Turquoise killifish, Nothobranchius furzeri, have an intrinsically short life span, with a median life span of <6 months and a maximum (90%) life span of 9 months. This short life span, which is unique among vertebrates, evolved naturally and has resulted in N. furzeri becoming a widely used laboratory model species in aging research and other disciplines. Here, we describe a protocol for the maintenance and breeding of the species under laboratory conditions. We provide details for egg incubation, hatching, everyday care of juvenile and adult fish, breeding and treatment of most common diseases. Emphasis is given to the fact that the requirements of N. furzeri substantially differ from those of other fish model taxa; N. furzeri live brief lives and in nature undergo nonaquatic embryo development, with consequences for their laboratory culture.

Intensive collection in southern Mozambique across and outside the potential range of Nothobranchius furzeri, the species with the shortest recorded life span among vertebrates used as a model in ageing research, revealed that, contrary to previous data, it is a widespread species. It occurs in small freshwater pools south of the Save River and north of the Incomati River, including basins of the Limpopo, Changane, Chefu, Mazimechopes and Vaneteze Rivers. During collection in February 2008 (the second part of the rainy season), populations were strongly female biased (mean, 28% of males across 19 populations), and there was a spatial pattern in female bias among metapopulations. Populations varied in the proportion of male colour morphs. Fourteen populations were composed exclusively of the red male phenotype, three populations of the yellow male phenotype and 12 populations were mixed. Overall, the red phenotype was more common, but there was strong geographical variation in morph proportion, with yellow males more abundant at the periphery and red male dominance in the centre of the range of N. furzeri in the Limpopo basin. Nothobranchius furzeri was sympatric with Nothobranchius orthonotus (35% of investigated pools) and Nothobranchius rachovii (27% of sites). Analysis of habitat use of N. furzeri is presented; N. furzeri was associated with pools containing a soft muddy substratum and turbid water.

BackgroundExtreme environmental conditions can give rise to extreme adaptations. We document growth, sexual maturation and fecundity in two species of African annual fish inhabiting temporary savanna pools.ResultsNothobranchius kadleci started to reproduce at the age of 17 days and size of 31 mm and Nothobranchius furzeri at 18 days and 32 mm. All four study populations demonstrated rapid growth rates of up to 2.72 mm/day (23.4% of their total length). Both species may produce diapausing embryos or embryos that are able to hatch in as few as 15 days, resulting in a minimum generation time as short as only one month. Incubation on the surface of damp peat moss results in high embryo survival (73%) and a high proportion of rapidly developing embryos (58%) that skip diapauses and hatch in less than 30 days. We further demonstrated that rapid growth and maturation do not compromise subsequent fecundity.ConclusionsOur data suggest that both species have the most rapid sexual maturation and minimum generation time of any vertebrate species, and that rapid maturity does not involve paedogenesis.

BackgroundEarly evolutionary theories of aging predict that populations which experience low extrinsic mortality evolve a retarded onset of senescence. Experimental support for this theory in vertebrates is scarce, in part for the difficulty of quantifying extrinsic mortality and its condition- and density-dependent components that –when considered- can lead to predictions markedly different to those of the “classical” theories. Here, we study annual fish of the genus Nothobranchius whose maximum lifespan is dictated by the duration of the water bodies they inhabit. Different populations of annual fish do not experience different strengths of extrinsic mortality throughout their life span, but are subject to differential timing (and predictability) of a sudden habitat cessation. In this respect, our study allows testing how aging evolves in natural environments when populations vary in the prospect of survival, but condition-dependent survival has a limited effect. We use 10 Nothobranchius populations from seasonal pools that differ in their duration to test how this parameter affects longevity and aging in two independent clades of these annual fishes.ResultsWe found that replicated populations from a dry region showed markedly shorter captive lifespan than populations from a humid region. Shorter lifespan correlated with accelerated accumulation of lipofuscin (an established age marker) in both clades. Analysis of wild individuals confirmed that fish from drier habitats accumulate lipofuscin faster also under natural conditions. This indicates faster physiological deterioration in shorter-lived populations.ConclusionsOur data provide a strong quantitative example of how extrinsic mortality can shape evolution of senescence in a vertebrate clade. Nothobranchius is emerging as a genomic model species. The characterization of pairs of closely related species with different longevities should provide a powerful paradigm for the identification of genetic variations responsible for evolution of senescence in natural populations.

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.

In ephemeral habitats, the same genotypes cope with unpredictable environmental conditions, favouring the evolution of developmental plasticity and alternative life-history strategies (ALHS). We tested the existence of intrapopulation ALHS in an annual killifish, Nothobranchius furzeri, inhabiting temporary pools. The pools are either primary (persisting throughout the whole rainy season) or secondary (refilled after desiccation of the initial pool), representing alternative niches. The unpredictable conditions led to the evolution of reproductive bet-hedging with asynchronous embryonic development. We used a common garden experiment to test whether the duration of embryonic period is associated with post-embryonic life-history traits. Fish with rapid embryonic development (secondary pool strategy, high risk of desiccation) produced phenotypes with more rapid life-history traits than fish with slow embryonic development (primary pool strategy). The fast fish were smaller at hatching but had larger yolk sac reserves. Their post-hatching growth was more rapid, and they matured earlier. Further, fast fish grew to a smaller body size and died earlier than slow fish. No differences in fecundity, propensity to mate or physiological ageing were found, demonstrating a combination of plastic responses and constraints. Such developmentally related within-population plasticity in life history is exceptional among vertebrates.

Life span and aging are substantially modified by natural selection. Across species, higher extrinsic (environmentally related) mortality (and hence shorter life expectancy) selects for the evolution of more rapid aging. However, among populations within species, high extrinsic mortality can lead to extended life span and slower aging as a consequence of condition-dependent survival. Using within-species contrasts of eight natural populations of Nothobranchius fishes in common garden experiments, we demonstrate that populations originating from dry regions (with short life expectancy) had shorter intrinsic life spans and a greater increase in mortality with age, more pronounced cellular and physiological deterioration (oxidative damage, tumor load), and a faster decline in fertility than populations from wetter regions. This parallel intraspecific divergence in life span and aging was not associated with divergence in early life history (rapid growth, maturation) or pace-of-life syndrome (high metabolic rates, active behavior). Variability across four study species suggests that a combination of different aging and life-history traits conformed with or contradicted the predictions for each species. These findings demonstrate that variation in life span and functional decline among natural populations are linked, genetically underpinned, and can evolve relatively rapidly.

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.