A Comprehensive DNA Barcode Reference for the Birds of Italy

The Italian peninsula and its Islands host about 551 bird species for a total of 702 taxa. More than a third are Species of European Conservation Concern or listed in critical IUCN categories. Of the 287 breeding bird species in Italy, many are of global and regional conservation concern. Nevertheless, knowledge on the genetic diversity of Italian species is still lacking as, before our study, the Barcode of Life Data system (BOLDSystem) hosted less than 350 sequences belonging to about 30 species. Curated reference DNA barcode libraries are fundamental not only in species-level identification but also in studies on evolution and diversity, identification of Management and Evolutionary Significant Units in conservation projects, eDNA metabarcoding surveys, and forensic analysis. Our study aimed to fill the existing gaps in the DNA barcode coverage of Italy's breeding bird species by sequencing the mitochondrial cytochrome c oxidase subunit I (COI) gene. The goal was to establish a comprehensive DNA barcode reference library for the Italian avifauna, starting from biological collections assembled by our research group since the early 2000s, comprising tissue and blood samples linked to voucher specimens and biometric data. To date, we have obtained about 650 DNA barcoding sequences from 280 species of Italian birds, sequencing 1 to 8 samples per species distributed across Italy. Our data leads to an increase of over 600% of species and 200% of sequences in BOLD, filling many gaps. Intraspecific variation agrees with literature, but high genetic distance was found in some species, suggesting a possible phylogeographic structure and an Italian hidden diversity. Future work will focus on completing the reference library by including all bird species occurring in Italy, such as wintering and migratory species, and by expanding the dataset to other markers (i.e. 12S rRNA), supporting future genetic applications for biodiversity.