Diagnostic Dloop fragment for Ovis species identification in metabarcoding studies

Accurate species identification is essential in metabarcoding diet studies, notably to distinguish domestic from wild species, as they have markedly different ecological and socio-economic implications. This is particularly true for the genus Ovis, where domestic sheep (Ovis aries) and European mouflon (O. musimon) often coexist but are indistinguishable with common mitochondrial markers due to insufficient variation. Misidentification of these taxa can skew carnivore predation assessments, notably for species like the grey wolf (Canis lupus), impacting wildlife conservation and management efforts. Here, we developed a diagnostic D-loop fragment that effectively differentiates O. aries and O. musimon haplotypes in diet metabarcoding studies. Using reference samples from 46 European mouflons and 46 domestic sheep collected in the French Alps, we identified a 178bp fragment (OvisCR) containing 43 SNPs, distinguishing four mouflon haplotypes and 35 sheep haplotypes. No haplotypes were shared between the two species, though one O. musimon haplotype was closely related to two O. aries haplotypes. The performance of the OvisCR fragment was tested using 115 wolf scat samples. We sequenced a general metabarcoding marker (a 60-140bp amplicon of the 12S region) alongside the new amplicon on the Ion Torrent platform to assess the ability of the latter to refine Ovis identification. Following sequencing, two bioinformatic pipelines (DADA2 and OBITools) were employed to compare their effectiveness in resolving Ovis operational taxonomic units (OTUs) or amplicon sequence variants (ASVs). The two pipelines demonstrated similar accuracy, with the OvisCR fragment achieving Ovis species-level assignment for more than 72% of total samples sequenced and more than 81% of scats with Ovis reads detected, regardless of the pipeline used. This new marker provides a robust and practical tool for metabarcoding applications, improving ecological inference in carnivore trophic studies and supporting biodiversity conservation management strategies. While effective, its full discriminatory power requires a thorough examination of local Ovis breeds due to near-neighbor haplotypes before broader geographic applicability.