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SummaryThe Sicilian wolf represented the only population of wolves living on a Mediterranean island until the first half of the twentieth century (1930s-1960s) 1–7. Previous studies hypothesised that they remained isolated from mainland wolves from the end of the Last Glacial Maximum (LGM) 8,9, until human persecutions led them to extinction 1–7.There are only seven known Sicilian wolf specimens from the 19th and 20th century preserved in museums in Italy and recent morphometric analyses assigned them to the new subspecies Canis lupus cristaldii10. To better understand the origins of the Sicilian wolf, and its relationship to other wolf populations, we sequenced four whole genomes (3.8×-11.6×) and five mitogenomes. We investigated the relationship between Sicilian wolves and other modern breeds to identify potential admixture. Furthermore, considering that the last land-bridge between Sicily and Italy disappeared after the LGM 11, around 17 kya, we explored the possibility that the Sicilian wolf retained ancestry from ancient wolf and dog lineages. Additionally, we explored whether the long-term isolation might have affected the genomic diversity, inbreeding levels and genetic load of the Sicilian wolf.Our findings show that the Sicilian wolves shared most ancestry with the modern Italian wolf population but are better modelled as admixed with European dog breeds, and shared traces of Eneolithic and Bronze age European dogs. We also find signatures of severe inbreeding and low genomic diversity at population and individual levels due to long-term isolation and drift, suggesting also low effective population size.

During historical times many local grey wolf (Canis lupus) populations underwent a substantial reduction of their sizes or became extinct. Among these, the wolf population once living in Sicily, the biggest island of the Mediterranean Sea, was completely eradicated by human persecution in the early decades of the XX century.In order to understand the genetic identity of the Sicilian wolf, we applied ancient DNA techniques to analyse the mitochondrial DNA of six specimens actually stored in Italian museums.We successfully amplified a diagnostic mtDNA fragment of the control region (CR) in four of the samples. Results showed that two samples shared the same haplotype, that differed by two substitutions from the currently most diffused Italian wolf haplotype (W14) and one substitution from the only other Italian haplotype (W16). The third sample showed a wolf-like haplotype never described before and the fourth a haplotype commonly found in dogs.Furthermore, all the wolf haplotypes detected in this study belonged to the mitochondrial haplogroup that includes haplotypes detected in all the known European Pleistocene wolves and in several modern southern European populations.Unfortunately, this endemic island population, bearing unique mtDNA variability, was definitively lost before it was possible to understand its taxonomic uniqueness and conservational value.