Genetic differentiation among populations of the South African scrub hare Lepus saxalilis was examined using hypervariable mitochondrial DNA control region I (CR-I) sequences. Neighbour-joining analysis revealed a pattern that did not correspond to the current subspecies delineations. The CR-I sequence data delimit scrub hares into three major maternal lineages. The three phylogenetic assemblages exhibited different geographical distributions. AMOVA analyses and exact tests for population differentiation confirmed this phylogeographic partitioning. One lineage (SW) was confined to the south-western Cape, the second lineage (N) was exclusively found in the northern part of South Africa and in the neighbouring Countries, and the third lineage (C) was predominant in the central parts of South Africa. This spatial distribution did not coincide with the ranges of the 10 described Subspecies covered by our sampling regime. The lineages C and N overlapped in an area including eastern parts of South Africa and Southern Namibia. The presence of both lineages in that area of overlap was interpreted as the result of secondary contact due to recent range expansions after the two lineages had undergone a population restriction approximately 18 000 years ago. Analyses of contemporary gene flow disclosed an exchange of migrants between N and C, which was biased towards a movement from C to N. The SW group represents a very distinct evolutionary lineage that has been isolated for more than 45 000 years. It does not exchange female migrants with the other two groups. Mismatch distribution analyses indicated sudden Population size expansions in the history of all three populations.
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