Mammalian Biology

, Volume 84, Issue 1, pp 30–34 | Cite as

A new mitochondrial haplotype confirms the distinctiveness of the Italian wolf (Canis lupus) population

  • Luca MontanaEmail author
  • Romolo Caniglia
  • Marco Galaverni
  • Elena Fabbri
  • Ettore Randi
Short communication


In the past century the Italian wolf has been repeatedly indicated as a distinct subspecies, Canis lupus italicus, due to its unique morphology and its distinctive mtDNA control region (CR) monomorphism. However, recent studies on wolf x dog hybridization in Italy documented the presence of a second mtDNA CR haplotype (W16), previously found only in wolves from Eastern Europe, casting doubts on the genetic uniqueness of the Italian wolves. To test whether this second haplotype belongs to the Italian wolf population, we genotyped 92 wolf DNA samples from Italy, Slovenia, Greece and Bulgaria at four mtDNA regions (control-region, ATP6, COIII and ND4 genes) and at 39 autosomal microsatellites. Results confirm the presence of two mtDNA multi-fragment haplotypes (WH14 and WH19) in the Italian wolves, distinct from all the other European wolves. Network analyses of the multi-fragment mtDNA haplotypes identified two strongly differentiated clades, with the Italian wolf WH14 and WH19 multi-fragment haplotypes rooted together. Finally, Bayesian clustering clearly assigned all the wolves sampled in Italy to the Italian population, regardless of the two different multi-fragment haplotypes. These results demonstrate that the W16 CR haplotype is part of the genetic pool of the Italian wolf population, reconfirming its distinctiveness from other European wolves. Overall, considering the presence of unique mtDNA and Y-linked haplotypes, the sharply different frequencies of genome-wide autosomal alleles and the distinct morphological features of Italian wolves, we believe that this population should be considered a distinct subspecies.


Canis lupus italicus Bottleneck Conservation genetics Isolation Mitochondrial DNA 


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  1. Altobello, G., 1921. Fauna d’Abruzzo e Molise. In: Mammiferi IV., pp. 38–45.Google Scholar
  2. Bandelt, H.J., Forster, P., Röhl, A., 1994. Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol 16, 37–48.CrossRefGoogle Scholar
  3. Björnerfeldt, S., Webster, M.T., Vilà, C., 2006. Relaxation of selective constraint on dog mitochondrial DNA following domestication. Genome Res 16, 990–994, Scholar
  4. Boggiano, F., Ciofi, C., Boitani, L., Formia, A., Grottoli, L., Natali, C., Ciucci, P., 2013. Detection of an East European wolf haplotype puzzles mitochondrial DNA monomorphism of the Italian wolf population. Mamm. Biol.-Z. Säugetierkd 78, 374–378, Scholar
  5. Breitenmoser, U., 1998. Large predators in the Alps: the fall and rise of man’s competitors. Biol. Conserv 83, 279–289.CrossRefGoogle Scholar
  6. Caniglia, R., Fabbri, E., Galaverni, M., Milanesi, P., Randi, E., 2014. Noninvasive sampling and genetic variability, pack structure, and dynamics in an expanding wolf population. J. Mammal 95, 41–59, Scholar
  7. Chapron, G., Kaczensky, P., Linnell, J.D.C., von Arx, M., Huber, Đ., Andrén, H., López-Bao, J.V., Adamec, M., Álvares, F., Anders, O., Balčiauskas, L., Balys, V., Bedo, P., Bego, F., Blanco, J.C., Breitenmoser, U., Brøseth, H., Bufka, L., Bunikyte, R., Ciucci, P., Dutsov, A., Engleder, T., Fuxjäger, C., Groff, C., Holmala, J., Jerina, K., Kluth, G., Knauer, F., Kojola, I., Kos, I., Krofel, M., Kubala, J., Kunovac, S., Kusak, J., Kutal, M., Liberg, O., Majić, A., Männil, P., Manz, R., Marboutin, E., Marucco, F., Melovski, D., Mersini, K., Mertzanis, Y., Mysłajek, R.W., Nowak, S., Odden, J., Ozolins, J., Palomero, G., Paunović, M., Persson, J., Potočnik, H., Quenette, P.Y., Rauer, G., Reinhardt, I., Rigg, R., Ryser, A., Salvatori, V., Skrbinšek, T., Stojanov, A., Swenson, J.E., Szemethy, L., Trajce, A., Tsingarska-Sedefcheva, E., Váňa, M., Veeroja, R., Wabakken, P., Wölfl, M., Wölfl, S., Zimmermann, F., Zlatanova, D., Boitani, L., 2014. Recovery of large carnivores in Europe’s modern human-dominated landscapes. Science 346, 1517–1519, Scholar
  8. Elson, J.L., Turnbull, D.M., Howell, N., 2004. Comparative genomics and the evolution of human mitochondrial DNA: assessing the effects of selection. Am. J. Hum. Genet 74, 229–238.CrossRefGoogle Scholar
  9. Evanno, G., Regnaut, S., Goudet, J., 2005. Detecting the number of clusters of individuals usingthe software STRUCTURE: asimulation study. Mol. Ecol 14, 2611–2620, Scholar
  10. Fabbri, E., Caniglia, R., Kusak, J., Galov, A., Gomerčić, T., Arbanasić, H., Huber, Đ., Randi, E., 2014. Genetic structure of expanding wolf (Canis lupus) populations in Italy and Croatia, and the early steps of the recolonization of the Eastern Alps. Mamm. Biol.-Z. Säugetierkd 79, 138–148, Scholar
  11. Falush, D., Stephens, M., Pritchard, J.K., 2003. Inference of population structure using multilocus genotype data: linked loci and correlated allele frequencies. Genetics 164, 1567–1587.PubMedPubMedCentralGoogle Scholar
  12. Fan, Z., Silva, P., Gronau, I., Wang, S., Armero, A.S., Schweizer, R.M., Ramirez, O., Pollinger, J., Galaverni, M., Ortega Del-Vecchyo, D., Du, L., Zhang, W., Zhang, Z., Xing, J., Vilà, C., Marques-Bonet, T., Godinho, R., Yue, B., Wayne, R.K., 2016. Worldwide patterns of genomic variation and admixture in gray wolves. Genome Res 26, 1–11, Scholar
  13. Fredrickson, R.J., Hedrick, P.W., Wayne, R.K., VonHoldt, B.M., Phillips, M.K., 2015. Mexican wolves are a valid subspecies and an appropriate conservation target. J. Hered 106, 415–416, Scholar
  14. Galaverni, M., Caniglia, R., Fabbri, E., Milanesi, P., Randi, E., 2016. One, no one, or one hundred thousand: how many wolves are there currently in Italy? Mamm. Res 61, 13–24, Scholar
  15. Gomerčić, T., Sindičić, M., Galov, A., Arbanasić, H., Kusak, J., Kocijan, I., Gomerčić, M.Đ., Huber, Đ., 2010. High genetic variability of the grey wolf (Canis lupus L.) population from Croatia as revealed by mitochondrial DNA control region sequences. Zool. Stud 49, 816–823.Google Scholar
  16. Gundry, R.L., Allard, M.W., Moretti, T.R., Honeycutt, R.L., Wilson, M.R., Monson, K.L., Foran, D.R., 2007. Mitochondrial DNA analysis of the domestic dog: control region variation within and among breeds.J. Forensic Sci 52, 562–572, Scholar
  17. Koblmüller, S., Vilà, C., Lorente-Galdos, B., Dabad, M., Ramirez, O., Marques-Bonet, T., Wayne, R.K., Leonard, J.A., 2016. Whole mitochondrial genomes illuminate ancient intercontinental dispersals of grey wolves (Canis lupus). J Biogeogr., 1–11, Scholar
  18. Kopelman, N.M., Mayzel, J., Jakobsson, M., Rosenberg, N.A., 2015. CLUMPAK: a program for identifying clustering modes and packaging population structure inferences across K. Mol. Ecol. Resour 15, 1179–1191, Scholar
  19. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452, Scholar
  20. Lucchini, V., Galov, A., Randi, E., 2004. Evidence of genetic distinction and long-term population decline in wolves (Canis lupus) in the Italian Apennines. Mol. Ecol 13, 523–536, Scholar
  21. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U. S. A 76, 5269–5273.CrossRefGoogle Scholar
  22. Nowak, R.M., Federoff, N.E., 2002. The systematic status of the Italian wolf Canis lupus. Acta Theriol 47, 333–338.CrossRefGoogle Scholar
  23. Pilot, M., Branicki, W., Jędrzejewski, W., Goszczynski, J., Jędrzejewska, B., Dykyy, I., Shkvyrya, M., Tsingarska, E., 2010. Phylogeographic history of grey wolves in Europe. BMC Evol. Biol 10, Scholar
  24. Pilot, M., Dabrowski, M.J., Hayrapetyan, V., Yavruyan, E.G., Kopaliani, N., Tsingarska, E., Bujalska, B., Kaminiski, S., Bogdanowicz, W., 2014. Genetic variability of the grey wolf Canis lupus in the caucasus in comparison with Europe and the Middle East: distinct or intermediary population? PLoS One 9, Scholar
  25. Ražen, N., Brugnoli, A., Castagna, C., Groff, C., Kaczensky, P., Kljun, F., Knauer, F., Kos, I., Krofel, M., Luštrik, R., Majić, A., Rauer, G., Righetti, D., Potočnik, H., 2016. Long-distance dispersal connects Dinaric-Balkanand Alpine grey wolf (Canis lupus) populations. Eur. J. Wildl. Res., 137–142, Scholar
  26. Randi, E., Lucchini, V., 2002. Detecting rare introgression of domestic dog genes into wild wolf (Canis lupus) populations by Bayesian admixture analyses of microsatellite variation. Conserv. Genet 3, 31–45, Scholar
  27. Randi, E., Lucchini, V., Christensen, M.F., Mucci, N., Funk, S.M., Dolf, G., Loeschcke, V., 2000. Mitochondrial DNA variability in Italian and east European wolves: detecting the consequences of small population size and hybridization. Conserv. Biol 14, 464–473, Scholar
  28. Randi, E., Alves, P.C., Carranza, J., Milosevic-Zlatanovi, S., Sfougaris, A., Mucci, N., 2004. Phylogeography of roedeer (Capreolus capreolus) populations: the effects of historical genetic subdivisions and recent nonequilibrium dynamics. Mol. Ecol 13, 3071–3083, Scholar
  29. Randi, E., Hulva, P., Fabbri, E., Galaverni, M., Galov, A., Kusak, J., Bigi, D., Bolfíková, B.C., Smetanová, M., Caniglia, R., 2014. Multilocus detection of wolf x dog hybridization in Italy, and guidelines for marker selection. PLoS One 9, Scholar
  30. Rutledge, L.Y., Patterson, B.R., White, B.N., 2010. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes. BMC Evol. Biol 10, 1–12.CrossRefGoogle Scholar
  31. Stronen, A.V., Jędrzejewska, B., Pertoldi, C., Demontis, D., Randi, E., Niedziałkowska, M., Pilot, M., Sidorovich, V.E., Dykyy, I., Kusak, J., Tsingarska, E., Kojola, I., Karamanlidis, A.A., Ornicans, A., Lobkov, V.A., Dumenko, V., Czarnomska, S.D., 2013. North-south differentiation and a region of high diversity in european wolves (Canis lupus) PLoS One 8, 1–9, Scholar
  32. Valière, N., Fumagalli, L., Gielly, L., Miquel, C., Lequette, B., Poulle, M., Weber, J., Arlettaz, R., Taberlet, P., 2003. Long-distance wolf recolonization of France and Switzerland inferred from non-invasive genetic sampling over a period of 10 years. Anim. Conserv 6, 83–92, Scholar
  33. Vilà, C., Amorim, I.R., Leonard, J.A., Posada, D., Castroviejo, J., Petrucci-Fonseca, F., Crandall, K.A., Ellegren, H., Wayne, R.K., 1999. Mitochondrial DNA phylogeography and population history of the grey wolf Canis lupus. Mol. Ecol 8, 2089–2103.CrossRefGoogle Scholar
  34. VonHoldt, B.M., Pollinger, J.P., Earl, D.A., Knowles, J.C., Boyko, A.R., Parker, H.G., Geffen, E., Pilot, M., Jedrzejewski, W., Jedrzejewska, B., Sidorovich, V.E., Greco, C., Randi, E., Musiani, M., Kays, R., Bustamante, C.D., Ostrander, E.A., Novembre, J., Wayne, R.K., 2011. Agenome-wide perspective onthe evolutionary history of enigmatic wolf-like canids. Genome Res 21, Scholar
  35. Weckworth, B.V., Dawson, N.G., Talbot, S.L., Cook, J.A., et al., 2015. Genetic distinctiveness of Alexander Archipelago Wolves (Canis lupus ligoni): reply to Cronin et al. (2015). J. Hered., 1–3, Scholar
  36. Zachos, F.E., Mattioli, S., Ferretti, F., Lorenzini, R., 2014. The unique Mesola red deer of Italy: taxonomic recognition (Cervus elaphus italicus nova ssp., Cervidae) would endorse conservation. Ital. J. Zool 81, 136–143, Scholar
  37. Zimen, E., Boitani, L., 1975. Number and distribution of wolves in Italy. Mamm. Biol.-Z. Säugetierkd 40, 102–112.Google Scholar

Copyright information

© Deutsche Gesellschaft für Säugetierkunde, e. V. DGS 2017

Authors and Affiliations

  • Luca Montana
    • 1
    • 2
    Email author
  • Romolo Caniglia
    • 1
  • Marco Galaverni
    • 1
  • Elena Fabbri
    • 1
  • Ettore Randi
    • 1
    • 3
  1. 1.Laboratorio di GeneticaIstituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA)Ozzano dell’Emilia, BolognaItaly
  2. 2.Département de BiologieUniversité de SherbrookeSherbrookeCanada
  3. 3.Department 18/Section of Environmental EngineeringAalborg UniversityAalborgDenmark

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