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

Zobrazit více v PubMed

Chapron, G. et al. Recovery of large carnivores in Europe’s modern human-dominated landscapes. PubMed DOI

Doan, K. et al. Evolutionary history of the extinct wolf population from France in the context of global phylogeographic changes throughout the Holocene. PubMed DOI

Angelici, F. M. et al. The sicilian wolf: genetic identity of a recently extinct insular population. PubMed DOI

Gómez-Sánchez, D. et al. On the path to extinction: inbreeding and admixture in a declining grey wolf population. PubMed DOI

Jarausch, A., Harms, V., Kluth, G., Reinhardt, I. & Nowak, C. How the west was won: genetic reconstruction of rapid wolf recolonization into Germany’s anthropogenic landscapes. PubMed DOI PMC

Dissegna, A. et al. How to choose? Comparing different methods to count wolf packs in a protected area of the Northern Apennines. PubMed DOI PMC

Marucco, F. et al. A multidisciplinary approach to estimating wolf population size for long-term conservation. PubMed DOI

Fabbri, E. et al. Genetic structure of expanding wolf ( DOI

Valière, N. et al. Long-distance wolf recolonization of France and Switzerland inferred from non-invasive genetic sampling over a period of 10 years. DOI

Stronen, A. V. et al. The use of museum skins for genomic analyses of temporal genetic diversity in wild species. DOI

Cilli, E. et al. Museomics provides insights into conservation and education: the instance of an African lion specimen from the Museum of Zoology Pietro Doderlein. DOI

Utzeri, V. J. et al. Ancient DNA re-opens the question of the phylogenetic position of the sardinian pika PubMed DOI PMC

Jansson, E., Harmoinen, J., Ruokonen, M. & Aspi, J. Living on the edge: reconstructing the genetic history of the Finnish wolf population. PubMed DOI PMC

Straube, N. et al. Successful application of ancient DNA extraction and library construction protocols to museum wet collection specimens. PubMed DOI

Suchan, T. et al. Hybridization capture using RAD probes (hyRAD), a new tool for performing genomic analyses on collection specimens. PubMed DOI PMC

Linck, E. B., Hanna, Z. R., Sellas, A. & Dumbacher, J. P. Evaluating hybridization capture with RAD probes as a tool for museum genomics with historical bird specimens. PubMed DOI PMC

Hansen, H. B. et al. Comparing ancient DNA preservation in petrous bone and tooth cementum. PubMed DOI PMC

Palkopoulou, E. et al. A comprehensive genomic history of extinct and living elephants. PubMed DOI PMC

Pinsky, M. L. et al. Genomic stability through time despite decades of exploitation in cod on both sides of the Atlantic. PubMed DOI PMC

Andrews, A. J. et al. Exploitation history of Atlantic bluefin tuna in the Eastern Atlantic and Mediterranean - insights from ancient bones. DOI

Dalén, L., Heintzman, P. D., Kapp, J. D. & Shapiro, B. Deep-time paleogenomics and the limits of DNA survival. PubMed DOI PMC

Zimen, E. & Boitani, L. Number and distribution of wolves in Italy.

Fabbri, E. et al. From the Apennines to the alps: colonization genetics of the naturally expanding Italian wolf ( PubMed DOI

Ražen, N. et al. Long-distance dispersal connects Dinaric-Balkan and Alpine grey wolf ( DOI

Hulva, P. et al. Wolves at the crossroad: fission–fusion range biogeography in the western carpathians and Central Europe. DOI

Hulva, P. et al. Genetic admixture between Central European and Alpine wolf populations.

Gervasi, V. et al. Estimating distribution and abundance of wide-ranging species with integrated spatial models: opportunities revealed by the first wolf assessment in South-central Italy. PubMed DOI PMC

Montana, L. et al. Combining phylogenetic and demographic inferences to assess the origin of the genetic diversity in an isolated wolf population. PubMed DOI PMC

vonHoldt, B. M. et al. A genome-wide perspective on the evolutionary history of enigmatic wolf-like canids. PubMed DOI PMC

Pilot, M. et al. Genome-wide signatures of population bottlenecks and diversifying selection in European wolves. PubMed DOI PMC

Silva, P. et al. Genomic evidence for the old divergence of Southern European wolf populations. PubMed PMC

Altobello, G. Fauna dell’Abruzzo e del Molise. Mammiferi. IV. I Carnivori (Carnivora). (1921).

Caniglia, R. et al. A standardized approach to empirically define reliable assignment thresholds and appropriate management categories in deeply introgressed populations. PubMed DOI PMC

Musto, C. et al. Men and wolves: anthropogenic causes are an important driver of wolf mortality in human-dominated landscapes in Italy.

Nowak, R. M. & Federoff, N. E. The systematic status of the Italian wolf DOI

Verginelli, F. et al. Mitochondrial DNA from prehistoric canids highlights relationships between dogs and south-east European wolves. PubMed DOI

Dufresnes, C. et al. Howling from the past: historical phylogeography and diversity losses in European grey wolves. PubMed DOI PMC

Ciucani, M. M. et al. Old wild wolves: ancient DNA survey unveils population dynamics in late pleistocene and Holocene Italian remains. PubMed DOI PMC

Iurino, D. A. et al. A Middle Pleistocene wolf from central Italy provides insights on the first occurrence of PubMed DOI PMC

Randi, E. et al. Multilocus detection of wolf x dog hybridization in Italy, and guidelines for marker selection. PubMed DOI PMC

Pinhasi, R. et al. Optimal ancient DNA yields from the inner ear part of the human petrous bone. PubMed DOI PMC

Koupadi, K. et al. Population dynamics in Italian canids between the late pleistocene and bronze age. PubMed DOI PMC

Dabney, J. et al. Complete mitochondrial genome sequence of a middle pleistocene cave bear reconstructed from ultrashort DNA fragments. PubMed DOI PMC

Cilli, E. et al. A multifaceted approach towards investigating childbirth deaths in double burials: anthropology, paleopathology and ancient DNA. DOI

Serventi, P. et al. Iron Age Italic population genetics: the Piceni from Novilara (8th–7th century BC). PubMed DOI

Caniglia, R. et al. Big bad wolf or man’s best friend? Unmasking a false wolf aggression on humans. PubMed DOI

Velli, E. et al. Guess who’s coming to dinner: molecular tools to reconstruct multilocus genetic profiles from wild canid consumption remains. PubMed DOI PMC

Anderson, T. M. et al. Molecular and evolutionary history of melanism in north American gray wolves. PubMed DOI PMC

Caniglia, R. et al. Black coats in an admixed wolf × dog pack is melanism an indicator of hybridization in wolves? DOI

Sundqvist, A-K., Ellegren, H., Olivier, M. & Vilà, C. Y chromosome haplotyping in scandinavian wolves ( PubMed DOI

Randi, E. et al. Mitochondrial DNA variability in Italian and east European wolves: detecting the consequences of small population size and hybridization. DOI

Fabbri, E. et al. From predation to management: monitoring wolf distribution and understanding depredation patterns from attacks on livestock.

Fabbri, E. et al. Genetic structure and expansion of golden jackals ( DOI

Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.

Librado, P. & Rozas, J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. PubMed DOI

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. PubMed DOI

Valière, N. Gimlet: a computer program for analysing genetic individual identification data. DOI

Pompanon, F., Bonin, A., Bellemain, E. & Taberlet, P. Genotyping errors: causes, consequences and solutions. PubMed DOI

Jombart, T. Adegenet: a R package for the multivariate analysis of genetic markers. PubMed DOI

Pritchard, J. K., Stephens, M. & Donnelly, P. Inference of population structure using multilocus genotype data. PubMed DOI PMC

Caniglia, R., Fabbri, E., Mastrogiuseppe, L. & Randi, E. Who is who? Identification of livestock predators using forensic genetic approaches. PubMed DOI

Kopelman, N. M., Mayzel, J., Jakobsson, M., Rosenberg, N. A. & Mayrose, I. Clumpak: a program for identifying clustering modes and packaging population structure inferences across K. PubMed DOI PMC

Caniglia, R., Fabbri, E., Galaverni, M., Milanesi, P. & Randi, E. Noninvasive sampling and genetic variability, pack structure, and dynamics in an expanding wolf population. DOI

Belkhir, K. GENETIX 4.05, population genetic software in Windows TM. (2004).

Hammer, Ø., Harper, D. A. T. & Ryan, P. D. PAST: paleontological statistics software package for education and data analysis.

Levine, M. A. Angela von den Driesch: A guide to the measurement of animal bone from archaeological sites. Peabody Museum Bulletin 1. Harvard: Peabody Museum of Archaeology and Ethnology, 138 pp., 62 figs. $5.00.

Khosravi, R., Kaboli, M., Imani, J. & Nourani, E. Morphometric variations of the skull in the Gray Wolf (Canis lupus) in Iran.

Engdal, V. A. Phenotypic variation in past and present Scandinavian wolves (

Andersone, Ž. & Ozoliņš, J. Craniometrical characteristics and dental anomalies in wolves DOI

Okarma, H. & Buchalczyk, T. Craniometrical characteristics of wolves DOI

Galaverni, M. et al. Disentangling timing of admixture, patterns of introgression, and phenotypic indicators in a hybridizing wolf population. PubMed DOI PMC

Ciucci, P., Lucchini, V., Boitani, L. & Randi, E. Dewclaws in wolves as evidence of admixed ancestry with dogs. DOI

Kreeger, T. J. The internal wolf: physiology, pathology, and pharmacology.

Sutter, N. B., Mosher, D. S., Gray, M. M. & Ostrander, E. A. Morphometrics within dog breeds are highly reproducible and dispute Rensch’s rule. PubMed DOI PMC

Trbojević, I. Sexual dimorphism and population differentiation of the wolf (

Cooper, A. & Poinar, H. N. Ancient DNA: do it right or not at all. PubMed DOI

Castelló, J. R.

Lopez, L., Turner, K. G., Bellis, E. S. & Lasky, J. R. Genomics of natural history collections for understanding evolution in the wild. PubMed DOI

Ciucani, M. M. et al. The extinct sicilian wolf shows a complex history of isolation and admixture with ancient dogs. PubMed DOI PMC

Leone, A. et al. Pliocene colonization of the Mediterranean by Great White Shark inferred from fossil records, historical jaws, phylogeographic and divergence time analyses. DOI

Lucchini, V., Galov, A. & Randi, E. Evidence of genetic distinction and long-term population decline in wolves ( PubMed DOI

Muñoz-Fuentes, V., Darimont, C. T., Paquet, P. C. & Leonard, J. A. The genetic legacy of extirpation and re-colonization in Vancouver Island wolves. DOI

Kardos, M. et al. Genomic consequences of intensive inbreeding in an isolated wolf population. PubMed DOI

Robinson, J. A. et al. Genomic signatures of extensive inbreeding in Isle Royale wolves, a population on the threshold of extinction. PubMed DOI PMC

Lobo, D., López-Bao, J. V. & Godinho, R. The population bottleneck of the Iberian wolf impacted genetic diversity but not admixture with domestic dogs: a temporal genomic approach. PubMed DOI

Aragno, P. et al. La Popolazione Di Lupo Nelle Regioni dell’Italia peninsulare 2020/2021. Relazione tecnica realizzata nell’ambito della convenzione ISPRA-Ministero della Transizione Ecologica Attività Di Monitoraggio Nazionale Nell’ambito del piano di Azione Del Lupo. (2022).

Battilani, D. et al. Beyond population size: whole-genome data reveal bottleneck legacies in the peninsular Italian wolf. PubMed PMC

Marucco, F., Pilgrim, K. L., Avanzinelli, E., Schwartz, M. K. & Rossi, L. Wolf dispersal patterns in the Italian Alps and implications for wildlife diseases spreading. PubMed DOI PMC

Stronen, A. V. et al. A reduced SNP panel to trace gene flow across southern European wolf populations and detect hybridization with other PubMed DOI PMC

Bergmann, C. Ueber die Verhaltnisse Der Warmeokonomie Der Thierezuihrer Grosse.

Musto, C. et al. First evidence of widespread positivity to anticoagulant rodenticides in grey wolves ( PubMed DOI