After centuries of decline and protracted bottlenecks, the peninsular Italian wolf population has naturally recovered. However, an exhaustive comprehension of the effects of such a conservation success is still limited by the reduced availability of historical data. Therefore, in this study, we morphologically and genetically analyzed historical and contemporary wolf samples, also exploiting the optimization of an innovative bone DNA extraction method, to describe the morphological variability of the subspecies and its genetic diversity during the last 30 years. We obtained high amplification and genotyping success rates for tissue, blood and also petrous bone DNA samples. Multivariate, clustering and variability analyses confirmed that the Apennine wolf population is genetically and morphologically well-distinguishable from both European wolves and dogs, with no natural immigration from other populations, while its genetic variability has remained low across the last three decades, without significant changes between historical and contemporary specimens. This study highlights the scientific value of well-maintained museum collections, demonstrates that petrous bones represent reliable DNA sources, and emphasizes the need to genetically long-term monitor the dynamics of peculiar wolf populations to ensure appropriate conservation management actions.

Biodiversity Management and Monitoring Office Majella National Park via Badia 28 Sulmona 67039 L'Aquila Italy

Department of Cultural Heritage Alma Mater Studiorum University of Bologna via degli Ariani 1 Ravenna 48121 Italy

Department of Preclinical Sciences and Pathology Faculty of Veterinary Medicine Norwegian University of Life Sciences Ås Norway

Department of Zoology Faculty of Science Charles University Viničná 7 Prague 2 128 43 Czech Republic

Department of Zoology Institute of Ecology and Earth Sciences University of Tartu J Liivi 2 Tartu 50409 Estonia

Faculty of Tropical AgriSciences Czech University of Life Sciences Kamýcká 129 Prague 16500 Czech Republic

Unit for Conservation Genetics Via Cà Fornacetta 9 Bologna 40064 Ozzano dell'Emilia Italy

Zoological Museum Italian Institute for Environmental Protection and Research Via Cà Fornacetta 9 Bologna 40064 Ozzano dell'Emilia Italy

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