Molecular Ecology (2006) 15, 1007–1020 doi: 10.1111/j.1365-294X.2006.02747.x MitochondrialBlackwell Publishing, Ltd. DNA reveals a strong phylogeographic structure in the badger across Eurasia J. MARMI,* F. LÓPEZ-GIRÁLDEZ,*† D. W. MACDONALD,‡ F. CALAFELL,* E. ZHOLNEROVSKAYA§ and X. DOMINGO-ROURA*† *Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Dr. Aiguader 80, 08003 Barcelona, Spain, †Genètica de la Conservació, Institut de Recerca i Tecnologia Agroalimentàries, Centre de Cabrils, Carretera de Cabrils s/n, 08348 Cabrils, Spain, ‡Wildlife Conservation Research Unit, Department of Zoology, University of Oxford, Tubney House, Tubney, Abingdon, OX13 5QL, UK, §Siberian Zoological Museum, Institute of Animal Systematics and Ecology, Siberian Division of the Russian Academy of Sciences, Frunze Street 11, 630091, Novosibirsk, Russia Abstract The badger, Meles meles, is a widely distributed mustelid in Eurasia and shows large geo- graphic variability in morphological characters whose evolutionary significance is unclear and needs to be contrasted with molecular data. We sequenced 512 bp of the mitochondrial DNA control region in 115 Eurasian badgers from 21 countries in order to test for the exist- ence of structuring in their phylogeography, to describe the genetic relationships among their populations across its widespread geographic range, and to infer demographic and biogeographic processes. We found that the Eurasian badger is divided into four groups regarding their mitochondrial DNA: Europe, Southwest Asia, North and East Asia, and Japan. This result suggests that the separation of badgers into phylogeographic groups was influenced by cold Pleistocene glacial stages and permafrost boundaries in Eurasia, and by geographic barriers, such as mountains and deserts. Genetic variation within phylogeo- graphic groups based on distances assuming the Tamura–Nei model with rate heterogeneity and invariable sites (dT-N range: 3.3–4.2) was much lower than among them (dT-N range: 10.7– 38.0), and 80% of the variation could be attributed to differences among regions. Spatial analysis of molecular variance (SAMOVA), median-joining network, and Mantel test did not detect genetic structuring within any of the phylogeographic groups with the exception of Europe, where 50% of variation was explained by differences among groups of populations. Our data suggest that the European, Southwest Asian, and North and East Asian badgers evolved separately since the end of Pliocene, at the beginnings of glacial ages, whereas Japanese badgers separated from continental Asian badgers during the middle Pleistocene. Endangered badgers from Crete Island, classified as Meles meles arcalus subspecies, were closely related to badgers from Southwest Asia. We also detected sudden demographic growth in European and Southwest Asian badgers that occurred during the Middle Pleistocene. Keywords: control region, Eurasia, Meles, mitochondrial DNA, phylogeography, postglacial colonization Received 27 October 2004; revision accepted 24 August 2005 collaris (Bryant et al. 1993). During the Pliocene, the Meles Introduction lineage evolved in the temperate forests of Asia, spreading The Eurasian badger, Meles meles, is classified within the west into Europe between the Late Pliocene and the Early mustelid subfamily Melinae (Wozencraft 1993; Macdonald Pleistocene (Neal & Cheeseman 1996). Badger forms very 2001) where it is closely related to the hog badger, Arctonyx similar to the modern Eurasian badger are found in the fossil record of Eurasia since the Middle Pleistocene (Kurten 1968; Correspondence: Josep Marmi, Present address: Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Petter 1971). Universitat Autònoma de Barcelona, 08193, Cerdanyola del Vallès, At the present time, the Eurasian badger is one of the Barcelona, Spain. Fax: +34935812106; E-mail: [email protected] most widely distributed mustelids. Its geographic range © 2006 Blackwell Publishing Ltd 1008 J. MARMI ET AL. includes forested and steppe areas in the Palearctic region louse, Trichodectes melis, or fleas, such as Paraceas melis) from the Iberian Peninsula in the west to the Japanese (Neal & Cheeseman 1996). archipelago in the east; and from Scandinavia and west Phylogeographic studies of mammalian species across Siberia in the north to Palestine, Iran, south China and Eurasia have begun to emerge (for example grey wolf, Tibet in the south (Heptner et al. 1967; Corbet 1978). Insular Canis lupus, Vilà et al. 1999; red deer, Cervus elaphus, Mahmut populations exist in Ireland, Britain, Sicily, Crete, Rhodes, et al. 2002 and Ludt et al. 2004; voles, Microtus sp., Jaarola & and Japan (Corbet 1978). Considerable differences in size, Searle 2002 and Brunhoff et al. 2003). Since the badger is coloration, and morphological characters are reported across extensively distributed across Eurasia, shows low disper- its geographic range. Main morphological differences are sion rates, and its maternal philopatry has been repeatedly found in dentition (e.g. the frequency of loss of first pre- reported (Neal & Cheeseman 1996; Revilla & Palomares molars, and the shape and proportions of first upper molar) 2002) (the two latter characteristics promoting allele and bones (e.g. skull and bacular structure) (Baryshnikov fixation and genetic drift (Melnick & Hoelzer 1992)), the & Potapova 1990; Lynch et al. 1997; Abramov 2002). regional structuring of its populations can offer insights Molecular data for badgers are scarce, but high levels of into colonization routes, barriers to dispersal and glacial genetic differentiation across regions were found com- refuges at a continental level not hitherto much explored. paring complete cytocrome b sequences from 17 Japanese, The objectives of this report are (i) to describe the genetic one Siberian and two European badgers (Kurose et al. 2001). variation in badgers across their distribution; (ii) to define Locally, badger populations can show from moderate levels phylogeographic groups and check if their mitochondrial of genetic variability (Bijlsma et al. 2000, using microsatel- sequences are geographically structured and follow a lites in The Netherlands and Denmark) to reduced variability pattern of isolation by distance; (iii) to explore geographic (Pertoldi et al. 2000, in Denmark using allozymes; Domingo- barriers to dispersal in Eurasia; and (iv) to draw inferences Roura et al. 2003, in the UK using microsatellite markers). on past demographic and biogeographic processes, such as The species has been killed for meat, fur, hair, and for possible population expansions or contractions, of this being a wild host of Mycobacterium bovis, which causes philopatric and widely distributed carnivore. bovine tuberculosis (Neal & Cheeseman 1996; Gallagher & Clifton-Hadley 2000). The Eurasian and hog badgers are Materials and methods classified as ‘Least Concern’ by the International Union for the Conservation of Nature and Natural Resources DNA extraction and sequencing (IUCN). Where they are monitored, Eurasian badger popu- lations appear to be either stable or increasing in Europe, One hundred and fifteen (115) badger samples from 21 although there are threats of local extinction in the Nether- countries (considered here as populations) throughout lands and Albania (Griffiths & Thomas 1997). Populations Eurasia were kindly provided by collaborators listed in from the islands of Crete and Rhodes, which have been Table S1 (Supplementary material). Animals came mainly classified as different subspecies (Meles meles arcalus and from road kills and were often museum specimens well Meles meles rhodius, respectively) from continental badgers, preserved frozen or in ethanol (none was killed for this are considered ‘Vulnerable’ (Karandinos 1992). The badger project). DNA was extracted from muscle, skin, ear, heart is strictly protected and considered ‘Endangered’ in Albania and blood tissues using standard phenol–chloroform (Zamir Dedej, personal communication). There is little protocols (Sambrook et al. 1989). Bone and teeth samples information about the status of the species in most parts of (13 out of 15 samples from the Siberian Zoological Museum continental Asia where, in general, it can be hunted. collected between 1937 and 1974) were powdered in a The quantification and distribution of genetic variability, coffee grinder, which was washed twice with lye and once and the understanding of population history and structure, with 96% ethanol between each sample. DNA from bone are crucial for improved management and conservation and teeth samples was extracted using DNeasy Tissue (Avise 1989). Extensive molecular data for Eurasian badger Extraction Kit (QIAGEN) according to modifications are needed to better understand the differences in mor- suggested by Iudica et al. (2001). To avoid contamination, phological characters found in this species and to elucidate extractions were done at a separate pre-PCR (polymerase if differences described according to morphology corre- chain reaction) room. Aerosol-barrier pipette tips were used. spond to genetic differences. In addition, the species shows One sample was handled at a time and negative controls variability in ecological adaptations, behaviour and social were included in the extraction procedure. systems (Kruuk 1989) and the clarification of evolutionary We amplified the complete mitochondrial DNA (mtDNA) relationships among badgers worldwide can offer insight control region in 16 samples