Turnover of sex chromosomes in the Lake Tanganyika cichlid tribe Tropheini (Teleostei: Cichlidae).

Sex chromosome replacement is frequent in many vertebrate clades, including fish, frogs, and lizards. In order to understand the mechanisms responsible for sex chromosome turnover and the early stages of sex chromosome divergence, it is necessary to study lineages with recently evolved sex chromosomes. Here we examine sex chromosome evolution in a group of African cichlid fishes (tribe Tropheini) which began to diverge from one another less than 4 MYA. We have evidence for a previously unknown sex chromosome system, and preliminary indications of several additional systems not previously reported in this group. We find a high frequency of sex chromosome turnover and estimate a minimum of 14 turnovers in this tribe. We date the origin of the most common sex determining system in this tribe (XY-LG5/19) near the base of one of two major sub-clades of this tribe, about 3.4 MY ago. Finally, we observe variation in the size of one sex-determining region that suggests independent evolution of evolutionary strata in species with a shared sex-determination system. Our results illuminate the rapid rate of sex chromosome turnover in the tribe Tropheini and set the stage for further studies of the dynamics of sex chromosome evolution in this group.

Lack of host specialization despite selective host use in brood parasitic cuckoo catfish.

Host-parasite dynamics involve coevolutionary arms races, which may lead to host specialization and ensuing diversification. Our general understanding of the evolution of host specialization in brood parasites is compromised by a restricted focus on bird and insect lineages. The cuckoo catfish (Synodontis multipunctatus) is an obligate parasite of parental care of mouthbrooding cichlids in Lake Tanganyika. Given the ecological and taxonomic diversity of mouthbrooding cichlids in the lake, we hypothesized the existence of sympatric host-specific lineages in the cuckoo catfish. In a sample of 779 broods from 20 cichlid species, we found four species parasitized by cuckoo catfish (with prevalence of parasitism of 2%-18%). All parasitized cichlids were from the tribe Tropheini, maternal mouthbrooders that spawn over a substrate (rather than in open water). Phylogenetic analysis based on genomic (ddRAD sequencing) and mitochondrial (Dloop) data from cuckoo catfish embryos showed an absence of host-specific lineages. This was corroborated by analyses of genetic structure and co-ancestry matrix. Within host species, parasitism was not associated with any individual characteristic we recorded (parent size, water depth), but was costly as parasitized parents carried smaller clutches of their own offspring. We conclude that the cuckoo catfish is an intermediate generalist and discuss costs, benefits and constraints of host specialization in this species and brood parasites in general.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Good Practices for Histological Analysis of the Annual Killifish Nothobranchius furzeri (Nothobranchiidae).

Paraffin histology is one of the most important and commonly used laboratory techniques enabling the study of the microscopic structure of animal and plant tissues. This technique uses paraffin wax, which in liquid form impregnates fixed and dehydrated tissues and allows the preparation of thin sections when solidified in blocks. This protocol on good practices in paraffin histology of Nothobranchius furzeri (Nothobranchiidae) summarizes the authors' current experience in terms of technique, evaluation, and interpretation of sectioned tissues. The steps that precede paraffin block preparation are also presented as they play a key role in maximizing the quality of examined sections. The paraffin technique as described only requires basic laboratory conditions to produce good-quality results. The description of staining methods is limited to Mayer's hematoxylin and eosin (H&E), the routinely used histological dye staining cell nuclei in blue-black (hematein) and cell cytoplasm and connective tissue fibers in shades of pink-red (eosin). Killifish specialists are encouraged to engage in the study of histology and histopathology, taking advantage of interdisciplinary cooperation.

Patterns and drivers of Nothobranchius killifish diversity in lowland Tanzania.

Temporary pools are seasonal wetland habitats with specifically adapted biota, including annual Nothobranchius killifishes that survive habitat desiccation as diapausing eggs encased in dry sediment. To understand the patterns in the structure of Nothobranchius assemblages and their potential in wetland conservation, we compared biodiversity components (alpha, beta, and gamma) between regions and estimated the role and sources of nestedness and turnover on their diversity. We sampled Nothobranchius assemblages from 127 pools across seven local regions in lowland Eastern Tanzania over 2 years, using dip net and seine nets. We estimated species composition and richness for each pool, and beta and gamma diversity for each region. We decomposed beta diversity into nestedness and turnover components. We tested nestedness in three main regions (Ruvu, Rufiji, and Mbezi) using the number of decreasing fills metric and compared the roles of pool area, isolation, and altitude on nestedness. A total of 15 species formed assemblages containing 1-6 species. Most Nothobranchius species were endemic to one or two adjacent regions. Regional diversity was highest in the Ruvu, Rufiji, and Mbezi regions. Nestedness was significant in Ruvu and Rufiji, with shared core (N. melanospilus, N. eggersi, and N. janpapi) and common (N. ocellatus and N. annectens) species, and distinctive rare species. Nestedness apparently resulted from selective colonization rather than selective extinction, and local species richness was negatively associated with altitude. The Nothobranchius assemblages in the Mbezi region were not nested, and had many endemic species and the highest beta diversity driven by species turnover. Overall, we found unexpected local variation in the sources of beta diversity (nestedness and turnover) within the study area. The Mbezi region contained the highest diversity and many endemic species, apparently due to repeated colonizations of the region rather than local diversification. We suggest that annual killifish can serve as a flagship taxon for small wetland conservation.

Individual experience as a key to success for the cuckoo catfish brood parasitism.

Brood parasites are involved in coevolutionary arms races with their hosts, whereby adaptations of one partner elicit the rapid evolution of counter-adaptations in the other partner. Hosts can also mitigate fitness costs of brood parasitism by learning from individual or social experience. In brood parasites, however, the role of learning can be obscured by their stealthy behaviour. Cuckoo catfish (Synodontis multipunctatus) parasitise clutches of mouthbrooding cichlids in Lake Tanganyika and are the only non-avian obligate brood parasites among vertebrates. We experimentally demonstrate that cuckoo catfish greatly enhance their efficiency in parasitising their hosts as they learn to overcome host defences. With increasing experience, cuckoo catfish increased their parasitism success by greater efficiency through improved timing and coordination of intrusions of host spawnings. Hence, within the coevolutionary arms races, brood parasites learn to overcome host defences during their lifetime.

The sources of sex differences in aging in annual fishes.

Intersexual differences in life span (age at death) and aging (increase in mortality risk associated with functional deterioration) are widespread among animals, from nematodes to humans. Males often live shorter than females, but there is substantial unexplained variation among species and populations. Despite extensive research, it is poorly understood how life span differences between the sexes are modulated by an interplay among genetic, environmental and social factors. The goal of our study was to test how sex differences in life span and ageing are modulated by social and environmental factors, and by intrinsic differences between males and females. To disentangle the complex basis of sex differences in life span and aging, we combined comparative data from sex ratios in 367 natural populations of four species of African annual killifish with experimental results on sex differences in life span and aging from eight laboratory populations tested in treatments that varied social and environmental conditions. In the wild, females consistently outlived males. In captivity, sex-specific mortality depended on social conditions. In social-housed experimental groups, male-biased mortality persisted in two aggressive species, but ceased in two placid species. When social and physical contacts were prevented by housing all fish individually, male-biased mortality ceased in all four species. This outcome held across benign and challenging environmental conditions. Fitting demographic survival models revealed that increased baseline mortality was primarily responsible for a shorter male life span in social-housing conditions. The timing and rate of aging were not different between the sexes. No marker of functional aging we recorded in our study (lipofuscin accumulation, proliferative changes in kidney and liver) differed between males and females, despite their previously confirmed association with functional aging in Nothobranchius killifish. We show that sex differences in life span and aging in killifish are driven by a combination of social and environmental conditions, rather than differential functional aging. They are primarily linked to sexual selection but precipitated through multiple processes (predation, social interference). This demonstrates how sex-specific mortality varies among species even within an ecologically and evolutionary discrete lineage and explains how external factors mediate this difference.

Lifespan and telomere length variation across populations of wild-derived African killifish.

Telomeres and telomerase prevent the continuous erosion of chromosome-ends caused by lifelong cell division. Shortened telomeres are associated with age-related pathologies. While short telomere length is positively correlated with increased lethality at the individual level, in comparisons across species short telomeres are associated with long (and not short) lifespans. Here, we tested this contradiction between individual and evolutionary patterns in telomere length using African annual killifish. We analysed lifespan and telomere length in a set of captive strains derived from well-defined wild populations of Nothobranchius furzeri and its sister species, N. kadleci, from sites along a strong gradient of aridity which ultimately determines maximum natural lifespan. Overall, males were shorter-lived than females, and also had shorter telomeres. Male lifespan (measured in controlled laboratory conditions) was positively associated with the amount of annual rainfall in the site of strain origin. However, fish from wetter climates had shorter telomeres. In addition, individual fish which grew largest over the juvenile period possessed shorter telomeres at the onset of adulthood. This demonstrates that individual condition and environmentally-driven selection indeed modulate the relationship between telomere length and lifespan in opposite directions, validating the existence of inverse trends within a single taxon. Intraindividual heterogeneity of telomere length (capable to detect very short telomeres) was not associated with mean telomere length, suggesting that the shortest telomeres are controlled by regulatory pathways other than those that determine mean telomere length. The substantial variation in telomere length between strains from different environments identifies killifish as a powerful system in understanding the adaptive value of telomere length.

Selection on Phalanx Development in the Evolution of the Bird Wing.

The frameshift hypothesis is a widely accepted model of bird wing evolution. This hypothesis postulates a shift in positional values, or molecular-developmental identity, that caused a change in digit phenotype. The hypothesis synthesized developmental and paleontological data on wing digit homology. The "most anterior digit" (MAD) hypothesis presents an alternative view based on changes in transcriptional regulation in the limb. The molecular evidence for both hypotheses is that the MAD expresses Hoxd13 but not Hoxd11 and Hoxd12. This digit I "signature" is thought to characterize all amniotes. Here, we studied Hoxd expression patterns in a phylogenetic sample of 18 amniotes. Instead of a conserved molecular signature in digit I, we find wide variation of Hoxd11, Hoxd12, and Hoxd13 expression in digit I. Patterns of apoptosis, and Sox9 expression, a marker of the phalanx-forming region, suggest that phalanges were lost from wing digit IV because of early arrest of the phalanx-forming region followed by cell death. Finally, we show that multiple amniote lineages lost phalanges with no frameshift. Our findings suggest that the bird wing evolved by targeted loss of phalanges under selection. Consistent with our view, some recent phylogenies based on dinosaur fossils eliminate the need to postulate a frameshift in the first place. We suggest that the phenotype of the Archaeopteryx lithographica wing is also consistent with phalanx loss. More broadly, our results support a gradualist model of evolution based on tinkering with developmental gene expression.

Embryo ecology: Developmental synchrony and asynchrony in the embryonic development of wild annual fish populations.

Embryo-environment interactions are of paramount importance during the development of all organisms, and impacts during this period can echo far into later stages of ontogeny. African annual fish of the genus Nothobranchius live in temporary pools and their eggs survive the dry season in the dry bottom substrate of the pools by entering a facultative developmental arrest termed diapause. Uniquely among animals, the embryos (encased in eggs) may enter diapause at three different developmental stages. Such a system allows for the potential to employ different regulation mechanisms for each diapause. We sampled multiple Nothobranchius embryo banks across the progressing season, species, and populations. We present important baseline field data and examine the role of environmental regulation in the embryonic development of this unique system. We describe the course of embryo development in the wild and find it to be very different from the typical development under laboratory conditions. Development across the embryo banks was synchronized within and across the sampled populations with all embryos entering diapause I during the rainy season and diapause II during the dry season. Asynchrony occurred at transient phases of the habitat, during the process of habitat desiccation, and at the end of the dry season. Our findings reveal the significance of environmental conditions in the serial character of the annual fish diapauses.

Histopathology of laboratory-reared Nothobranchius fishes: Mycobacterial infections versus neoplastic lesions.

Short-lived killifishes of the genus Nothobranchius Peters, 1868 (Cyprinodontiformes) are considered promising model organisms for biomedical research on ageing and tumorigenesis. We conducted histopathological analysis of 411 adult individuals from three Nothobranchius species to study details on spontaneous age-related neoplastic lesions. Light microscopy based on H&E and toluidine blue-stained sections revealed (a) non-proliferative liver changes with pronounced vacuolation of hepatocytes; (b) proliferation of kidney haemopoietic tissue contributing to excretory system damage; (c) proliferation of splenic mononuclear haemoblasts accompanied by reduced erythropoiesis; (d) proliferation of mononuclear cell aggregates in the liver parenchyma; and (e) rare occurrence of hepatocellular adenomas. Ziehl-Neelsen (ZN) staining revealed that the proliferative lesions are a host defence response to mycobacterial infections manifested by activation of the mononuclear phagocytic system and atypical granulomatous inflammatory reaction. 16S rRNA analysis identified three species of Mycobacterium in our samples. Our findings turn attention to lesions which mimic neoplasms by their gross appearance and question the light microscopic interpretation of lesions unless differential ZN staining is included. Beyond the limitations of our morphological approach, the intensity of mycobacterial infections is a challenging opportunity for research into the molecular-genetic background of the mononuclear phagocytic system reaction in Nothobranchius killifish.

Spontaneous adenocarcinoma of the gas gland in Nothobranchius fishes.

Nothobranchius fishes (Cyprinodontiformes), known for their genetically encoded extremely compressed lifespan, are considered an excellent vertebrate model for the research of aging. Unlike the rapid accumulation of data concerning their biology, ecology and genome, knowledge of their age-related diseases, including tumours, is still very limited. This Note reports spontaneous neoplastic lesions in the swim bladder gas glands of Nothobranchius furzeri, N. kadleci and N. orthonotus. Based on light and transmission electron microscopy, the neoplastic proliferation of gas gland cells was classified as adenocarcinoma. There was a concurrent proliferation of haemopoietic cells in the kidney interstitium in all individuals diagnosed with this type of primary neoplasia.

Genetic diversity of a widespread annual killifish from coastal Tanzania.

BACKGROUND: African annual killifishes (Nothobranchius spp.) are adapted to seasonally desiccating habitats (ephemeral pools), surviving dry periods as dormant eggs. Given their peculiar life history, geographic aspects of their diversity uniquely combine patterns typical for freshwater taxa (river basin structure and elevation gradient) and terrestrial animals (rivers acting as major dispersal barriers). However, our current knowledge on fine-scale inter-specific and intra-specific genetic diversity of African annual fish is limited to a single, particularly dry region of their distribution (subtropical Mozambique). Using a widespread annual killifish from coastal Tanzania and Kenya, we tested whether the same pattern of genetic divergence pertains to a wet equatorial region in the centre of Nothobranchius distribution. RESULTS: In populations of Nothobranchius melanospilus species group across its range, we genotyped a part of mitochondrial cytochrome oxidase subunit 1 (COI) gene (83 individuals from 22 populations) and 10 nuclear microsatellite markers (251 individuals from 16 populations). We found five lineages with a clear phylogeographic structure but frequent secondary contact. Mitochondrial lineages were largely congruent with main population genetic clusters identified on microsatellite markers. In the upper Wami basin, populations are isolated as a putative Nothobranchius prognathus, but include also a population from a periphery of the middle Ruvu basin. Other four lineages (including putative Nothobranchius kwalensis) coexisted in secondary contact zones, but possessed clear spatial pattern. Main river channels did not form apparent barriers to dispersal. The most widespread lineage had strong signal of recent population expansion. CONCLUSIONS: We conclude that dispersal of a Nothobranchius species from a wet part of the genus distribution (tropical lowland) is not constrained by main river channels and closely related lineages frequently coexist in secondary contact zones. We also demonstrate contemporary connection between the Ruvu and Rufiji river basins. Our data do not provide genetic support for existence of recently described cryptic species from N. melanospilus complex, but cannot resolve this issue.

Author Correction: From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

From taxonomic deflation to newly detected cryptic species: Hidden diversity in a widespread African squeaker catfish.

Cryptic genetic diversity and erroneous morphological species determination represent frequent problems in biodiversity research. Here, examination of 138 specimens of Synodontis (Mochokidae, Siluriformes) from the Nile River and Lake Turkana revealed the presence of both S. schall-like and S. frontosus-like morphotypes, with a phenotypic gradient between them. We concluded phylogenetic and population genetic analyses based on two mitochondrial and one nuclear marker including 131 coxI (565 bp), 96 cytb (973 bp) and 19 RAG2 (896 bp) sequences from the Nile-Turkana population, plus additional GenBank data of Synodontis spp. Whilst nuclear data were inconclusive, mitochondrial sequences suggested that both morphotypes and intermediate forms are conspecific. The results imply probable synonymy of S. frontosus with S. schall. Conversely, a strong biogeographical signal was revealed among widely distributed and supposedly conspecific S. schall-like catfish of the Nilo-Sudanian ichthyological province. Synodontis schall sensu stricto (=Eastern clade), as defined by type locality in the Nile, is apparently restricted to the eastern part of the Nilo-Sudanian ichthyological province (e.g. Nile, Turkana, Chad). Synodontis schall Western clade (Senegambia, Niger, Chad) most probably represents a cryptic taxon, unrecognized thus far due to the absence of distinctive morphological differences.

Morphological and molecular characterization of Apatemon sp. infecting killifish in Mozambique.

Strigeid trematodes of the genus Apatemon Szidat, 1928 are intestinal parasites of fish-eating birds, utilizing various fish species as second intermediate hosts. In this study, we report morphometrical and molecular characterization of Apatemon sp. metacercariae parasitizing killifish Nothobranchius furzeri (Cyprinodontiformes: Nothobranchiidae) in south-east Mozambique. Metacercariae obtained from the cerebral cavity of killifish and two adult individuals isolated from experimentally infected ducklings were used for detailed morphological and molecular description, both resulting in generic affiliation to Apatemon. This is the first molecularly confirmed record of this trematode genus in Africa. Considering the morphological variability and wide host range of individual Apatemon species, the combination of both morphological and molecular analyses is indispensable for valid identification of this parasite. The results of our molecular analysis together with phylogenetic reconstruction indicated the presence of a new African lineage, reflecting potentially high diversity within the genus Apatemon comparable with other digenean genera.

Rapid growth and large body size in annual fish populations are compromised by density-dependent regulation.

We tested the effect of population density on maximum body size in three sympatric species of annual killifishes Nothobranchius spp. from African ephemeral pools. We found a clear negative effect of population density on body size, limiting their capacity for extremely fast development and rapid growth. This suggests that density-dependent population regulation and the ephemeral character of their habitat impose contrasting selective pressures on the life history of annual killifishes.

Limited scope for reproductive senescence in wild populations of a short-lived fish.

Senescence in wild populations was long considered negligible but current evidence suggests that it is widespread in natural populations of mammals and birds, affecting the survival and reproductive output of older individuals. In contrast, little is known about reproductive senescence in species with asymptotic growth that can keep increasing their reproductive output as they grow older and larger. Using a cross-sectional study, we tested age-related decline in fecundity and relative allocation to reproduction in five wild populations of an annual killifish, Nothobranchius furzeri (Cyprinodontiformes). We did not detect any decline in absolute female egg production over their short lifespan in the wild. Relative fecundity (egg production controlled for female body mass) tended to decrease with age. This effect was driven primarily by a single population that survived 17 weeks, almost twice as long as the median persistence of the other four study populations. There was no decrease in relative ovary mass while in males, relative testes mass actually increased with age. Intra-population variation in relative ovary mass increased in older females suggesting heterogeneity in individual trajectories of female reproductive allocation. Overall, we demonstrate that annual killifish do not experience significant age-related decline in reproductive functions during their very short lifespan in the wild despite the marked deterioration of gonad tissue detected in captivity.

Success of cuckoo catfish brood parasitism reflects coevolutionary history and individual experience of their cichlid hosts.

Obligate brood parasites manipulate other species into raising their offspring. Avian and insect brood parasitic systems demonstrate how interacting species engage in reciprocal coevolutionary arms races through behavioral and morphological adaptations and counteradaptations. Mouthbrooding cichlid fishes are renowned for their remarkable evolutionary radiations and complex behaviors. In Lake Tanganyika, mouthbrooding cichlids are exploited by the only obligate nonavian vertebrate brood parasite, the cuckoo catfish Synodontis multipunctatus. We show that coevolutionary history and individual learning both have a major impact on the success of cuckoo catfish parasitism between coevolved sympatric and evolutionarily naïve allopatric cichlid species. The rate of cuckoo catfish parasitism in coevolved Tanganyikan hosts was 3 to 11 times lower than in evolutionarily naïve cichlids. Moreover, using experimental infections, we demonstrate that parasite egg rejection in sympatric hosts was much higher, leading to seven times greater parasite survival in evolutionarily naïve than sympatric hosts. However, a high rejection frequency of parasitic catfish eggs by coevolved sympatric hosts came at a cost of increased rejection of their own eggs. A significant cost of catfish parasitism was universal, except for coevolved sympatric cichlid species with previous experience of catfish parasitism, demonstrating that learning and individual experience both contribute to a successful host response.

Longitudinal demographic study of wild populations of African annual killifish.

The natural history of model organisms is often overlooked despite its importance to correctly interpret the outcome of laboratory studies. Ageing is particularly understudied in natural populations. To address this gap, we present lifetime demographic data from wild populations of an annual species, the turquoise killifish, Nothobranchius furzeri, a model species in ageing research, and two other species of coexisting annual killifishes. Annual killifish hatch synchronously, have non-overlapping generations, and reproduce daily after reaching sexual maturity. Data from 13 isolated savanna pools in southern Mozambique demonstrate that the pools supporting killifish populations desiccated 1-4 months after their filling, though some pools persisted longer. Declines in population size over the season were stronger than predicted, because they exceeded the effect of steady habitat shrinking on population density that, contrary to the prediction, decreased. Populations of N. furzeri also became more female-biased with progressing season suggesting that males had lower survival. Nothobranchius community composition did not significantly vary across the season. Our data clearly demonstrate that natural populations of N. furzeri and its congeners suffer strong mortality throughout their lives, with apparent selective disappearance (condition-dependent mortality) at the individual level. This represents selective force that can shape the evolution of lifespan, and its variation across populations, beyond the effects of the gradient in habitat persistence.