNA metabarcoding to investigate the extent to which three syntopic species of migratory songbirds-Acadian Flycatcher, Louisiana Waterthrush, and Wood Thrush-breeding in Appalachian riparian habitats (Pennsylvania, USA) exploit and partition aquatic prey subsidies as a nestling food resource. Despite substantial differences in adult foraging strategies, nearly every nestling in this study consumed aquatic taxa, suggesting that aquatic subsidies are an important prey resource for Neotropical migrants nesting in riparian habitats. While our results revealed significant interspecific dietary niche divergence, the diets of Acadian Flycatcher and Wood Thrush nestlings were strikingly similar and exhibited significantly more overlap than expected. These results suggest that the dietary niches of Neotropical migrants with divergent foraging strategies may converge due to the opportunistic provisioning of non-limiting prey resources in riparian habitats. In addition to providing the first application of DNA metabarcoding to investigate diet in a community of Neotropical migrants, this study emphasizes the importance of aquatic subsidies in supporting breeding songbirds and improves our understanding of how anthropogenic disturbances to riparian habitats may negatively impact long-term avian conservation.

Identifying the composition of avian diets is a critical step in characterizing the roles of birds within ecosystems. However, because birds are a diverse taxonomic group with equally diverse dietary habits, gaining an accurate and thorough understanding of avian diet can be difficult. In addition to overcoming the inherent difficulties of studying birds, the field is advancing rapidly, and researchers are challenged with a myriad of methods to study avian diet, a task that has only become more difficult with the introduction of laboratory techniques to dietary studies. Because methodology drives inference, it is important that researchers are aware of the capabilities and limitations of each method to ensure the results of their study are interpreted correctly. However, few reviews exist which detail each of the traditional and laboratory techniques used in dietary studies, with even fewer framing these methods through a bird-specific lens. Here, we discuss the strengths and limitations of morphological prey identification, DNA-based techniques, stable isotope analysis, and the tracing of dietary biomolecules throughout food webs. We identify areas of improvement for each method, provide instances in which the combination of techniques can yield the most comprehensive findings, introduce potential avenues for combining results from each technique within a unified framework, and present recommendations for the future focus of avian dietary research.

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.

Streams and their surrounding riparian habitats are linked by reciprocal exchanges of insect prey essential to both aquatic and terrestrial consumers. Aquatic insects comprise a large proportion of total prey in riparian habitats and are opportunistically exploited by terrestrial insectivores; however, several species of songbirds are known to preferentially target aquatic prey via specialized foraging strategies. For these songbirds, reduced availability of aquatic insects via stream acidification may result in compensatory changes in provisioning during the nesting period, thereby influencing both adult and nestling diet composition. In this study, we used DNA metabarcoding to test the hypothesis that an obligate riparian Neotropical migratory songbird, the Louisiana Waterthrush (Parkesia motacilla), expands its diet to compensate for the loss of preferred aquatic prey taxa (primarily pollution-sensitive Ephemeroptera, Plecoptera, and Trichoptera) as a result of stream acidification. Our results revealed that both adult and nestling waterthrush exhibited an increase in dietary richness and niche breadth resulting from the consumption of terrestrial prey taxa in acidified riparian habitats. In contrast, compensatory dietary shifts were not observed in syntopic Neotropical migrant species known to primarily provision terrestrial prey taxa. In addition to providing support for our hypothesis that waterthrush compensate for stream acidification and aquatic prey limitations by expanding their diet, our findings highlight the vulnerability of Louisiana Waterthrush to anthropogenic disturbances that compromise stream quality or reduce the availability of pollution-sensitive aquatic insects.

Providing taxonomically precise dietary characterisations for freshwater fish species is critical for gaining a deeper understanding of the trophic dynamics present in freshwater ecosystems. However, our current understanding of freshwater trophic ecology has relied almost entirely upon direct observation of foraging attempts or morphological identification of partially digested prey. Due to the limitations of morphological dietary characterisations of soft‐bodied arthropod prey, these techniques offer dietary descriptions that can lack satisfactory taxonomic resolution and may bias our interpretations of freshwater food webs. Recent advancements in DNA‐based prey identification have allowed for species‐level prey characterisations for many terrestrial insectivores, although these techniques have seldom been applied to understand the diets of freshwater fish. This study used DNA metabarcoding with high‐throughput, next‐generation sequencing to provide species‐level descriptions of prey composition for three naturally reproducing, syntopic freshwater trout species. Our study supports previous findings that suggested that brook trout (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) are generalist predators that display a high degree of seasonal dietary flexibility. Prey composition varied significantly across sampling periods, with detection frequency of terrestrial prey greater in the spring/summer period compared to the autumn period. Pollution‐sensitive aquatic macroinvertebrates were detected frequently across both sampling periods, highlighting the importance of high‐quality streams that support such arthropod prey. DNA metabarcoding also detected a high richness of soft‐bodied, Lepidoptera prey species, a taxonomic group that has been largely underrepresented in previous trout dietary studies that used traditional morphological techniques. This study demonstrates the applicability of dietary DNA metabarcoding for the detection and species‐level identification of arthropods found in freshwater fish lavage samples and highlights the importance of taxonomically precise techniques when attempting to better understand trophic interactions within freshwater communities.

The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA-based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces, and specialized training in molecular and computational techniques; limitations effectively excluding field researchers, underfunded programs, and citizen scientists from contributing to cutting-edge science. It is for these reasons that we have designed a simplified, inexpensive method for field-based molecular organism detection – FINDeM (Field-deployable Isothermal Nucleotide-based Detection Method). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body-heat inducible reactions – recombinase polymerase amplification and a CRISPR-cas12a fluorescent reporter assay – to amplify and detect target DNA, respectively. Here, we demonstrate FINDeM in detecting Batrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single-digit DNA copies from epidermal swabs in under one hour using low-cost supplies and field-friendly equipment.

Background Diet analysis is essential to understanding the functional role of large bird species in food webs. Morphological analysis of regurgitated bird pellet contents is time intensive and may underestimate biodiversity. DNA metabarcoding has the ability to circumvent these issues, but has yet to be done. Methods We present a pilot study using DNA metabarcoding of MT-RNR1 and MT-CO1 markers to determine the species of origin and prey of 45 pellets collected in Qinghai and Gansu Provinces, China. Results We detected four raptor species [Eurasian Eagle Owl (Bubo bubo), Saker Falcon (Falco cherrug), Steppe Eagle (Aquila nipalensis), and Upland Buzzard (Buteo hemilasius)] and 11 unique prey species across 10 families and 4 classes. Mammals were the greatest detected prey class with Plateau Pika (Ochotona curzoniae) being the most frequent. Observed Shannon’s and Simpson’s diversity for Upland Buzzard were 1.089 and 0.479, respectively, while expected values were 1.312 ± 0.266 and 0.485 ± 0.086. For Eurasian Eagle Owl, observed values were 1.202 and 0.565, while expected values were 1.502 ± 0.340 and 0.580 ± 0.114. Interspecific dietary niche partitioning between the two species was not detected. Conclusions Our results demonstrate successful use of DNA metabarcoding for understanding diet via a novel noninvasive sample type to identify common and uncommon species. More work is needed to understand how raptor diets vary locally, and the mechanisms that enable exploitation of similar dietary resources. This approach has wide ranging applicability to other birds of prey, and demonstrates the power of using DNA metabarcoding to study species noninvasively.

While an increasing number of studies are adopting molecular and chemical methods for dietary characterization, these studies often employ only one of these laboratory‐based techniques; this approach may yield an incomplete, or even biased, understanding of diet due to each method's inherent limitations. To explore the utility of coupling molecular and chemical techniques for dietary characterizations, we applied DNA metabarcoding alongside stable isotope analysis to characterize the dietary niche of breeding Louisiana waterthrush (Parkesia motacilla), a migratory songbird hypothesized to preferentially provision its offspring with pollution‐intolerant, aquatic arthropod prey. While DNA metabarcoding was unable to determine if waterthrush provision aquatic and terrestrial prey in different abundances, we found that specific aquatic taxa were more likely to be detected in successive seasons than their terrestrial counterparts, thus supporting the aquatic specialization hypothesis. Our isotopic analysis added greater context to this hypothesis by concluding that breeding waterthrush provisioned Ephemeroptera and Plecoptera, two pollution‐intolerant, aquatic orders, in higher quantities than other prey groups, and expanded their functional trophic niche when such prey were not abundantly provisioned. Finally, we found that the dietary characterizations from each approach were often uncorrelated, indicating that the results gleaned from a diet study can be particularly sensitive to the applied methodologies. Our findings contribute to a growing body of work indicating the importance of high‐quality, aquatic habitats for both consumers and their pollution‐intolerant prey, while also demonstrating how the application of multiple, laboratory‐based techniques can provide insights not offered by either technique alone.