Studies in evolution, ecology and conservation are increasingly based on genetic and genomic inferences. With increased focus on molecular approaches, ethical concerns about destructive or more invasive techniques need to be considered, with a push for minimally invasive sampling to be optimised. Buccal swabs have been increasingly used to collect DNA in a number of taxa, including amphibians. However, DNA yield and purity from swabs is often low, limiting its use. In this study we compare different types of swabs, preservation method and storage, and DNA extraction technique in three case studies to assess the optimal approach for recovering DNA in anurans. Out of the five different types of swab that we tested, Isohelix MS-02 and Rapidry swabs generated higher DNA yields than other swabs. When comparing storage buffers, ethanol is a better preservative than a non-alcoholic alternative. Dried samples resulted in similar or better final DNA yields than ethanol-fixed samples if kept cool. DNA extraction via a Qiagen™ DNeasy Blood and Tissue Kit and McHale’s salting out extraction method resulted in similar DNA yields but the Qiagen™ kit extracts contained less contamination. We also found that samples produce better DNA recovery if frozen as soon as possible after collection. We provide recommendations for sample collection and extraction under different conditions, including budgetary considerations, size of individual sampled, access to cold storage facilities, and DNA extraction methodology. Maximising efficacy of all of these factors for better DNA recovery will allow buccal swabs to be used for genetic and genomic studies in a range of vertebrates.

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.

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.