A powerful long metabarcoding method for the determination of complex diets from faecal analysis of the European pond turtle (Emys orbicularis, L. 1758)

Abstract

High‐throughput sequencing has become an accurate method for the identification of species present in soil, water, faeces, gut or stomach contents. However, information at the species level is limited due to the choice of short barcodes and based on the idea that DNA is too degraded to allow longer sequences to be amplified. We have therefore developed a long DNA metabarcoding method based on the sequencing of short reads followed by de novo assembly, which can precisely identify the taxonomic groups of organisms associated with complex diets, such as omnivorous individuals. The procedure includes 11 different primer pairs targeting the COI gene, the large subunit of the ribulose‐1,5‐bisphosphate carboxylase gene, the maturase K gene, the 28S rRNA and the trnL‐trnF chloroplastic region. We validated this approach using 32 faeces samples from an omnivorous reptile, the European pond turtle (Emys orbicularis, L. 1758). This metabarcoding approach was assessed using controlled experiments including mock communities and faecal samples from captive feeding trials. The method allowed us to accurately identify prey DNA present in the diet of the European pond turtles to the species level in most of the cases (82.4%), based on the amplicon lengths of multiple markers (168–1,379 bp, average 546 bp), and produced by de novo assembly. The proposed approach can be adapted to analyse various diets, in numerous conservation and ecological applications. It is consequently appropriate for detecting fine dietary variations among individuals, populations and species as well as for the identification of rare food items.

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair’s skink (Leiolopisma telfairii)

“…Our study represents one of only a few complete dietary analyses of omnivores using DNA metabarcoding (but see De Barba et al, 2014; Ducotterd et al, 2021; Robeson et al, 2018; Silva et al, 2019) and the first study examining the omnivorous diet of a threatened endemic reptile. We found that Telfair's skinks consume a few species regularly and many species rarely.…”

Impact of invading species on biodiversity: Diet study of the green whip snake’s (Hierophis viridiflavus, L. 1789) in Switzerland

“…Genomic DNA extraction was performed on approximately 100 mg of material, with the QIAamp® PowerFecal® DNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. The concentration and the quality of the extracted DNA were evaluated using a NanoDrop™ 1000 spectrophotometer (Thermo Fischer Scientific, Switzerland) and a Qubit® 3.0 Fluorometer (Thermo Fischer Scientific) (Ducotterd et al, 2021).…”

“…We used the long metabarcoding method developed by (Ducotterd et al, 2021) from which we only retained the combination of primers pairs targeting the mitochondrial-encoded cytochrome oxidase subunit I (COI) gene. Namely, ModRepCOI-F and ModRepCOI-R (Reeves et al, 2018), COI-CO2 and COI-CO4 (Che et al, 2012), ODO-LCO1490d and ODO-HCO2198d (Dijkstra et al, 2014) and mlCOIintF and jgHCO2198 (Leray et al, 2013).…”

“…Nowadays, this limitation is being increasingly overcome by the use of long reads sequencing technologies and genome assembly softwares, which produce longer contigs. Thereby, such approaches as the long metabarcoding method developed by (Ducotterd et al, 2020), the use of universal primers and de novo assembly of short DNA reads allow the formation of long contigs and the accurate specific determination of ingested prey, even on degraded material (Ducotterd et al, 2020(Ducotterd et al, , 2021 The green whip snake (Hierophis viridiflavus, L. 1789) is a Colubrid snake widespread in Western Europe. This diurnal, thermophilic species is known to be a terrestrial racer with high energy requirements represented by two phenotypically distinct subspecies (Lelièvre et al, 2012a).…”

“…Considering this feeding behaviour of Hierophis viridiflavus and its coexistence with endangered species of reptiles in the canton of Vaud, it was essential to study the diet of these introduced populations. For this purpose, a long metabarcoding method (Ducotterd et al, 2020(Ducotterd et al, , 2021 was used in this study to overcome the lack of taxonomic resolution accuracy, known to be the main limitation of this approach. The main aim of this study was to assess whether these introduced populations induced competition or predation on local communities according to sites and time, with a particular interest in threatened herpetofauna.…”