When to use next generation sequencing or diagnostic PCR in diet analyses.

Next-generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co-amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co-amplified, NGS is better suited to discover the range of possible prey, than for comparing co-occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost-efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample-sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.

A broadly applicable COI primer pair and an efficient single-tube amplicon library preparation protocol for metabarcoding.

The nucleotide variation in the cytochrome c oxidase subunit I (COI) gene makes it ideal for assigning sequences to species. However, this variability also makes it difficult to design truly universal primers. Here, we present the forward primer "Sauron-S878," specifically designed to facilitate library preparation for metabarcoding. This primer is modified to improve the coverage of terrestrial species compared to the primer mCOIintF, optimized for aquatic systems, which raised the in silico coverage from 74.4% to 98.3% of available NCBI sequences (perfect match in 3' region, up to three mismatches in remaining primer). When paired with the reverse primer "jgHCO2198" (fragment length ~313 bp), these primers amplified 98.4% of 255 tested DNA extracts from various taxa, which are better than many other common COI barcoding primers. Furthermore, a single-tube protocol was developed, wherein these primers amplify the target gene, and attach MIDs and Illumina sequencing adapters in one reaction. This eliminates the need for re-amplification or enzymatic ligation during library preparation while keeping the flexibility to modularly combine primers and MIDs. Using the single-tube approach, three replicates of three mock samples were sequenced on a MiSeq platform with no adverse effects compared to commercial Nextera indexing kits. From this run, 75% of all included taxa could be recovered, with no considerable bias among taxonomic groups. Despite the fact that 98.4% of the extracts were confirmed to amplify in vitro, this number was lower than expected. A reason for this discrepancy was a clear link between the relative concentration of a specific DNA type in the template and the number of returned reads for this DNA. We would argue that such a bias may be especially problematic in metabarcoding where samples usually contain trace DNA in unknown amounts. However, how this affects the completeness of metabarcoding results has yet been poorly investigated.

Habitat heterogeneity induces rapid changes in the feeding behaviour of generalist arthropod predators.

The "habitat heterogeneity hypothesis" predicts positive effects of structural complexity on species coexistence. Increasing habitat heterogeneity can change the diversity (number of species, abundances) and the functional roles of communities. The latter, however, is not well understood as species and individuals may respond very differently and dynamically to a changing environment.Here, we experimentally test how habitat heterogeneity affects generalist arthropod predators, including epigaeic spiders, carabid and staphylinid beetles, under natural conditions by assessing their diversity and directly measuring their trophic interactions (which provide a proxy for their functional roles). The experiment was conducted in spring barley fields in Southern Sweden where habitat heterogeneity was manipulated by increasing within-field plant diversity.Increased habitat heterogeneity triggered rapid changes in the feeding behaviour of generalist predators characterized by lower trophic specialization at both network (H2', degree of interaction specialization in the entire network) and species level (d', degree of interaction specialization at the species level). We presume that this is because spatial separation resulted in relaxed competition and allowed an increased overlap in resources used among predator species. Predators collected from heterogenous habitats also showed greater individual-level dietary variability which might be ascribed to relaxed intraspecific competition.Our results provide conclusive evidence that habitat heterogeneity can induce rapid behavioural responses independent of changes in diversity, potentially promoting the stability of ecosystem functions. A plain language summary is available for this article.