Molecular Ecology (2002) 11, 1717–1729 © 2002 Blackwell Science Ltd Blackwell Science, Ltd Late glacial history of the cold-adapted freshwater fish Cottus gobio, revealed by microsatellites B. HÄNFLING,* B. HELLEMANS,† F. A. M. VOLCKAERT † and G. R. CARVALHO * *Molecular Ecology and Fisheries Genetics Laboratory, Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK, †Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Ch. de Beriotstraat 32, B-3000 Leuven, Belgium Abstract The distribution of genetic diversity at 10 highly polymorphic microsatellite loci within the European freshwater fish, Cottus gobio, L. was examined. The sampling range comprised a large geographical scale including lineages known to be highly divergent at both mito- chondrial DNA (mtDNA) and allozymes. An analysis of genetic variability within popula- tions showed that expected heterozygosity and allelic richness could be explained largely by current effective population sizes. Evidence was found, however, that historical processes predating the last major glaciation affected allelic richness. In addition to confirming the large-scale patterns from earlier studies, the microsatellite data revealed new insights into recent processes by analysing genetic structure within ancient lineages defined by mtDNA data. Stepwise mutation model (SMM) and nonSMM-based methods demonstrated a clear genetic structuring within the Northwestern European lineage comprising popula- tions from Britain and Belgium, and within the Central European lineage populations from the rivers Danube, Elbe and Main. Supported by an analysis of genetic variability within populations these results showed that the bullhead populations most probably persisted throughout the last major glaciation within the British Isles and within the drain- ages of the rivers Elbe and Main. Such observations provide the first genetic evidence for a glacial refugium in such close proximity to the European glacial margins. Keywords: Cottidae, Europe, phylogeography, Pleistocene, sculpin, northern refugia Received 7 November 2001; revision received 25 April 2002; accepted 7 May 2002 Introduction Knowledge on the global impact of Quaternary climate and geography on the genetic structure of species and popula- tions has increased dramatically (Avise 2000; Hewitt 2000). Climatic fluctuations in addition to geographical and geo- logical modifications have affected the natural range and habitat of most species markedly throughout the Pleistocene. During cold periods, most species shifted southwards into so-called glacial refugia, whereas interglacials were followed by northerly range expansions (Hewitt 1996). The effects of glacial events have left a genomic signature, resulting in concordant phylogeographical structures among many species. For example, Hewitt (1999) identified three general phylogeographical patterns for terrestrial animal and plant species in Europe. Furthermore, population genetic theory predicts that processes associated with range shifts during the Pleistocene history of a species such as fragmentation, range expansion and reduced population size, will impact genetic variability. A loss of genetic diversity during post- glacial population expansion has been modelled (Ibrahim et al. 1996), and has also been shown empirically, for example in North American salmonid fishes (Bernatchez & Wilson 1998). Recently, an increasing number of papers have invest- igated the phylogeography of European primary fresh- water fishes using mitochondrial DNA (mtDNA) sequence analysis (Durand et al. 1999; Nesbø et al. 1999; Kotlik & Berrebi 2001) revealing in all cases distinct clades that dif- ferentiated hundreds of thousands of years ago. One such case is the bullhead, Cottus gobio, a small freshwater fish widely distributed across Europe (Lelek 1987), for which seven major clades of mtDNA haplotype lineage have been described (Englbrecht et al. 2000; Volckaert et al. 2002) across Europe. Although the precise dating of clade origins Correspondence: B. Hänfling. Fax: + 44 1482 465458; E-mail: b.haenfling@biosci.hull.ac.uk