Many studies have looked at the phylogeographic patterns of species, primarily using mitochondrial DNA sequence comparisons, and linked the patterns seen today to severe climatic changes in the past [1, 2]. During the Pleistocene (approximately 2.6 million to 12,000 years ago), Europe experienced cyclical glacial and interglacial periods, with the last glacial period ending approximately 10,000 years ago . These fluctuations in climate had profound effects on species distributions, and during glacial periods temperate species in Europe are thought to have been forced south into warmer refugial areas, primarily in Iberia, Italy and the Balkans, although other smaller cryptic refugia have also been proposed [3–6]. Following the retreat of the glaciers towards the North, species were able to recolonise the now warmer and more habitable northern regions of Europe. These patterns of range contraction and expansion have shaped the genetic diversity in modern populations through a combination of genetic drift and gene flow.
Within Europe, genetic studies on terrestrial species typically find divergent clades which represent the refugial origins of the focal species during the glacial periods [3, 4]. Many taxa show a deep split between Eastern and Western European clades, which likely reflects two main refugial origins in Iberia and the Italo-Balkan region , and generally speaking, we now have a good understanding of the routes of post-glacial expansions for various terrestrial species [1, 4](Hewitt, 1999, Hewitt, 2004). Furthermore, we also have a good understanding of how these expansions occurred; theory suggests that long-distance migrants become founders of new populations [7, 8], replacing the previous orthodoxy which suggested that expansions were a result of substantial genetic drift [9, 10]. A loss of genetic variability typically accompanies such range expansions, with populations that are further from a refugium tending to have lower genetic diversity .
However, carnivores in particular seem to show discordant patterns, with some species showing strong phylogeographical structuring, whilst others show no evidence of structuring. For example, population divergence is found in brown bears in Europe and North America [2, 11] and black bears in North America , whilst no such phylogeographical structuring is found in North American coyotes . Likewise, little evidence for partitioning of haplotypes at a regional or even continental scale has been found for another carnivore, the grey wolf [14, 15]. It seems probable that species with relatively high dispersal rates, combined with high adaptability to a range of habitats (i.e. high capabilities for migration and gene flow) may show less phylogeographical structuring. The swift fox, kit fox and arctic fox have also been shown to have little within-species phylogeographical structuring [16, 17], however these species are refined to relatively small, specific habitat regions and so might not be expected to show structure within such restricted areas (see http://www.canids.org for distribution maps).
We present a phylogeographical study within Europe of a species that is widespread throughout the Northern hemisphere; the red fox (Vulpes vulpes). Previous studies on the red fox have indicated that three distinct subclades exist in North America, reflecting two recent colonisation events from North to South, and one widespread clade representing an earlier colonisation event from South to North . However, an allozyme and cytochrome b study of modern red fox from ten locations in Southern Europe and Israel revealed little geographical structure in this limited region . Our study provides a substantially wider geographical coverage of Europe, and includes ancient DNA samples dating approximately up to 40,000 years old, allowing us to add a temporal component to the study. In this study we aimed to assess the evidence for separate clades that might be associated with different refugial origins, and whether there is any evidence for a population bottleneck and subsequent expansion (as might be related to the Last Glacial Maximum). In addition, we considered the impact of genetic drift, by looking for patterns of isolation by distance and time-associated changes in haplotypes. To approach these questions, we examined variation in two mitochondrial DNA (mtDNA) genes, cytochrome b and the control region. Cytochrome b (Cytb) is relatively conserved, and as such can be used to resolve deeper splits, whilst the control region harbours higher diversity and can provide higher resolution [20, 21]. The use of two mtDNA gene fragments allows us to have more confidence in the results, and to help to resolve any potential ambiguities. Mitochondrial DNA is the most appropriate marker in this instance as the mutation rate is rapid enough to provide variability over the time scales in question, and recombination is very unusual, enabling straightforward interpretation of the patterns observed .