The study of dietary niche partitioning is of great importance for understanding community structure and species coexistence, particularly if these are threatened. Here we used DNA metabarcoding from faeces to assess the diet of four threatened steppe birds (two bustards and two sandgrouse), with the aim of better understanding their dietary requirements, trophic interactions and potential threats. We found seasonal and interspecific differences in their plant diet, with greater importance of cultivated plants during autumn and winter (around 50% of their diet) than spring. Despite differences, plants of the genus Convolvulus and of the family Brassicaceae were frequently consumed by all species. In spring, poppies were a considerable part of their diet, and could be used as a source of carotenoids or for their anti-parasitic properties. Furthermore, the results also showed a dietary niche partitioning between species, with a marked segregation between bustards and, to a lesser extent, between sandgrouse. Diet similarity was generally higher between species from different orders that occur in mixed-species flocks (bustard - sandgrouse) than between species from the same order. This partitioning was probably related to a stratification in habitat use rather than to specialization and might prevent competition to some extent. However, the homogenization of trophic resources resulting from agricultural intensification could pose an important threat, particularly during autumn, when weeds are scarcer and the most abundant trophic resource are sown seeds, which are often treated with pesticides.

Agriculture expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from faecal eDNA. To help understand the impact of agricultural expansion, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the faeces of farmland birds. The capacity to identify diet and parasites of two existing and three developed mini-barcodes was tested “in silico” in relation to the behaviour of a reference eukaryotic primer set. Of the mini-barcodes tested, MiniB18S_81, one of our designs, showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa sequences. This primer pair was tested on faeces samples from five different steppe bird species. According to our data, this barcode shows good taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes and Apicomplexa, at the order level. The mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasitology of birds, essential for conservation and management.