Abstract
DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187–387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.
Highlights
Analysis of trophic interactions facilitates our understanding of community ecology and ecosystem functioning
Current approaches to molecular analysis of herbivory are generally unable to identify the majority of plants to the species level across a range of families, using amplicons short enough to detect degraded DNA recovered from faecal samples
The most widely applied DNA barcode currently used to study herbivory, the P6 loop of the chloroplast trnL (UAA) gene, has nearly universal priming sites allowing extremely high taxonomic coverage[22], and allows about 50% of taxa to be identified to species[27]
Summary
Analysis of trophic interactions facilitates our understanding of community ecology and ecosystem functioning. Large herbivores in particular are recognised as keystone consumers[1,12] and determining their diets can be critical to understanding their impact on plant communities and the wider food web This is relevant in the light of recent rewilding efforts, including the introduction of non-native species as ecological replacements (analogues) for extinct taxa to restore ecosystem function, or the conservation or reintroduction of native species[1,12]. General primers for ITS2 have been designed for priming sites within the more conserved flanking regions of 5.8S and 26S35,38 This presents a problem for dietary studies since the resultant amplicon length (approximately 387–547 bp using S2F and S3R35) is potentially too great to be reliably detected in semi-digested samples. ITS2 presents challenges in interpretation due to the presence of paralogous gene copies and the potential for co-amplification of non-target fungal amplicons[36]
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