Phylogeography and historical demography play crucial roles in explaining species distributions and are instrumental in revealing episodes of fragmentation and subsequent expansions connected to environmental and climatic fluctuations [1, 2]. Most phylogeographic research has been focused on the Northern Hemisphere, often linked to the Pleistocene glacial cycles. Our knowledge of biogeographical patterns and processes in the Southern Hemisphere is relatively poor , and it is especially marked in non-forest biomes. In sub-Saharan Africa studies on large mammals living in savannah have identified large-scale phylogeographic patterns and provided a basic understanding of the major biogeographic regions at a continental scale , though studies with small-scale resolution are still rare [5–8].
In Africa, Pleistocene climatic fluctuations have resulted in more arid conditions during glacial periods, as large masses of water were deposited in continental glaciers, alternating with more mesic conditions during interglacials (termed pluvials in the tropics). Thus African ecosystems have undergone a series of aridification events, accompanied by spreading savannah habitats, since the Pliocene, with relatively wetter periods between 2.7-2.5 Mya, 1.9-1.7 Mya and 1.1-0.9 Mya . Further aridification is documented in the late Pleistocene and, notably, the last interpluvial (130–114 kya) was coincident with a series of extreme droughts in eastern Africa . Studies on large savannah-dwelling mammals demonstrated their less pronounced phylogeographic structure and high level of genetic variation in Southern Africa, suggesting a long-standing persistence of large savannahs in this part of the continent (reviewed in ). However, research from temperate areas in the northern latitudes led to understanding that large and highly mobile animals are not ideal taxa to specify the location and character of past refugia (e.g. ). On the contrary, species with very low dispersal ability and strong dependence on particular habitat have provided much more detailed picture of phylogeographical patterns and processes, for example the identification of unexpected cryptic refugia (e.g. ) or “refugia within refugia” (e.g. [8, 13]).
Killifishes (Cyprinodontiformes) are an ancient fish clade with a predominantly Gondwanan distribution and high species richness in Africa . African killifish (family Nothobranchiidae) include several clades that show radiations in specific habitat conditions, such as small rain forest streams, margins of large rivers or brackish zones . A notable clade, comprising the genus Nothobranchius (59 species; ) from East Africa and related monotypic genera Pronothobranchius and Fundulosoma from West Africa, has adapted to a unique environment of temporary savannah pools . The pools inhabited by Nothobranchius are completely separated from river systems and typically not inhabited by other fishes except lungfish (Protopterus spp.). The pools are strictly seasonal and filled with rainwater at the beginning of the rainy season. Pools desiccate during the dry season, with the duration of pools depending on local climate and ranging from 3–11 months [15, 17, 18]. In some years, a major flooding may connect several pools via flooded savannah and may also connect pools with intermittent streams, though such events appear to be rare. Importantly, specific soil conditions (the presence of alluvial vertisols) are necessary for the occurrence of Nothobranchius [17, 19], limiting dispersal and occurrence of Nothobranchius across the savannah to specific habitat patches. Therefore, population processes affecting genetic structure of Nothobranchius are predicted to be a combination of processes that are unique to Nothobranchius and these affecting terrestrial and aquatic taxa.
All Nothobranchius species are annual. Fish hatch soon after a pool fills with water [15, 20], grow rapidly, achieve sexual maturity within a few weeks , and reproduce daily thereafter . Adult lifespan is limited by habitat desiccation, with the following generation surviving in the form of diapaused embryos in desiccation-resistant egg envelopes encased in dry mud . Populations of Nothobranchius seldom co-occur with other teleost fish species . Dispersal by Nothobranchius may be limited to occasional large-scale floods in years with unusually high rainfall, which may transport live fish among savannah pools. Alternatively, eggs encased in mud may be carried attached to the bodies of large herbivores, as reported for aquatic invertebrates and macrophytes , or possibly by waterbirds.
Here we use Nothobranchius furzeri Jubb to investigate phylogeographic patterns and demographic processes in this group of annual, savannah-adapted fish. Its rapid development, short lifespan and age-dependent deterioration of physiological functions have made it a valuable vertebrate model organism in ageing research . In contrast to that, little is known about natural populations . Nothobranchius furzeri is distributed in southern Mozambique , with a single locality known in Zimbabwe (Sazale Pan at Gona Re Zhou National Park, Chefu basin, close to the Mozambican border) . Within its geographical range this species is restricted to pools associated with vertisol soils, while it is absent from pools associated with sandy and lateritic soils [17, 19]. Previous research on the phylogenetic relationships of Mozambican Nothobranchius included samples from several N. furzeri populations . The mitochondrial marker COI (cytochrome oxidase I) suggested high spatial structuring within the range of all species investigated, but the three nuclear sequences used in the study (GHITM, Cx32.2, PNP) had insufficient power to resolve intraspecific relationships.
Here, we provide the first detailed study of the phylogeographic patterns and demographic processes in N. furzeri, covering the entire distribution range of the species using a combination of mitochondrial gene CYTB (cytochrome b) and 13 nuclear microsatellite markers. Specifically, we tested following hypotheses: (1) N. furzeri is composed of three reproductively isolated clades (sensu ), with no secondary contact among them. (2) The main divergence was influenced by Plio-Pleistocene climate changes with allopatric diversification due to savannah fragmentation during pluvials. (3) The populations of savannah-dwelling Nothobranchius fishes have expanded during last interpluvial, together with their preferred habitat. (4) Genetic diversity will be lower and genetic structure higher on the range periphery, where populations are exposed to more frequent extinction-recolonization processes associated with strong genetic drift.