An intronic transposon insertion associates with a trans-species color polymorphism in Midas cichlid fishes

Polymorphisms have fascinated biologists for a long time, but their genetic underpinnings often remain elusive. Here, we aim to uncover the genetic basis of the gold/dark polymorphism that is eponymous of Midas cichlid fish (Amphilophus spp.) adaptive radiations in Nicaraguan crater lakes. While most Midas cichlids are of the melanic “dark morph”, about 10% of individuals lose their melanic pigmentation during their ontogeny and transition into a conspicuous “gold morph”. Using a new haplotype-resolved long-read assembly we discover an 8.2 kb, transposon-derived inverted repeat in an intron of an undescribed gene, which we term goldentouch in reference to the Greek myth of King Midas. The gene goldentouch is differentially expressed between morphs, presumably due to structural implications of inverted repeats in both DNA and/or RNA (cruciform and hairpin formation). The near-perfect association of the insertion with the phenotype across independent populations suggests that it likely underlies this trans-specific, stable polymorphism.Peer reviewe

Identifying Prognostic Factors for Well-Differentiated Metastatic Pancreatic Neuroendocrine Tumours: A Retrospective International Multicentre Cohort Study

Pancreatic neuroendocrine tumours (pNETs) represent rare neoplasms of all NETs often presenting without functional activity. Many sporadic non-functioning pNET patients are already metastatic at the time of diagnosis, and the therapeutic approach to such patients is mostly palliative. In this international, multicentre, retrospective cohort study, we assessed the prognostic value of a set of anthropometric, clinical, biochemical, radiological and pathological parameters at baseline and the impact of the therapeutic strategies on the survival of patients with sporadic grade 1/2, stage IV, non-functioning pNETs. Three hundred and twelve consecutive patients diagnosed between 1993 and 2010 were included. The median overall survival (OS) was 6.6 years and survival at 5 and 10 years was 62 and 34% respectively. On univariate analysis, Eastern Cooperative Oncology Group (ECOG) status > 2, grade 2, bilobar hepatic metastases, synchronous metastases, and high chromogranin A, alkalinephosphatase and lactic-dehydrogenase were associated with a significant reduction of OS. Palliative/curative surgery and loco-regional hepatic interventions were significant factors improving OS. On multivariate analysis, ECOG status >= 2, synchronous metastases, Ki-67 >= 10%, and high alkalinephosphatase correlated significantly with an increased risk of death. Both palliative/curative surgery and loco-regional hepatic interventions had a positive impact on OS. Although most parameters did not prove to be independent OS predictors at multivariate analysis, they showed a tendency towards that. Future prospective studies including larger patient populations may give greater clarity. We believe the integration of these parameters has the potential to provide a reliable prognostic score for the stratification of patients with sporadic well-differentiated metastatic non-functioning pNETs

Contrasting signatures of genomic divergence during sympatric speciation

Population genomic analyses of Midas cichlid fishes in young Nicaraguan crater lakes suggest that sympatric speciation is promoted by polygenic architectures. The transition from 'well-marked varieties' of a single species into 'well-defined species'-especially in the absence of geographic barriers to gene flow (sympatric speciation)-has puzzled evolutionary biologists ever since Darwin(1,2). Gene flow counteracts the buildup of genome-wide differentiation, which is a hallmark of speciation and increases the likelihood of the evolution of irreversible reproductive barriers (incompatibilities) that complete the speciation process(3). Theory predicts that the genetic architecture of divergently selected traits can influence whether sympatric speciation occurs(4), but empirical tests of this theory are scant because comprehensive data are difficult to collect and synthesize across species, owing to their unique biologies and evolutionary histories(5). Here, within a young species complex of neotropical cichlid fishes (Amphilophus spp.), we analysed genomic divergence among populations and species. By generating a new genome assembly and re-sequencing 453 genomes, we uncovered the genetic architecture of traits that have been suggested to be important for divergence. Species that differ in monogenic or oligogenic traits that affect ecological performance and/or mate choice show remarkably localized genomic differentiation. By contrast, differentiation among species that have diverged in polygenic traits is genomically widespread and much higher overall, consistent with the evolution of effective and stable genome-wide barriers to gene flow. Thus, we conclude that simple trait architectures are not always as conducive to speciation with gene flow as previously suggested, whereas polygenic architectures can promote rapid and stable speciation in sympatry.Peer reviewe

Evolutionary Genetics of Coloration in Cichlids

Why are there so many species? Or, why aren’t there more species? The factors that lead to biodiversity have always been a matter of curiosity for biologists and the general public alike. Although the main patterns and the driving forces of speciation are now reasonably well-understood, there is still much to investigate. The genetic basis of speciation has a central role in testing the current theory of speciation but our knowledge is scarce and biased towards a few model organisms. How well these model organisms represent the speciation continuum is debatable. Methodological advances now allow biologists to look beyond traditional model organisms and to assess the workings of speciation in systems that, while not being genetic models are well-recognized model systems for speciation research. Cichlids are probably the most famous textbook example of speciation. The cichlid problem, i.e. existence of flocks of thousands of recently diverged species in isolated lakes, has occupied evolutionary biologists for decades. The question “why are there so many species of cichlids?” is clearly a difficult, perhaps even an unanswerable one. Nevertheless, there is consensus in the community that the extreme variation in coloration patterns, characteristic of cichlid fishes, contributes directly to these explosive speciation rates. The goals of the present thesis are to investigate the genetics of two cichlid color traits that could constitute speciation phenotypes. Aiming at providing resources to foster further forward genetic studies, a genetic map of the most investigated African cichlid, A. burtoni was constructed. The map comprises over 200 markers within 25 linkage groups. In addition to anonymous markers, genes known to influence adaptation and speciation in cichlids were also placed on the map. By including members of the Hox clusters, it was also found that the different clusters map to different linkage groups, which supports the origin of these clusters during the whole genome duplication. The present doctoral thesis also reports on two case-studies of the genetics of color-traits thought to influence speciation in cichlids. The first are eggspots. These are interesting biological traits characteristic of African cichlids. They are color patterns on the anal fins that mimic eggs and play several roles in the mating of cichlids of the haplochromine lineage. Many have suggested that eggspots are key evolutionary innovations that influence the speciation rates of haplochromines. The genetics and sexual advertisement functions of the numbers of eggspots were investigated using selective breeding and behavioral experiments, respectively. The number of eggspots was found to harbor extensive additive genetic variation and to respond asymmetrically to selection. There was also a correlated response of body size. Furthermore, no evidence was found for the previously proposed sexual advertisement functions. All of this is inconsistent with the view that such trait is under strong directional sexual selection. It seems more likely that either non-adaptive processes currently shape the distribution of eggspot numbers or that selection acting on a correlated trait does. The second case-study concerns the gold/normal polychromatism in the Midas cichlid species of the genus Amphilophus. Gold or normal colored adults exist in several different populations and species in Nicaragua. All fish start their lives with a grayish (normal) color pattern and most individuals maintain their melanophores (dark pigment cells) throughout their lives. Others undergo morphological color change due to a process of melanophore cell death and exhibit a distinctive white-to-gold coloration in adulthood. It was shown that the color morphs mate assortatively and have diverged significantly in at least two lakes. In the present thesis, the genetic architecture of the gold phenotype is shown to be a simple one. This incipient speciation phenotype is determined by a dominant allele at a single gene. It was found that the gold locus might also control mating preference. In order to identify the causative gene, a number of approaches were taken, including forward-genetics (family- and population-based mapping), candidate-gene and transcriptomic experiments. The genomic region harboring the gold locus contains only a few positional candidate-genes out of which a single one has a known pigmentation function. Significant genotype-phenotype association was found in this region in an independent, field-collected population sample. Known color-genes that determine similar phenotypes in other organisms were either unrelated to or appear to be downstream targets of the gold locus. Interestingly, the genes that showed an expression pattern consistent with a role in the process of morphological color change are also related to human pigmentation disorders (e.g. skin cancer, psoriasis). One curious and unexpected result was that heterozygous gold fish undergo color change later in life than homozygous fish. This observation has implications for the evolutionary dynamics of this allele in the wild. In summary, the present results contribute towards filling the knowledge gaps of cichlid pigmentation and speciation genetics. By questioning previous conceptions, confirming theoretical expectations and generating new and exciting hypotheses, the present thesis illustrates the importance of understanding the genetic bases of ecologically-relevant phenotypes

Evolution of sex chromosomes in the genus Eigenmannia (Teleostei: Gymnotiformes)

Cromossomos sexuais evoluíram repetidas vezes independentemente nos grandes grupos de vertebrados. Sistemas sexuais altamente diferenciados e antigos são caracterizados por grandes diferenças morfológicas e de conteúdo gênico entre os dois cromossomos homólogos onde a recombinação é restrita a uma pequena região homóloga. Os sistemas recentes característicos de peixes caracterizam-se pela similaridade entre os cromossomos X e Y (ou Z e W), nos quais as diferenças observadas freqüentemente envolvem a presença de heterocromatina, translocações e inversões. A recombinação ocorre entre o par sexual na maior parte de sua extensão, sendo inibida apenas na região diretamente relacionada com a determinação sexual. Notavelmente, sistemas diferentes de determinação podem ser encontrados em espécies, ou mesmo populações. O gênero Eigenmannia compreende grupos de espécies crípticas do ponto de vista morfológico que exibem variação no número cromossômico e podem apresentar sistemas sexuais XY ou ZW, incluindo sistemas múltiplos (com translocação Y-autossomo). Estes sistemas estão entre os mais recentes descritos (<16ma) e estão dispostos de forma desordenada em árvores de relações filogenéticas, sugerindo origens múltiplas. No presente estudo, a técnicas de pintura cromossômica usando sondas obtidas por microdissecção de cromossomos sexuais foram empregadas para testar a homologia de dois sistemas XY encontrados nos citótipos (ou espécies) E. virescens e E. sp.2. Os resultados mostram que, de fato, ambos são não homólogos. A fusão Y-autossomo provavelmente ocorreu após a separação de E. sp.2 com sua espécie irmã, E. sp.1 uma vez que um evento de fusão independente, envolvendo um dos cromossomos homólogos ao Y, foi detectado em E. sp.1. A hibridação in sit&#956; do cromossomo X de E. virescens em sua população mais próxima (também com 38 cromossomos, mas sem cromossomos sexuais heteromórficos) mostrou que o cromossomo X é homólogo a um par de acrocêntricos, condizente com o modelo proposto de diferenciação por acúmulo de heterocromatina. Essa heterocromatina foi caracterizada e mostrou um padrão complexo de seqüências CG-ricas. Dois fragmentos de DNA repetitivo GC-ricos presentes no cromossomo X foram isolados e seqüenciados. Não foram detectadas similaridades em comparações com bases de dados e entre os fragmentos obtidos. Estes mostraram-se concentrados nas regiões cromomicina-positivas de E. virescens, incluindo regiões periteloméricas de sete pares e os dois maiores blocos heterocromáticos (nos cromossomos X e par n. 8), além de um cromossomo acrocêntrico, possivelmente o Y. Curiosamente, essas seqüências foram detectadas em apenas três pares cromossômicos na população mais próxima, incluindo um par acrocêntrico de morfologia semelhante à condição ancestral do X, sugerindo que processos dinâmicos de expansão e homogenização genômica ocorreram após a separação dessas populaçõesSex chromosomes have evolved independently several times in all major groups of vertebrates. Highly differentiated sex chromosomes are characterized by extensive differences in morphology and gene content, whereas recombination is restricted to a small homologous region. Recent sex chromosomes are characteristic of fish, and display a high level of homology between X and Y (or Z and W) chromosomes, recombination is restricted only in a small sex determining region. Notably, different sex chromosome systems can be found in closely related groups, such as species or even populations. The genus Eigenmannia comprises a group of morphologically cryptic species that display a variety of diploid numbers and different sex chromosome systems, including XY, ZW and a multiple XY system (with a Y-autosome fusion). These systems are among the most recent known (<16ma) and occur with a lack of phylogenetic pattern, whereas frequently populations bearing heteromorphic sex chromosomes are closest related to populations displaying no sex chromosomes. In the present study, chromosome painting using probes derived from the microdissection of two different sex chromosomes where used to investigate the homology of both systems. Results show that, in fact, they are non-homologous and evolved independently. The Y-autosome hypothesis gained further support from the observation that a chromosome homologous to the Y in a close population is involved in yet a different fusion event. The X chromosome present in the E. virescens karyotype was found to be homologous to acrocentric chromosomes in all populations analyzed, thus supporting the notion that its differentiations is mainly due to the accumulation of heterochromatin. The X heterochromatic block was shown to form a complex pattern of GC-rich sequences, different from what was previously described. Two GC-rich fragments were isolated and sequenced; both showed no similarities to known sequences and to one another. These sequences were shown to be concentrated viii on the two largest heterochromatic blocks, those of the X and n.8 chromosomes besides peri-telomeric regions of seven additional pairs and the putative Y. Curiously, these sequences were detected in only three pairs in the closest population, including an acrocentric pair morphologically similar to undifferentiated sex pair. This suggests that dynamic evolutionary processes of expansion and genomic homogenization have occurred after the separation of these populations

The evolutionary genomics of Cichlid fishes : explosive speciation and adaptation in the postgenomic era

With more than 1,500 species, cichlid fishes provide textbook examples of recent and diverse adaptive radiations, rapid rates of speciation, and the parallel evolution of adaptive phenotypes among both recently and distantly related lineages. This extraordinary diversity has attracted considerable interest from researchers across several biological disciplines. Their broad phenotypic variation coupled with recent divergence makes cichlids an ideal model system for understanding speciation, adaptation, and phenotypic diversification. Genetic mapping, genome-wide analyses, and genome projects have flourished in the past decade and have added new insights on the question of why there are so many cichlids. These recent findings also show that the sharing of older DNA polymorphisms is extensive and suggest that linage sorting is incomplete and that adaptive introgression played a role in the African radiation. Here, we review the results of genetic and genomic research on cichlids in the past decade and suggest some potential avenues to further exploit the potential of the cichlid model system to provide a better understanding of the genomics of adaptation and speciation

Rapid and parallel adaptive evolution of the visual system of Neotropical Midas cichlid fishes

Midas cichlid fish are a Central American species flock containing 13 described species that has been dated to only few thousand years old, a historical timescale infrequently associated with speciation. Their radiation involved the colonization of several clear water crater lakes from two turbid great lakes. Therefore, Midas cichlids have been subjected to widely varying photic conditions during their radiation. Being a primary signal relay for information from the environment to the organism, the visual system is under continuing selective pressure and a prime organ system for accumulating adaptive changes during speciation, particularly in the case of dramatic shifts in photic conditions. Here, we characterize the full visual system of Midas cichlids at organismal and genetic levels, to determine what types of adaptive changes evolved within the short time span of their radiation. We show that Midas cichlids have a diverse visual system with unexpectedly high intra- and interspecific variation in color vision sensitivity and lens transmittance. Midas cichlid populations in the clear crater lakes have convergently evolved visual sensitivities shifted towards shorter wavelengths compared to the ancestral populations from the turbid great lakes. This divergence in sensitivity is driven by changes in chromophore usage, differential opsin expression, opsin coexpression, and to a lesser degree by opsin coding sequence variation. The visual system of Midas cichlids has the evolutionary capacity to rapidly integrate multiple adaptations to changing light environments. Our data may indicate that, in early stages of divergence, changes in opsin regulation could precede changes in opsin coding sequence evolution

Eggspot Number and Sexual Selection in the Cichlid Fish <em>Astatotilapia burtoni</em>

<div><p>Sexual selection on male coloration is one of the main mechanisms proposed to explain the explosive speciation rates in East African cichlid fish. True eggspots are color patterns characteristic of the most species-rich lineage of cichlids, the Haplochromini, and have been suggested to be causally related to the speciation processes. Eggspots are thought to have originated by sensory exploitation and subsequently gained several roles in sexual advertisement. However, for most of these functions the evidence is equivocal. In addition, the genetic architecture of this trait still is largely unknown. We conducted bidirectional selective breeding experiments for eggspot numbers in the model cichlid, <em>Astatotilapia burtoni.</em> After two generations, low lines responded significantly, whereas the high lines did not. Body size was both phenotypically and genotypically correlated with eggspot number and showed correlated response to selection. Males with higher numbers of eggspots were found to sire larger offspring. Despite the potential to act as honest indicators of fitness, the behavioral experiments showed no evidence of a role in either intra- or inter-sexual selection. Visual-based female preference was instead explained by courtship intensity. The evolution of this trait has been interpreted in light of adaptive theories of sexual selection, however the present and published results suggest the influence of non-adaptive factors such as sensory exploitation, environmental constraints and sexual antagonism.</p> </div

Species-specific differences in adaptive phenotypic plasticity in an ecologically relevant trophic trait : hypertrophic lips in midas cichlid fishes

The spectacular species richness of cichlids and their diversity in morphology, coloration, and behavior have made them an ideal model for the study of speciation and adaptive evolution. Hypertrophic lips evolved repeatedly and independently in African and Neotropical cichlid radiations. Cichlids with hypertrophic lips forage predominantly in rocky crevices and it has been hypothesized that mechanical stress caused by friction could result in larger lips through phenotypic plasticity. To test the influence of the environment on the size and development of lips, we conducted a series of breeding and feeding experiments on Midas cichlids. Full-sibs of Amphilophus labiatus (thick-lipped) and Amphilophus citrinellus (thin-lipped) each were split into a control group which was fed food from the water column and a treatment group whose food was fixed to substrates. We found strong evidence for phenotypic plasticity on lip area in the thick-lipped species, but not in the thin-lipped species. Intermediate phenotypic values were observed in hybrids from thick- and thin-lipped species reared under “control” conditions. Thus, both a genetic, but also a phenotypic plastic component is involved in the development of hypertrophic lips in Neotropical cichlids. Moreover, species-specific adaptive phenotypic plasticity was found, suggesting that plasticity is selected for in recent thick-lipped species

Data from: Species-specific differences in adaptive phenotypic plasticity in an ecologically relevant trophic trait: hypertrophic lips in Midas cichlid fishes

The spectacular species richness of cichlids and their diversity in morphology, coloration, and behaviour have made them an ideal model for the study of speciation and adaptive evolution. Hypertrophic lips evolved repeatedly and independently in African and Neotropical cichlid radiations. Cichlids with hypertrophic lips forage predominantly in rocky crevices and it has been hypothesized that mechanical stress caused by friction could result in larger lips through phenotypic plasticity. To test the influence of the environment on the size and development of lips, we conducted a series of breeding and feeding experiments on Midas cichlids. Full-sibs of Amphilophus labiatus (thick-lipped) and A. citrinellus (thin-lipped) each were split into a control group which was fed food from the water column and a treatment group whose food was fixed to substrates. We found strong evidence for phenotypic plasticity on lip area in the thick-lipped species, but not in the thin-lipped species. Intermediate phenotypic values were observed in hybrids from thick- and thin-lipped species reared under “control” conditions. Thus, both a genetic, but also a phenotypic plastic component is involved in the development of hypertrophic lips in Neotropical cichlids. Moreover, species-specific adaptive phenotypic plasticity was found, suggesting that plasticity is selected for in recent thick-lipped species