Molecular parallelism in the evolution of a master sex-determining role for the anti-Mullerian hormone receptor 2 gene (amhr2) in Midas cichlids.

The evolution of sex chromosomes and their differentiation from autosomes is a major event during genome evolution that happened many times in several lineages. The repeated evolution and lability of sex-determination mechanisms in fishes makes this a well-suited system to test for general patterns in evolution. According to current theory, differentiation is triggered by the suppression of recombination following the evolution of a new master sex-determining gene. However, the molecular mechanisms that establish recombination suppression are known from few examples, owing to the intrinsic difficulties of assembling sex-determining regions (SDRs). The development of forward-genetics and long-read sequencing have generated a wealth of data questioning central aspects of the current theory. Here, we demonstrate that sex in Midas cichlids is determined by an XY system, and identify and assemble the SDR by combining forward-genetics, long-read sequencing and optical mapping. We show how long-reads aid in the detection of artefacts in genotype-phenotype mapping that arise from incomplete genome assemblies. The male-specific region is restricted to a 100-kb segment on chromosome 4 that harbours transposable elements and a Y-specific duplicate of the anti-Mullerian receptor 2 gene, which has evolved master sex-determining functions repeatedly. Our data suggest that amhr2Y originated by an interchromosomal translocation from chromosome 20 to 4 pre-dating the split of Midas and Flier cichlids. In the latter, it is pseudogenized and translocated to another chromosome. Duplication of anti-Mullerian genes is a common route to establishing new sex determiners, highlighting the role of molecular parallelism in the evolution of sex determination.

Diving into divergence: Differentiation in swimming performances, physiology and gene expression between locally-adapted sympatric cichlid fishes.

Sympatric speciation occurs without geographical barriers and is thought to often be driven by ecological specialization of individuals that eventually diverge genetically and phenotypically. Distinct morphologies between sympatric populations occupying different niches have been interpreted as such differentiating adaptive phenotypes, yet differences in performance and thus likely adaptiveness between them were rarely tested. Here, we investigated if divergent body shapes of two sympatric crater lake cichlid species from Nicaragua, one being a shore-associated (benthic) species while the other prefers the open water zones (limnetic), affect cruising (Ucrit ) and sprinting (Usprint ) swimming abilities - performances particularly relevant to their respective lifestyles. Furthermore, we investigated species differences in oxygen consumption (MO2 ) across different swimming speeds and compare gene expression in gills and white muscle at rest and during exercise. We found a superior cruising ability in the limnetic Amphilophus zaliosus compared to the benthic Amphilophus astorquii, while sprinting was not different, suggesting that their distinct morphologies affect swimming performance. Increased cruising swimming ability in A. zaliosus was linked to a higher oxygen demand during activity (but not rest), indicating different metabolic rates during exercise - a hypothesis supported by coinciding gene expression patterns of gill transcriptomes. We identified differentially expressed genes linked to swimming physiology, regulation of swimming behaviour and oxygen intake. A combination of physiological and morphological differences may thus underlie adaptations to these species' distinct niches. This complex ecological specialization probably resulted in morphological and physiological trade-offs that contributed to the rapid establishment and maintenance of divergence with gene flow.

Incipient speciation driven by hypertrophied lips in Midas cichlid fishes?

Sympatric speciation has been debated in evolutionary biology for decades. Although it has gained in acceptance recently, still only a handful of empirical examples are seen as valid (e.g. crater lake cichlids). In this study, we disentangle the role of hypertrophied lips in the repeated adaptive radiations of Nicaraguan crater lake cichlid fish. We assessed the role of disruptive selection and assortative mating during the early stages of divergence and found a functional trade-off in feeding behaviour between thick- and thin-lipped ecotypes, suggesting that this trait is a target of disruptive selection. Thick-lipped fish perform better on nonevasive prey at the cost of a poorer performance on evasive prey. Using enclosures in the wild, we found that thick-lipped fish perform significantly better in rocky than in sandy habitats. We found almost no mixed pairs during two breeding seasons and hence significant assortative mating. Genetic differentiation between ecotypes seems to be related to the time since colonization, being subtle in L. Masaya (1600 generations ago) and absent in the younger L. Apoyeque (<600 generations ago). Genome-wide differentiation between ecotypes was higher in the old source lakes than in the young crater lakes. Our results suggest that hypertrophied lips might be promoting incipient sympatric speciation through divergent selection (ecological divergence in feeding performance) and nonrandom mating (assortative mating) in the young Nicaraguan crater lakes. Nonetheless, further manipulative experiments are needed in order to confirm the role of hypertrophied lips as the main cue for assortative mating.

Incipient sympatric speciation in Midas cichlid fish from the youngest and one of the smallest crater lakes in Nicaragua due to differential use of the benthic and limnetic habitats?

Understanding how speciation can occur without geographic isolation remains a central objective in evolutionary biology. Generally, some form of disruptive selection and assortative mating are necessary for sympatric speciation to occur. Disruptive selection can arise from intraspecific competition for resources. If this competition leads to the differential use of habitats and variation in relevant traits is genetically determined, then assortative mating can be an automatic consequence (i.e., habitat isolation). In this study, we caught Midas cichlid fish from the limnetic (middle of the lake) and benthic (shore) habitats of Crater Lake Asososca Managua to test whether some of the necessary conditions for sympatric speciation due to intraspecific competition and habitat isolation are given. Lake As. Managua is very small (<900 m in diameter), extremely young (maximally 1245 years of age), and completely isolated. It is inhabited by, probably, only a single endemic species of Midas cichlids, Amphilophus tolteca. We found that fish from the limnetic habitat were more elongated than fish collected from the benthic habitat, as would be predicted from ecomorphological considerations. Stable isotope analyses confirmed that the former also exhibit a more limnetic lifestyle than the latter. Furthermore, split-brood design experiments in the laboratory suggest that phenotypic plasticity is unlikely to explain much of the observed differences in body elongation that we observed in the field. Yet, neutral markers (microsatellites) did not reveal any genetic clustering in the population. Interestingly, demographic inferences based on RAD-seq data suggest that the apparent lack of genetic differentiation at neutral markers could simply be due to a lack of time, as intraspecific competition may only have begun a few hundred generations ago.

Multispecies Outcomes of Sympatric Speciation after Admixture with the Source Population in Two Radiations of Nicaraguan Crater Lake Cichlids.

The formation of species in the absence of geographic barriers (i.e. sympatric speciation) remains one of the most controversial topics in evolutionary biology. While theoretical models have shown that this most extreme case of primary divergence-with-gene-flow is possible, only a handful of accepted empirical examples exist. And even for the most convincing examples uncertainties remain; complex histories of isolation and secondary contact can make species falsely appear to have originated by sympatric speciation. This alternative scenario is notoriously difficult to rule out. Midas cichlids inhabiting small and remote crater lakes in Nicaragua are traditionally considered to be one of the best examples of sympatric speciation and lend themselves to test the different evolutionary scenarios that could lead to apparent sympatric speciation since the system is relatively small and the source populations known. Here we reconstruct the evolutionary history of two small-scale radiations of Midas cichlids inhabiting crater lakes Apoyo and Xiloá through a comprehensive genomic data set. We find no signs of differential admixture of any of the sympatric species in the respective radiations. Together with coalescent simulations of different demographic models our results support a scenario of speciation that was initiated in sympatry and does not result from secondary contact of already partly diverged populations. Furthermore, several species seem to have diverged simultaneously, making Midas cichlids an empirical example of multispecies outcomes of sympatric speciation. Importantly, however, the demographic models strongly support an admixture event from the source population into both crater lakes shortly before the onset of the radiations within the lakes. This opens the possibility that the formation of reproductive barriers involved in sympatric speciation was facilitated by genetic variants that evolved in a period of isolation between the initial founding population and the secondary migrants that came from the same source population. Thus, the exact mechanisms by which these species arose might be different from what had been thought before.

Parallel evolution of Nicaraguan crater lake cichlid fishes via non-parallel routes.

Fundamental to understanding how biodiversity arises and adapts is whether evolution is predictable in the face of stochastic genetic and demographic factors. Here we show rapid parallel evolution across two closely related but geographically isolated radiations of Nicaraguan crater lake cichlid fishes. We find significant morphological, ecological and genetic differentiation between ecomorphs in sympatry, reflected primarily in elongated versus high-bodied shape, differential ecological niche use and genetic differentiation. These eco-morphological divergences are significantly parallel across radiations. Based on 442,644 genome-wide single nucleotide polymorphisms, we identify strong support for the monophyly of, and subsequent sympatric divergence within, each radiation. However, the order of speciation differs across radiations; in one lake the limnetic ecomorph diverged first while in the other a benthic ecomorph. Overall our results demonstrate that complex parallel phenotypes can evolve very rapidly and repeatedly in similar environments, probably due to natural selection, yet this evolution can proceed along different evolutionary genetic routes.

Genomic signatures of divergent selection and speciation patterns in a 'natural experiment', the young parallel radiations of Nicaraguan crater lake cichlid fishes.

Divergent selection is the main driving force in sympatric ecological speciation and may also play a strong role in divergence between allopatric populations. Characterizing the genome-wide impact of divergent selection often constitutes a first step in unravelling the genetic bases underlying adaptation and ecological speciation. The Midas cichlid fish (Amphilophus citrinellus) species complex in Nicaragua is a powerful system for studying evolutionary processes. Independent colonizations of isolated young crater lakes by Midas cichlid populations from the older and great lakes of Nicaragua resulted in the repeated evolution of adaptive radiations by intralacustrine sympatric speciation. In this study we performed genome scans on two repeated radiations of crater lake species and their great lake source populations (1030 polymorphic AFLPs, n âˆ¼ 30 individuals per species). We detected regions under divergent selection (0.3% in the crater lake Xiloá flock and 1.7% in the older crater lake Apoyo radiation) that might be responsible for the sympatric diversifications. We find no evidence that the same genomic regions have been involved in the repeated evolution of parallel adaptations across crater lake flocks. However, there is some genetic parallelism apparent (seven out of 51 crater lake to great lake outlier loci are shared; 13.7%) that is associated with the allopatric divergence of both crater lake flocks. Interestingly, our results suggest that the number of outlier loci involved in sympatric and allopatric divergence increases over time. A phylogeny based on the AFLP data clearly supports the monophyly of both crater lake species flocks and indicates a parallel branching order with a primary split along the limnetic-benthic axis in both radiations.

Rapid sympatric ecological differentiation of crater lake cichlid fishes within historic times.

BACKGROUND: After a volcano erupts, a lake may form in the cooled crater and become an isolated aquatic ecosystem. This makes fishes in crater lakes informative for understanding sympatric evolution and ecological diversification in barren environments. From a geological and limnological perspective, such research offers insight about the process of crater lake ecosystem establishment and speciation. In the present study we use genetic and coalescence approaches to infer the colonization history of Midas cichlid fishes (Amphilophus cf. citrinellus) that inhabit a very young crater lake in Nicaragua-the ca. 1800 year-old Lake Apoyeque. This lake holds two sympatric, endemic morphs of Midas cichlid: one with large, hypertrophied lips (approximately 20% of the total population) and another with thin lips. Here we test the associated ecological, morphological and genetic diversification of these two morphs and their potential to represent incipient speciation. RESULTS: Gene coalescence analyses [11 microsatellite loci and mitochondrial DNA (mtDNA) sequences] suggest that crater lake Apoyeque was colonized in a single event from the large neighbouring great lake Managua only about 100 years ago. This founding in historic times is also reflected in the extremely low nuclear and mitochondrial genetic diversity in Apoyeque. We found that sympatric adult thin- and thick-lipped fishes occupy distinct ecological trophic niches. Diet, body shape, head width, pharyngeal jaw size and shape and stable isotope values all differ significantly between the two lip-morphs. The eco-morphological features pharyngeal jaw shape, body shape, stomach contents and stable isotopes (delta15N) all show a bimodal distribution of traits, which is compatible with the expectations of an initial stage of ecological speciation under disruptive selection. Genetic differentiation between the thin- and thick-lipped population is weak at mtDNA sequence (FST = 0.018) and absent at nuclear microsatellite loci (FST < 0.001). CONCLUSIONS: This study provides empirical evidence of eco-morphological differentiation occurring very quickly after the colonization of a new and vacant habitat. Exceptionally low levels of neutral genetic diversity and inference from coalescence indicates that the Midas cichlid population in Apoyeque is much younger (ca. 100 years or generations old) than the crater itself (ca. 1 800 years old). This suggests either that the crater remained empty for many hundreds of years after its formation or that remnant volcanic activity prevented the establishment of a stable fish population during the early life of the crater lake. Based on our findings of eco-morphological variation in the Apoyeque Midas cichlids, and known patterns of adaptation in Midas cichlids in general, we suggest that this population may be in a very early stage of speciation (incipient species), promoted by disruptive selection and ecological diversification.