MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 32 (2004) 788–798 www.elsevier.com/locate/ympev Phylogeographic history of the yellow-necked fieldmouse (Apodemus flavicollis) in Europe and in the Near and Middle East J.R. Michaux,a,b,* R. Libois,a E. Paradis,b and M.-G. Filippuccic a Unite de Recherches Zoogeographiques, Institut de Zoologie, Quai Van Beneden, 22, 4020 Liege, Belgium b Laboratoire de Paleontologie-cc064, Institut des Sciences de l’Evolution (UMR 5554-CNRS), Universite Montpellier II, Place E. Bataillon, 34095, Montpellier Cedex 05, France c Dipartimento di Biologia, Universita di Roma ‘‘Tor Vergata’’ Via della Ricerca Scientifica, 00133 Rome, Italy Received 25 July 2003; revised 3 February 2004 Available online 15 April 2004 Abstract The exact location of glacial refugia and the patterns of postglacial range expansion of European mammals are not yet com- pletely elucidated. Therefore, further detailed studies covering a large part of the Western Palearctic region are still needed. In this order, we sequenced 972 bp of the mitochondrial DNA cytochrome b (mtDNA cyt b) from 124 yellow-necked fieldmice (Apodemus flavicollis) collected from 53 European localities. The aims of the study were to answer the following questions: • Did the Mediterranean peninsulas act as the main refuge for yellow-necked fieldmouse or did the species also survive in more easterly refugia (the Caucasus or the southern Ural) and in Central Europe? • What is the role of Turkey and Near East regions as Quaternary glacial refuges for this species and as a source for postglacial recolonisers of the Western Palearctic region? The results provide a clear picture of the impact of the quaternary glaciations on the genetic and geographic structure of the fieldmouse. This species survived the ice ages in two main refuges, the first one in the Italo-Balkan region; the second one in Turkey and the Near East regions. It is from the Balkan refuge that it recolonised all European regions at the end of the last glaciation. The Turkish and Near East populations are distinct from the European ones and they did not recolonise the Palearctic region probably because: (i) they were blocked by the Black Sea and the Caucasus, (ii) the long term presence of fieldmice populations in the Balkans prevented their expansion. These are genetically differentiated from the European and Russian ones and could be described as a particular subspecies. This result emphasises the importance of Turkey and the Near and Middle East regions as a refuge for Palearctic mammals. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Apodemus flavicollis; Phylogeography; Mitochondrial DNA; Western palearctic region; Near east region 1. Introduction ice age, about 16,000 years ago (Hewitt, 2001; Michaux et al., 2003). However, some authors consider that this Recent molecular phylogeographic studies in Europe model of exclusively southern European glacial refugia suggest that the majority of temperate species of is not universal and that some species such as the mammals survived the Quaternary glaciations in Med- brown bear (Ursus arctos) (Taberlet et al., 1998) or iterranean refugia such as the Iberian, the Italian, and different small mammal species (Clethrionomys glareo- the Balkan peninsulas (Hewitt, 1999, 2001; Michaux lus, Sorex minutus, and Sorex araneus, Bilton et al., et al., 2003; Taberlet et al., 1998). From these regions, 1998; Microtus arvalis, Jaarola and Searle, 2002; they recolonised northern Europe at the end of the last Microtus oeconomus, Brunhoff et al., 2003) also sur- vived in more easterly refugia (the Caucasus or the southern Urals) and in Central Europe. Nevertheless, in * Corresponding author. Fax : +32-43-66-59-45. order to find the exact location of glacial refugia and to E-mail address: [email protected] (J.R. Michaux). elucidate the patterns of postglacial range expansion of 1055-7903/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2004.02.018 J.R. Michaux et al. / Molecular Phylogenetics and Evolution 32 (2004) 788–798 789 European mammals, further detailed studies covering a 2. Material and methods large part of the Western Palearctic region are still needed. 2.1. Biological material Moreover, to the exception of some data obtained on the hedgehog (Santucci et al., 1998; Seddon et al., A total of 124 A. flavicollis coming from 53 localities 2001), the woodmouse (Michaux et al., 2003) and the widespread throughout their distribution area have been lesser white-toothed shrew (Taberlet et al., 1998), very analysed (Table 1, Fig. 1). little information exists concerning the role of Turkey and Near East regions as refugia for European mam- 2.2. Laboratory methods mals during glaciations as well as their role as a source for postglacial recolonisers of the Western Palearctic DNA was extracted from ethanol-preserved tissue region. following Sambrook et al. (1989). Tissues were taken These studies showed that: from the Apodemus tissue collection of J.R. Michaux • The Oriental regions were probably a refuge for these (JRM-numbers) and the mammal tissue collection species during Quaternary glaciations. housed at the Institut des Sciences de lÕEvolution de • Turkish and Near Eastern populations seem to be Montpellier (Catzeflis, 1991; T-numbers). more or less genetically differentiated from the Euro- A large portion of the cytochrome b (972 bp) was pean ones. This suggests that they were separated at amplified using the universal PCR primers L7 (50-A different historical periods probably following allo- CCAATGACATGAAAAATCATCGTT-30) and H16 patric isolations, which were reinforced by the pres- (50-A CATGAATYGGAGGYCAACCWG-30) (Kocher ence of strong biogeographic barriers such as the et al., 1989). Amplification reactions were carried out in Black Sea and the Caucasus. However, these different 2 Â 50 ll volumes including 25 ll of each 2 lM primer, historical factors as well as the impact of such bioge- 20 ll of 1 mM dNTP, 10 llof10Â reaction buffer, 10 ll ographic barriers were not well determined, due to of purified water, and 0.2 llof5U/ll Promega Taq sampling bias in the Oriental populations. Therefore, DNA polymerase. Approximately 200 ng of DNA ex- the eastern regions need to be examined more thor- tract was used per PCR amplification. Amplifications oughly. were performed in a Labover PTC100 thermal cycler • The three studied species do not seem to have re- employing 33 cycles (20 s at 94 °C, 30 s at 50 °C, and colonised Northern Europe from Oriental popula- 1 min 30 s at 68 °C) with a final extension cycle of 10 min tions after the last ice age, probably because of at 68 °C. PCR products were purified using the Ultra- the presence of the Black Sea and the Caucasus, free DA Amicon kit (Millipore). Both strands were but also because they were probably prevented by sequenced using a Bigdye terminator (Applied Biosys- the more rapid expansion of the Iberian, Italian tems) sequencing kit and run on an ABI 310 (Applied or Balkan genomes, that filled the northern areas Biosystems) automated sequencer. first. In order to contribute to a better knowledge of the 2.3. Sequence alignment phylogeography of European mammals, a small rodent, the yellow-necked fieldmouse (Apodemus flavicollis), has Previously published (Michaux et al., 2003) cyto- been studied all over the Western Palearctic region, chrome b sequences for A. flavicollis (n ¼ 3: ABO32853, with a particular emphasis on Turkey and the Near AF159392, and AF127539), Apodemus sylvaticus (n ¼ 2: East. It has several advantages for phylogeographic AJ311148 and AJ311149), and Apodemus mystacinus studies. This species is a forest species that has been (n ¼ 2: AJ311146 and AJ311147) were downloaded present in the Western Palearctic region for the past from GenBank (see Table 1) and aligned with the new three millions years (My) (Michaux and Pasquier, sequences using the ED editor (MUST package, 1974). Therefore, like the forests, it probably survived Philippe, 1993). during the Quaternary glaciations through important fluctuations in its distribution area. The yellow-necked 2.4. Sequence analyses fieldmouse is relatively common, easy to catch and widespread all over the Western Palearctic region as The aligned sequences were analysed by distance well as in the Near East regions. Finally, the evolu- (neighbour joining, NJ; Saitou and Nei, 1987) and tionary history of the genus Apodemus is well known maximum parsimony (MP) (Fitch, 1971) methods. The (Michaux and Pasquier, 1974; Michaux et al., 1997; distance analyses were performed using the ME criterion, Michaux et al., 2002; Serizawa et al., 2000; Suzuki KimuraÕs 2 parameter (K2P) and General Time Revers- et al., 2000), this genus being well represented in the ible (GTR) models (chosen with Modeltest 3.0, Posada fossil data. Therefore, calibration of a molecular clock and Crandall, 1998). These analyses were developed is possible for this genus. assuming a gamma distribution for substitution rates 790 J.R. Michaux et al. / Molecular Phylogenetics and Evolution 32 (2004) 788–798 Table 1 Geographic distribution and references of Apodemus tissues Geographic origin Total number Sample symbols Tissue sample or GenBank accession of animals (see Figs. 1–3) numbers A. flavicollis Austria Vorarlberg 3 A JRM-202 to JRM-204 Belarus Berezina Biosphere reserve 3 Bel JRM-313 to JRM-315 Belgium Ardennes 1 B JRM-332 Czech Republic Kasperske 4 CZ1 JRM-373, JRM-377, JRM-378, JRM-379 Nove Mlyny, Beeclav (Bohemia) 1 CZ2 JRM-255 Estonia Tallin 2 Es JRM-316,