Dietary variation among individuals and cohorts can have a major impact on how populations adapt to environmental variation. Although variation in diet between cohorts and across habitats has been well studied in many taxa that feed on few or easily observed food items, the same is not true for most birds, especially smaller generalist passerines whose feeding habits are predominantly cryptic. For these and similar species, DNA metabarcoding can be used to good effect but remains underutilised. Here we used DNA metabarcoding with next-generation sequencing to assess spatio-temporal dietary variation among age and sex cohorts of the great tit Parus major, a model species in avian ecology. We identified animal prey throughout the year, and plants in winter, across different habitat fragments, achieving sample completeness of >90% for most analyses. There was considerable variation in dietary richness and composition among seasons and years, and diet composition varied between coniferous and mixed-deciduous habitats. Invertebrate prey were more species rich in mixed-deciduous habitats than in coniferous, primarily in winter, but when plants were also included there were no differences between habitat types in species richness. “Rare” species dominated the diet accounting for 30% of prey items across all samples. In winter, first year birds consumed more invertebrate species than adults. Beech (Fagus) was also almost twice as prevalent in the diets of first years (96% vs 52% in adults). Most differences between ages and sexes seemed driven primarily by differences in rare species, which we speculate is caused by divergent foraging modes and microhabitat use. We discuss the likely mechanisms for the patterns observed, all of which suggest considerable spatio-temporal variation in diet among cohorts. Our results also highlight that understanding the role of food in driving phenological phenomena requires consideration of dietary variation over space, time and between consumers.

Nutrient-specific foraging is the ecological theory that generalist consumers select food resources based on their nutritional content. While laboratory experiments support this idea, it has yet to be demonstrated in invertebrates in the field. We combined dietary DNA metabarcoding with prey availability data and macronutrient content in the field to analyze nutrient-based prey choice. We show that spider nutrient intake and prey choice deviates from what we would expect if individuals randomly chose their prey. Through a novel nutrient-based taxonomy and null modelling, we reveal a stable average macronutrient intake. There was disproportionate foraging for different macronutrients by individual spiders. Although, this might be expected as individual prey are biased toward particular nutrients and individual spiders were at different stages of nutrient balancing when collected. This finding suggests that spiders are redressing nutritional deficits to obtain a target nutrient intake, as would be expected of nutrient-specific foraging. This evidence for nutrient-specific foraging under field conditions is a significant advance, extending our understanding beyond lab-based behavioral assays to now resolving complex real-world systems.

Avian diet can be affected by site-specific variables, such as habitat, as well as intrinsic factors such as sex. This can lead to dietary niche separation, which reduces competition between individuals, as well as impacting how well avian species can adapt to environmental variation. Estimating dietary niche separation is challenging, due largely to difficulties in accurately identifying food taxa consumed. Consequently, there is limited knowledge of the diets of woodland bird species, many of which are undergoing serious population declines. Here, we show the effectiveness of multi-marker faecal metabarcoding to provide in-depth dietary analysis of a declining passerine, the Hawfinch (Coccothraustes coccothraustes). We collected faecal samples from (n=262) UK Hawfinches prior to, and during the breeding seasons in 2016-2019. We detected 49 and 90 plant and invertebrate taxa, respectively. We found Hawfinch diet varied spatially, as well as between sexes, indicating broad dietary plasticity and the ability of Hawfinches to utilise multiple resources within their foraging environments.

Eurasian otters are apex predators of freshwater ecosystems and a recovering species across much of their European range; investigating the dietary variation of this predator over time and space therefore provides opportunities to identify changes in freshwater trophic interactions and factors influencing the conservation of otter populations. Here we sampled faeces from 300 dead otters across England and Wales between 2007 and 2016, conducting both morphological analysis of prey remains and dietary DNA metabarcoding. Comparison of these methods showed that greater taxonomic resolution and breadth could be achieved using DNA metabarcoding but combining data from both methodologies gave the most comprehensive dietary description. All otter demographics exploited a broad range of taxa and variation primarily reflected changes in prey distributions and availability across the landscape. This study provides novel insights into the trophic generalism and adaptability of otters across Britain, which is likely to have aided their recent population recovery, and may increase their resilience to future environmental changes.

1. Generalist invertebrate predators are sensitive to weather conditions, but the relationship between their trophic interactions and weather is poorly understood. This study investigates how weather affects the identity and frequency of spider trophic interactions as mediated by prey community structure, web characteristics and density-independent prey choice. 2. Spiders and their locally available prey were collected from barley fields in Wales, UK from April to September 2017-2018. The gut contents of 300 spiders were screened using DNA metabarcoding, analysed via multivariate models, and compared against prey availability using null models. 3. Spiders' trophic interactions changed over time and with weather conditions, primarily related to concomitant changes in their prey communities. Spiders did, however, appear to mitigate the effects of structural changes in prey communities through changing prey preferences according to prevailing weather conditions, possibly facilitated by adaptive web construction. 4. Using these findings, we demonstrate that prey choice data collected under different weather conditions can be used to refine inter-annual predictions of spider trophic interactions, although prey abundance was secondary to diversity in driving the diet of these spiders. By improving our understanding of the interaction between trophic interactions and weather, we can better predict how ecological networks are likely to change in response to variation in weather conditions and, more urgently, global climate change.

The diet of an individual animal is subject to change over time, both in response to short-term food fluctuations and over longer time scales as an individual ages and meets different challenges over its life cycle. A metabarcoding approach was used to elucidate the diet of different life stages of a songbird, the Eurasian reed warbler (Acrocephalus scirpaceus) over the summer breeding season of 2017. The faeces of adult, juvenile and nestling warblers were screened for invertebrate DNA, enabling identification of prey species. Dietary analysis was coupled with monitoring of Diptera in the field using yellow sticky traps. Seasonal changes in warbler diet were subtle whereas age class had a greater influence on overall diet composition. Age classes showed high dietary overlap, but significant dietary differences were mediated through the selection of prey, i) from different taxonomic groups, ii) with different habitat origins (aquatic versus terrestrial) and iii) of different average approximate sizes. Our results highlight the value of metabarcoding data for enhancing ecological studies of insectivores in dynamic environments.

Understanding the role diet plays in the structure of food webs is vital, and dietary knowledge is key for conservation management success. There is limited knowledge of the diets of woodland bird species, due largely to difficulties in accurately identifying plant and invertebrate taxa being consumed. Here, we show the effectiveness of multi-marker faecal metabarcoding to provide the most in-depth dietary analysis of a generalist passerine, the Hawfinch (Coccothraustes coccothraustes, Linnaeus), to date. Faecal samples were obtained from 2016-2019 from Hawfinch populations prior to and during the breeding season throughout the UK. DNA was extracted from 263 samples and amplified using Internal Transcribed Spacer 2 (ITS2) and cytochrome C oxidase subunit I (COI) barcodes. Using high-throughput sequencing (HTS), we identified 49 and 97 ITS2 and COI zero radius operational taxonomic units (zOTUs) respectively which equated to reputed dietary items. The herbivorous element of Hawfinch diet was dominated by naturally occurring taxa such as beech (Fagus sylvatica, Linnaeus), hornbeam (Carpinus betulus, Linnaeus) and oak (Quercus sp., Linnaeus). The most taxon rich and commonly recorded invertebrate taxon identified was Lepidoptera. We found Hawfinch diet varied spatially, as well as between sexes. Hawfinch showed broad dietary plasticity and utilised multiple resources within their foraging environments. Our study shows the potential of multi-marker DNA metabarcoding to reveal subtle dietary differences, but also highlights the challenges of studying omnivorous species using metabarcoding methods.

Metabarcoding provides a powerful tool for investigating biodiversity and trophic interactions, but the high sensitivity of this methodology makes it vulnerable to errors, resulting in artefacts in the final data. Metabarcoding studies thus often utilise minimum sequence copy thresholds (MSCTs) to remove artefacts that remain in datasets; however, there is no consensus on best practice for the use of MSCTs. To mitigate erroneous reporting of results and inconsistencies, this study discusses and provides guidance for best-practice filtering of metabarcoding data for the ascertainment of conservative and accurate data. The most common MSCTs identified in the literature were applied to example datasets of Eurasian otter (Lutra lutra) and cereal crop spider (Araneae: Linyphiidae and Lycosidae) diets. Changes in both the method and threshold value considerably affected the resultant data. Of the MSCTs tested, it was concluded that the optimal method for the examples given combined a sample-based threshold with removal of maximum taxon contamination, providing stringent filtering of artefacts whilst retaining target data. Choice of threshold value differed between datasets due to variation in artefact abundance and sequencing depth, thus studies should employ controls (mock communities, negative controls with no DNA and unused MID-tag combinations) to select threshold values appropriate for each individual study.