Aim We compared the genetic variability and phylogeographical structure of three sympatric clades of annual killifishes (the Nothobranchius furzeri complex, N. orthonotus complex and N. rachovii complex) inhabiting annually desiccating savanna pools. Hypotheses on the mechanisms affecting intraspecific structure and speciation were tested. Location Temporary pools in Mozambique (Africa). Methods The study is based on spatially detailed samples covering the entire range of all three species complexes. A set of 12–13 microsatellites (1638 individuals, 96 populations) and cytochrome b sequences (463 fish, 152 populations) were used as genetic markers. Phylogenetic and population genetic approaches were used to describe the spatial genetic structure and to test the respective roles of river channels and river basins on diversification. Results Profound genetic differentiation among populations was evident; some populations located only a few kilometres apart were genetically very distinct, suggesting a significant role of genetic drift and low dispersal ability. Large rivers (Zambezi, Save, Limpopo) formed major barriers to gene flow, with minor differences among the three complexes. Further, the demographic expansion of previously isolated lineages was often limited by the river channel, and rivers were also confirmed as factors affecting speciation events. River basins and elevational gradient had a smaller, but non‐negligible, role in population structuring. Main conclusions River channels are the main barriers to gene flow in Nothobranchius fishes. The study demonstrated low dispersal ability and congruence in the phylogeographical pattern of all three complexes. Cases where Nothobranchius appear to have crossed river channels result from the dynamics of river morphology rather than from rare dispersal events. This conclusion is supported by simultaneous crossing events across lineages. A further division, also consistent among the three complexes, was detected between drier inland and wetter coastal areas. The phylogeographical pattern of Nothobranchius is unique in that it combines features of both aquatic and terrestrial taxa.

BackgroundIntraspecific genetic variation of African fauna has been significantly affected by pronounced climatic fluctuations in Plio-Pleistocene, but, with the exception of large mammals, very limited empirical data on diversity of natural populations are available for savanna-dwelling animals. Nothobranchius furzeri is an annual fish from south-eastern Africa, inhabiting discrete temporary savannah pools outside main river alluvia. Their dispersal is limited and population processes affecting its genetic structure are likely a combination of those affecting terrestrial and aquatic taxa. N. furzeri is a model taxon in ageing research and several populations of known geographical origin are used in laboratory studies. Here, we analysed the genetic structure, diversity, historical demography and temporal patterns of divergence in natural populations of N. furzeri across its entire distribution range.ResultsGenetic structure and historical demography of N. furzeri were analysed using a combination of mitochondrial (partial cytochrome b sequences, 687 bp) and nuclear (13 microsatellites) markers in 693 fish from 36 populations. Genetic markers consistently demonstrated strong population structuring and suggested two main genetic groups associated with river basins. The split was dated to the Pliocene (>2 Mya). The northern group inhabits savannah pools across the basin of the intermittent river Chefu in south-western Mozambique and eastern Zimbabwe. The southern group (from southernmost Mozambique) is subdivided, with the River Limpopo forming a barrier (maximum divergence time 1 Mya). A strong habitat fragmentation (isolated temporary pools) is reflected in significant genetic structuring even between adjacent pools, with a major influence of genetic drift and significant isolation-by-distance. Analysis of historical demography revealed that the expansion of both groups is ongoing, supported by frequent founder effects in marginal parts of the range and evidence of secondary contact between Chefu and Limpopo populations.ConclusionsWe demonstrated: (1) ancient (pre-Pleistocene) divergence between the two main N. furzeri lineages, their recent secondary contact and lack of reproductive isolation; (2) important genetic structuring attributed to the fragmented nature of their environment and isolation-by-distance, suggesting that dispersal is limited, occurs over short distances and is not directly associated with river routes; (3) an apparent role of the River Limpopo as a barrier to dispersal and gene flow.

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