Metabarcoding provides a powerful tool for ecological studies of biodiversity and trophic interactions (Deiner et al., 2017;Taberlet et al., 2018). By combining high-throughput sequencing (HTS) with DNA barcoding, large volumes of high-resolution data can be generated from many samples simultaneously (Taberlet et al., 2018). As an accurate means of detecting and identifying not just common species, but also cryptic and rare species, metabarcoding has in many cases superseded traditional methods such as morphological analysis

1. Money spiders (Linyphiidae) are an important component of conservation biological control in cereal crops, but they rely on alternative prey when pests are not abundant, such as between cropping cycles. To optimally benefit from these generalist predators, prey choice dynamics must first be understood. 2. Money spiders and their locally available prey were collected from cereal crops 2 weeks pre-and post-harvest. Spider gut DNA was amplified with two novel metabarcoding primer pairs designed for spider dietary analysis, and sequenced. 3. The combined general and spider-exclusion primers successfully identified prey from 15 families in the guts of the 46 linyphiid spiders screened, whilst avoiding amplification of Erigone spp. The primers show promise for application to the diets of other spider families such as Agelenidae and Pholcidae. 4. Distinct invertebrate communities were identified pre-and post-harvest, and changes in spider diet and, to a lesser extent, prey choice reflected this. Spiders were found to consume one another more than expected, indicating their propensity towards intraguild predation, but also consumed common pest families. 5. Changes in spider prey choice may redress prey community changes to maintain a consistent dietary intake. Consistent provision of alternative prey via permanent refugia should be considered to sustain effective conservation biocontrol.

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 migratory songbird, the Eurasian reed warbler ( Acrocephalus scirpaceus ) over the 2017 summer breeding season in Somerset, the United Kingdom. The feces of adult, juvenile, and nestling warblers were screened for invertebrate DNA, enabling the 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 vs. 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.

This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Introduced species can exert disproportionately negative effects on island ecosystems, but their potential role as food for native consumers is poorly studied. Telfair's skinks are endemic omnivores living on Round Island, Mauritius, a globally significant site of biodiversity conservation. We aimed to determine the dietary diversity and key trophic interactions of Telfair's skinks, whether introduced species are frequently consumed, and if diet composition changes seasonally between male and female skinks. We used DNA metabarcoding of skink fecal samples to identify animals (COI) and plants (ITS2) consumed by skinks. There were 389 dietary presence counts belonging to 77 dietary taxa found across the 73 Telfair's skink fecal samples. Introduced taxa were cumulatively consumed more frequently than other categories, accounting for 49.4% of all detections, compared to cryptogenic (20.6%), native (20.6%), and endemic taxa (9.5%). The most frequently consumed introduced species was the ant, Pheidole megacephala, present in 40% of samples. Blue latan palm, Latania loddigesii, was the most frequently consumed endemic species, present in 33% of samples but was only detected in the dry season, when fruits are produced. We found a strong seasonal difference in diet composition explained by the presence of certain plant species solely or primarily in one season and a marked increase in the consumption of animal prey in the dry season. Male and female skinks consumed several taxa at different frequencies. These results present a valuable perspective on the role of introduced species in the trophic network of their invaded ecosystem. Both native and introduced species provide nutritional resources for skinks, and this may have management implications in the context of species conservation and island restoration.

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 feces from 300 dead otters across England and Wales between 2007 and 2016, conducting both morphological analyses 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 likely 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.

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.

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.