A pilot study on the application of DNA metabarcoding for non-invasive diet analysis in the Italian hare

The Italian hare is endemic to central and southern Italy and currently designated as vulnerable by the International Union for the Conservation of Nature. Despite the worrying status, few studies have explored its ecology. In this scenario, improving knowledge of food preferences is of primary importance. Here, we present a non-invasive pilot study set to analyse the diet composition of the Italian hare from southern Italy, starting from fecal pellets, and using, for the first time on this species, DNA metabarcoding and next generation sequencing. The findings indicated that this approach provides reliable qualitative and semi-quantitative information, allowing the characterization of the hare diet and its seasonal variation using 22 fecal samples. In a single experiment, through time and cost-effective screening of multiple DNA metabarcodes, we detected a broad diversity of plants (99 taxa). Unlike traditional methods, this approach can identify items that leave no solid remains or that simply are lacking in diagnostic taxonomic features. Moreover, it starts from small amounts of input fecal material, useful in investigation on elusive and vulnerable species. The major guideline for future applications is to use a barcode short enough to allow amplification of environmental degraded DNA and the selection of the universal primers for PCR amplification.