Annual Nothobranchius fishes are distributed in East and Southern Africa and inhabit ephemeral pools filled during the monsoon season. Nothobranchius show extreme life-history adaptations: embryos survive by entering diapause and they are the vertebrates with the fastest maturation and the shortest lifespan. The distribution of Nothobranchius overlaps with the East Africa Rift System. The geological and paleoclimatic history of this region is known in detail: in particular, aridification of East Africa and expansion of grassland habitats started 8 Mya and three humid periods between 3 and 1 Mya are superimposed on the longer-term aridification. These climatic oscillations are thought to have shaped evolution of savannah African mammals. We reconstructed the phylogeny of Nothobranchius and dated the different stages of diversification in relation to these paleoclimatic events.
We sequenced one mitochondrial locus and five nuclear loci in 63 specimens and obtained a robust phylogeny. Nothobranchius can be divided in four geographically separated clades whose boundaries largely correspond to the East Africa Rift system. Statistical analysis of dispersal and vicariance identifies a Nilo-Sudan origin with southwards dispersion and confirmed that these four clades are the result of vicariance events In the absence of fossil Nothobranchius, molecular clock was calibrated using more distant outgroups (secondary calibration). This method estimates the age of the Nothobranchius genus to be 8.3 (6.0 – 10.7) My and the separation of the four clades 4.8 (2.7-7.0) Mya. Diversification within the clades was estimated to have started ~3 Mya and most species pairs were estimated to have an age of 0.5-1 My.
The mechanism of Nothobranchius diversification was allopatric and driven by geographic isolation. We propose a scenario where diversification of Nothobranchius started in rough coincidence with aridification of East Africa, establishment of grassland habitats and the appearance of the typical African bovid fauna of the savannah. Although confidence intervals for the estimated ages of the four Nothobranchius clades are quite large, this scenario is compatible with the biology of extant Nothobranchius that are critically dependent on savannah habitats. Therefore, Nothobranchius diversification might have been shaped by the same paleoclimatic events that shaped African ungulate evolution.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0210-3) contains supplementary material, which is available to authorized users.
Killifish; allopatric speciation; life history evolution; evolution of aging; Africa biogeography
Annual fish of the genus Nothobranchius show large variations in lifespan and expression of age-related phenotypes between closely related populations. We studied N. kadleci and its sister species N. furzeri GRZ strain, and found that N.kadleci is longer-lived than the N. furzeri. Lipofuscin and apoptosis measured in the liver increased with age in N. kadleci with different profiles: lipofuscin increased linearly, while apoptosis declined in the oldest animals. More lipofuscin (P < 0.001) and apoptosis (P < 0.001) was observed in N. furzeri than in N. kadleci at 16w age. Lipofuscin and apoptotic cells were then quantified in hybrids from the mating of N. furzeri to N. kadleci. F1 individuals showed heterosis for lipofuscin but additive effects for apoptosis. These two age-related phenotypes were not correlated in F2 hybrids. Quantitative trait loci analysis of 287 F2 fish using 237 markers identified two QTL accounting for 10% of lipofuscin variance (P < 0.001) with overdominance effect. Apoptotic cells revealed three significant- and two suggestive QTL explaining 19% of variance (P < 0.001), showing additive and dominance effects, and two interacting loci. Our results show that lipofuscin and apoptosis are markers of different age-dependent biological processes controlled by different genetic mechanisms.
Nothobranchius; lifespan; lipofuscin; apoptosis; quantitative trait loci; aging
Intraspecific 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.
Genetic 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.
We 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.
Temporary pool; Phylogeography; Population genetics; Cyprinodontiformes; Senescence; Pluvials; Pleistocene climate changes; Dispersal; Founder effect; Killifish
Early 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.
We 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.
Our 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.
Ageing theory; Life history; Trade off; Nothobranchius; Lipofuscin
The African annual fish Nothobranchius furzeri has over recent years been established as a model species for ageing-related studies. This is mainly based on its exceptionally short lifespan and the presence of typical characteristics of vertebrate ageing. To substantiate its role as an alternative vertebrate ageing model, a transcript catalogue is needed, which can serve e.g. as basis for identifying ageing-related genes.
To build the N. furzeri transcript catalogue, thirteen cDNA libraries were sequenced using Sanger, 454/Roche and Solexa/Illumina technologies yielding about 39 Gb. In total, 19,875 protein-coding genes were identified and annotated. Of these, 71% are represented by at least one transcript contig with a complete coding sequence. Further, transcript levels of young and old fish of the strains GRZ and MZM-0403, which differ in lifespan by twofold, were studied by RNA-seq. In skin and brain, 85 differentially expressed genes were detected; these have a role in cell cycle control and proliferation, inflammation and tissue maintenance. An RNA-seq experiment for zebrafish skin confirmed the ageing-related relevance of the findings in N. furzeri. Notably, analyses of transcript levels between zebrafish and N. furzeri but also between N. furzeri strains differed largely, suggesting that ageing is accelerated in the short-lived N. furzeri strain GRZ compared to the longer-lived strain MZM-0403.
We provide a comprehensive, annotated N. furzeri transcript catalogue and a first transcriptome-wide insight into N. furzeri ageing. This data will serve as a basis for future functional studies of ageing-related genes.
Nothobranchius furzeri; Model fish species; Ageing; Transcriptome assembly; Transcript catalogue; Gene expression; RNA-seq
A comparison of the diversity of bacterial communities in the larval midgut and adult gut of the European forest cockchafer (Melolontha hippocastani) was carried out using approaches that were both dependent on and independent of cultivation. Clone libraries of the 16S rRNA gene revealed 150 operational taxonomic units (OTUs) that belong to 11 taxonomical classes and two other groups that could be classified only to the phylum level. The most abundant classes were β, δ and γ-proteobacteria, Clostridia, Bacilli, Erysipelotrichi and Sphingobacteria. Although the insect’s gut is emptied in the prepupal stage and the beetle undergoes a long diapause period, a subset of eight taxonomic classes from the aforementioned eleven were found to be common in the guts of diapausing adults and the larval midguts (L2, L3). Moreover, several bacterial phylotypes belonging to these common bacterial classes were found to be shared by the larval midgut and the adult gut. Despite this, the adult gut bacterial community represented a subset of that found in the larvae midgut. Consequently, the midgut of the larval instars contains a more diverse bacterial community compared to the adult gut. On the other hand, after the bacteria present in the larvae were cultivated, eight bacterial species were isolated. Moreover, we found evidence of the active role of some of the bacterial species isolated in food digestion, namely, the presence of amylase and xylanolytic properties. Finally, fluorescence in situ hybridization allowed us to confirm the presence of selected species in the insect gut and through this, their ecological niche as well as the metagenomic results. The results presented here elucidated the heterogeneity of aerobic and facultative bacteria in the gut of a holometabolous insect species having two different feeding habits.
Melanin-concentrating hormone receptor 1 (MCHR1) plays a significant role in regulation of energy balance, food intake, physical activity and body weight in humans and rodents. Several association studies for human obesity showed contrary results concerning the SNPs rs133072 (G/A) and rs133073 (T/C), which localize to the first exon of MCHR1. The variations constitute two main haplotypes (GT, AC). Both SNPs affect CpG dinucleotides, whereby each haplotype contains a potential methylation site at one of the two SNP positions. In addition, 15 CpGs in close vicinity of these SNPs constitute a weak CpG island. Here, we studied whether DNA methylation in this sequence context may contribute to population- and age-specific effects of MCHR1 alleles in obesity.
We analyzed DNA methylation of a 315 bp region of MCHR1 encompassing rs133072 and rs133073 and the CpG island in blood samples of 49 individuals by bisulfite sequencing. The AC haplotype shows a significantly higher methylation level than the GT haplotype. This allele-specific methylation is age-dependent. In young individuals (20–30 years) the difference in DNA methylation between haplotypes is significant; whereas in individuals older than 60 years it is not detectable. Interestingly, the GT allele shows a decrease in methylation status with increasing BMI, whereas the methylation of the AC allele is not associated with this phenotype. Heterozygous lymphoblastoid cell lines show the same pattern of allele-specific DNA methylation. The cell line, which exhibits the highest difference in methylation levels between both haplotypes, also shows allele-specific transcription of MCHR1, which can be abolished by treatment with the DNA methylase inhibitor 5-aza-2′-deoxycytidine.
We show that DNA methylation at MCHR1 is allele-specific, age-dependent, BMI-associated and affects transcription. Conceivably, this epigenetic regulation contributes to the age- and/or population specific effects reported for MCHR1 in several human obesity studies.
The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.
A genomic analysis of the annual fish Nothobranchius furzeri, a vertebrate with the shortest known life span in captivity and which may provide a new model organism for aging research.
The annual fish Nothobranchius furzeri is the vertebrate with the shortest known life span in captivity. Fish of the GRZ strain live only three to four months under optimal laboratory conditions, show explosive growth, early sexual maturation and age-dependent physiological and behavioral decline, and express aging related biomarkers. Treatment with resveratrol and low temperature significantly extends the maximum life span. These features make N. furzeri a promising new vertebrate model for age research.
To contribute to establishing N. furzeri as a new model organism, we provide a first insight into its genome and a comparison to medaka, stickleback, tetraodon and zebrafish. The N. furzeri genome contains 19 chromosomes (2n = 38). Its genome of between 1.6 and 1.9 Gb is the largest among the analyzed fish species and has, at 45%, the highest repeat content. Remarkably, tandem repeats comprise 21%, which is 4-12 times more than in the other four fish species. In addition, G+C-rich tandem repeats preferentially localize to centromeric regions. Phylogenetic analysis based on coding sequences identifies medaka as the closest relative. Genotyping of an initial set of 27 markers and multi-locus fingerprinting of one microsatellite provides the first molecular evidence that the GRZ strain is highly inbred.
Our work presents a first basis for systematic genomic and genetic analyses aimed at understanding the mechanisms of life span determination in N. furzeri.
Several lines of evidence indicate that the central cannabinoid receptor 1 (CNR1) as well as the major endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH), N-acylethanolamine-hydrolyzing acid amidase (NAAA) and monoglyceride lipase (MGLL) are implicated in mediating the orexigenic effects of cannabinoids. The aim of this study was to analyse whether nucleotide sequence variations in the CNR1, FAAH, NAAA and MGLL genes are associated with anorexia nervosa (AN).
We analysed the association of a previously described (AAT)n repeat in the 3' flanking region of CNR1 as well as a total of 15 single nucleotide polymorphisms (SNPs) representative of regions with restricted haplotype diversity in CNR1, FAAH, NAAA or MGLL in up to 91 German AN trios (patient with AN and both biological parents) using the transmission-disequilibrium-test (TDT). One SNP was additionally analysed in an independent case-control study comprising 113 patients with AN and 178 normal weight controls. Genotyping was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ARMS-PCR or using 3730xl capillary sequencers.
The TDT revealed no evidence for association for any of the SNPs or the (AAT)n repeat with AN (all two-sided uncorrected p-values > 0.05). The lowest p-value of 0.11 was detected for the A-allele of the CNR1 SNP rs1049353 for which the transmission rate was 59% (95% confidence interval 47%...70%). Further genotyping of rs1049353 in 113 additional independent patients with AN and 178 normal weight controls could not substantiate the initial trend for association (p = 1.00).
As we found no evidence for an association of genetic variation in CNR1, FAAH, NAAA and MGLL with AN, we conclude that genetic variations in these genes do not play a major role in the etiology of AN in our study groups.
NOD2 is an innate immune receptor for the bacterial cell wall component muramyl-dipeptide. Mutations in the leucine-rich repeat region of NOD2, which lead to an impaired recognition of muramyl-dipeptide, have been associated with Crohn disease, a human chronic inflammatory bowel disease. Tissue specific constitutive and inducible expression patterns of NOD2 have been described that result from complex regulatory events for which the molecular mechanisms are not yet fully understood.
We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa in vivo. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy.
The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.
DGAT2 is a promising candidate gene for obesity because of its function as a key enzyme in fat metabolism and because of its localization on chromosome 11q13, a linkage region for extreme early onset obesity detected in our sample.
We performed a mutation screen in 93 extremely obese children and adolescents and 94 healthy underweight controls. Association studies were performed in samples of up to 361 extremely obese children and adolescents and 445 healthy underweight and normal weight controls. Additionally, we tested for linkage and performed family based association studies at four common variants in the 165 families of our initial genome scan.
The mutation screen revealed 15 DNA variants, four of which were coding non-synonymous exchanges: p.Val82Ala, p.Arg297Gln, p.Gly318Ser and p.Leu385Val. Ten variants were synonymous: c.-9447A > G, c.-584C > G, c.-140C > T, c.-30C > T, IVS2-3C > G, c.812A > G, c.920T > C, IVS7+23C > T, IVS7+73C > T and *22C > T. Additionally, the small biallelic trinucleotide repeat rs3841596 was identified. None of the case control and family based association studies showed an association of investigated variants or haplotypes in the genomic region of DGAT2.
In conclusion, our results do not support the hypothesis of an important role of common genetic variation in DGAT2 for the development of obesity in our sample. Anyhow, if there is an influence of genetic variation in DGAT2 on body weight regulation, it might either be conferred by the less common variants (MAF < 0.1) or the detected, rare non-synonymous variants.
Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents.
By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays.
Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.
Defensins are important components of innate immunity to combat bacterial and viral infections, and can even elicit antitumor responses. Clusters of defensin (DEF) genes are located in a 2 Mb range of the human chromosome 8p23.1. This DEF locus, however, represents one of the regions in the euchromatic part of the final human genome sequence which contains segmental duplications, and recalcitrant gaps indicating high structural dynamics.
We find that inter- and intraindividual genetic variations within this locus prevent a correct automatic assembly of the human reference genome (NCBI Build 34) which currently even contains misassemblies. Manual clone-by-clone alignment and gene annotation as well as repeat and SNP/haplotype analyses result in an alternative alignment significantly improving the DEF locus representation. Our assembly better reflects the experimentally verified variability of DEF gene and DEF cluster copy numbers. It contains an additional DEF cluster which we propose to reside between two already known clusters. Furthermore, manual annotation revealed a novel DEF gene and several pseudogenes expanding the hitherto known DEF repertoire. Analyses of BAC and working draft sequences of the chimpanzee indicates that its DEF region is also complex as in humans and DEF genes and a cluster are multiplied. Comparative analysis of human and chimpanzee DEF genes identified differences affecting the protein structure. Whether this might contribute to differences in disease susceptibility between man and ape remains to be solved. For the determination of individual DEF gene repertoires we provide a molecular approach based on DEF haplotypes.
Complexity and variability seem to be essential genomic features of the human DEF locus at 8p23.1 and provides an ongoing challenge for the best possible representation in the human reference sequence. Dissection of paralogous sequence variations, duplicon SNPs ans multisite variations as well as haplotypes by sequencing based methods is the way for future studies of interindividual DEF locus variability and its disease association.
The African annual fish Nothobranchius furzeri emerged as a new model for age research over recent years. Nothobranchius furzeri show an exceptionally short lifespan, age-dependent cognitive/behavioral decline, expression of age-related biomarkers, and susceptibility to lifespan manipulation. In addition, laboratory strains differ largely in lifespan. Here, we set out to study the genetics of lifespan determination. We crossed a short- to a long-lived strain, recorded lifespan, and established polymorphic markers. On the basis of genotypes of 411 marker loci in 404 F2 progeny, we built a genetic map comprising 355 markers at an average spacing of 5.5 cM, 22 linkage groups (LGs) and 1965 cM. By combining marker data with lifespan values, we identified one genome-wide highly significant quantitative trait locus (QTL) on LG 9 (P < 0.01), which explained 11.3% of the F2 lifespan variance, and three suggestive QTLs on LG 11, 14, and 17. We characterized the highly significant QTL by synteny analysis, because a genome sequence of N. furzeri was not available. We located the syntenic region on medaka chromosome 5, identified candidate genes, and performed fine mapping, resulting in a c. 40% reduction of the initial 95% confidence interval. We show both that lifespan determination in N. furzeri is polygenic, and that candidate gene detection is easily feasible by cross-species analysis. Our work provides first results on the way to identify loci controlling lifespan in N. furzeri and illustrates the potential of this vertebrate species as a genetic model for age research.
lifespan; Nothobranchius furzeri; genetic linkage map; QTL mapping; synteny