To pool or not to pool: Pooled metabarcoding does not affect estimates of prey diversity in spider gut content analysis

Gut content metabarcoding has provided important insights into the food web ecology of spiders, the most dominant terrestrial arthropod predators. In small invertebrates, like spiders, gut content analysis is often performed on whole body DNA extracts of individual predators, from which prey sequences are selectively enriched and sequenced. Since many spider species are generalist predators, large numbers of samples comprising individual spider specimens must be analysed to recover an exhaustive image of a spider species' prey spectrum, which is costly and time‐consuming. Pooled processing of bulk samples of multiple specimens has been suggested to reduce the necessary workload and cost while still recovering a representative estimate of the prey diversity. However, it is still unclear if pooling approaches lead to bias in recovering the prey spectrum and if the results are comparable with data from individually processed spiders. Here, we test the effects of metabarcoding pooled spider gut content on the recovered taxonomic diversity and composition of prey. Using a newly adapted primer pair, which efficiently enriches COI barcode sequences of diverse arthropod prey groups while suppressing spider amplification, we test if pooling leads to reduced taxonomic diversity or skewed estimates of prey composition. Our results show that pooling and individual processing recover highly correlated taxonomic diversity and composition of prey. The only exception are very rare prey items which were less well recovered by pooling. Our results support pooling as a cost‐effective and time‐efficient approach to recover the diet of generalist predators for population‐level studies of spider trophic interactions. To reduce the cost and workload of large‐scale gut content metabarcoding studies, we tested the performance of pooled samples versus individually processed samples in terms of information completeness. Pooled samples and individually processed samples recovered highly correlated taxonomic diversity, composition and richness of prey. Pooling is a valuable method of approaching large‐ scale metabarcoding projects targeting spider gut content, that introduces no significant bias to the results.