New insights into the origin and the genetic status of the Balkan donkey from Serbia L. J. Stanisic 1 , J. M. Aleksic 2 , V. Dimitrijevic 3 , P. Simeunovic 4 , U. Glavinic 1 , J. Stevanovic 1 and Z. Stanimirovic 1 1 Department of Reproduction, Fertility and Artificial Insemination, Faculty of Veterinary Medicine, University of Belgrade, Bul. oslobodjenja 18, PO Box 310, 11000 Belgrade, Serbia. 2 Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade, Vojvode Stepe 444a, PO Box 23, 11010 Belgrade, Serbia. 3 Department of Animal Breeding and Genetics, Faculty of Veterinary Medicine, University of Belgrade, Bul. oslobodjenja 18, PO Box 310, 11000 Belgrade, Serbia. 4 Department of Farm Animal Diseases, Faculty of Veterinary Medicine, University of Belgrade, Bul. oslobodjenja 18, PO Box 310, 11000 Belgrade, Serbia. Summary The Balkan donkey (Equus asinus L.) is commonly regarded as a large-sized, unselected, unstructured and traditionally managed donkey breed. We assessed the current genetic status of the three largest E. asinus populations in the central Balkans (Serbia) by analysing the variability of nuclear microsatellites and the mitochondrial (mtDNA) control region of 77 and 49 individuals respectively. We further analysed our mtDNA dataset along with 209 published mtDNA sequences of ancient and modern individuals from 19 European and African populations to provide new insights into the origin and the history of the Balkan donkey. Serbian donkey populations are highly genetically diverse at both the nuclear and mtDNA levels despite severe population decline. Traditional Balkan donkeys in Serbia are rather heterogeneous; we found two groups of individuals with similar phenotypic features, somewhat distinct nuclear backgrounds and different proportions of mtDNA haplotypes belonging to matrilineal Clades 1 and 2. Another group, characterized by larger body size, different coat colour, distinct nuclear gene pool and predominantly Clade 2 haplotypes, was delineated as the Banat donkey breed. The maternal landscape of the large Balkan donkey population is highly heterogeneous and more complex than previously thought. Given the two independent domestication events in donkeys, multiple waves of introductions into the Balkans from Greece are hypoth- esized. Clade 2 donkeys probably appeared in Greece prior to those belonging to Clade 1, whereas expansion and diversification of Clade 1 donkeys within the Balkans predated that of Clade 2 donkeys. Keywords Equus asinus, the Balkans, genetic diversity, genetic structuring, mitochondrial control region, nuclear microsatellites, phylogeography Introduction The donkey, Equus asinus L., Equidae, was domesticated ~7000 years BC in the arid regions of north-eastern Africa (Beja-Pereira et al. 2004; Marshall 2007; Rossel et al. 2008; Rosenbom et al. 2015). Analysis of variability within the mitochondrial DNA (mtDNA) control region revealed two distinct matrilineal lineages, Clade 1 and Clade 2, and thus, provided evidence for two independent domestication events of donkeys (Beja-Pereira et al. 2004; Kimura et al. 2011). Beja-Pereira et al. (2004) further suggest that Clades 1 and 2 trace their origin to the historic Nubian wild donkey (E. asinus africanus; Heuglin & Fitzinger, 1866) and the Somali wild donkey (E. asinus somaliensis; Noack, 1884) respectively. However, Kimura et al. (2011) and Kefena et al. (2014) recently found that Clade 2 donkeys may be descendants of a yet unrecognized extinct wild population rather than Somali wild donkeys. The Balkan donkey is commonly regarded as a large- sized, unselected, unstructured and traditionally managed donkey breed found in the Balkans (Kugler et al. 2008). P erez-Pardal et al. (2014) recently studied mtDNA variabil- ity in 10 local populations of the Balkan donkey and did not Address for correspondence J. M. Aleksic, Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade, Vojvode Stepe 444a, PO Box 23, 11010 Belgrade, Serbia. E-mails: aleksic_jelena@yahoo.com.au; jelena.aleksic@imgge.bg.ac.rs Accepted for publication 15 June 2017 doi: 10.1111/age.12589 1 © 2017 Stichting International Foundation for Animal Genetics