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 ～ 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.     

Are sympatrically speciating Midas cichlid fish special? Patterns of morphological and genetic variation in the closely related species Archocentrus centrarchus

Established empirical cases of sympatric speciation are scarce, although there is an increasing consensus that sympatric speciation might be more common than previously thought. Midas cichlid fish are one of the few substantiated cases of sympatric speciation, and they formed repeated radiations in crater lakes. In contrast, in the same environment, such radiation patterns have not been observed in other species of cichlids and other families of fish. We analyze morphological and genetic variation in a cichlid species (Archocentrus centrarchus) that co‐inhabits several crater lakes with the Midas species complex. In particular, we analyze variation in body and pharyngeal jaw shape (two ecologically important traits in sympatrically divergent Midas cichlids) and relate that to genetic variation in mitochondrial control region and microsatellites. Using these four datasets, we analyze variation between and within two Nicaraguan lakes: a crater lake where multiple Midas cichlids have been described and a lake where the source population lives. We do not observe any within‐lake clustering consistent across morphological traits and genetic markers, suggesting the absence of sympatric divergence in A. centrarchus. Genetic differentiation between lakes was low and morphological divergence absent. Such morphological similarity between lakes is found not only in average morphology, but also when analyzing covariation between traits and degree of morphospace occupation. A combined analysis of the mitochondrial control region in A. centrarchus and Midas cichlids suggests that a difference between lineages in the timing of crater lake colonization cannot be invoked as an explanation for the difference in their levels of diversification. In light of our results, A. centrarchus represents the ideal candidate to study the genomic differences between these two lineages that might explain why some lineages are more likely to speciate and diverge in sympatry than others.     

Genomic architecture of ecologically divergent body shape in a pair of sympatric crater lake cichlid fishes

Determining the genetic bases of adaptations and their roles in speciation is a prominent issue in evolutionary biology. Cichlid fish species flocks are a prime example of recent rapid radiations, often associated with adaptive phenotypic divergence from a common ancestor within a short period of time. In several radiations of freshwater fishes, divergence in ecomorphological traits — including body shape, colour, lips and jaws — is thought to underlie their ecological differentiation, specialization and, ultimately, speciation. The Midas cichlid species complex (Amphilophus spp.) of Nicaragua provides one of the few known examples of sympatric speciation where species have rapidly evolved different but parallel morphologies in young crater lakes. This study identified significant QTL for body shape using SNPs generated via ddRAD sequencing and geometric morphometric analyses of a cross between two ecologically and morphologically divergent, sympatric cichlid species endemic to crater Lake Apoyo: an elongated limnetic species (Amphilophus zaliosus) and a high‐bodied benthic species (Amphilophus astorquii). A total of 453 genome‐wide informative SNPs were identified in 240 F₂ hybrids. These markers were used to construct a genetic map in which 25 linkage groups were resolved. Seventy‐two segregating SNPs were linked to 11 QTL. By annotating the two most highly supported QTL‐linked genomic regions, genes that might contribute to divergence in body shape along the benthic–limnetic axis in Midas cichlid sympatric adaptive radiations were identified. These results suggest that few genomic regions of large effect contribute to early stage divergence in Midas cichlids.     

Incipient speciation in sympatric Nicaraguan crater lake cichlid fishes: sexual selection versus ecological diversification.

The growing body of empirical evidence for sympatric speciation has been complemented by recent theoretical treatments that have identified evolutionary conditions conducive to speciation in sympatry. The Neotropical Midas cichlid (Amphilophus citrinellum) fits both of the key characteristics of these models, with strong assortative mating on the basis of a colour polymorphism coupled with trophic and ecological differentiation derived from a polymorphism in their pharyngeal jaws. We used microsatellite markers and a 480 bp fragment of the mitochondrial DNA control region to study four polymorphic populations of the Midas cichlid from three crater lakes and one large lake in Nicaragua in an investigation of incipient sympatric speciation. All populations were strongly genetically differentiated on the basis of geography. We identified strong genetic separation based on colour polymorphism for populations from Lake Nicaragua and one crater lake (Lake Apoyo), but failed to find significant genetic structuring based on trophic differences and ecological niche separation in any of the four populations studied. These data support the idea that sexual selection through assortative mating contributes more strongly or earlier during speciation in sympatry than ecological separation in these cichlids. The long-term persistence of divergent cichlid ecotypes (as measured by the percentage sequence divergence between populations) in Central American crater lakes, despite a lack of fixed genetic differentiation, differs strikingly from the patterns of extremely rapid speciation in the cichlids in Africa, including its crater lakes. It is unclear whether extrinsic environmental factors or intrinsic biological differences, e.g. in the degree of phenotypic plasticity, promote different mechanisms and thereby rates of speciation of cichlid fishes from the Old and New Worlds.     

Crater lake cichlids individually specialize along the benthic–limnetic axis

A common pattern of adaptive diversification in freshwater fishes is the repeated evolution of elongated open water (limnetic) species and high‐bodied shore (benthic) species from generalist ancestors. Studies on phenotype‐diet correlations have suggested that population‐wide individual specialization occurs at an early evolutionary and ecological stage of divergence and niche partitioning. This variable restricted niche use across individuals can provide the raw material for earliest stages of sympatric divergence. We investigated variation in morphology and diet as well as their correlations along the benthic‐limnetic axis in an extremely young Midas cichlid species, Amphilophus tolteca, endemic to the Nicaraguan crater lake Asososca Managua. We found that A. tolteca varied continuously in ecologically relevant traits such as body shape and lower pharyngeal jaw morphology. The correlation of these phenotypes with niche suggested that individuals are specialized along the benthic‐limnetic axis. No genetic differentiation within the crater lake was detected based on genotypes from 13 microsatellite loci. Overall, we found that individual specialization in this young crater lake species encompasses the limnetic‐ as well as the benthic macro‐habitat. Yet there is no evidence for any diversification within the species, making this a candidate system for studying what might be the early stages preceding sympatric divergence.     

Recent speciation between sympatric Tanganyikan cichlid colour morphs

Lake Tanganyika, Africa's oldest lake, harbours an impressive diversity of cichlid fishes. Although diversification in its radiating groups is thought to have been initially rapid, cichlids from Lake Tanganyika show little evidence for ongoing speciation. In contrast, examples of recent divergence among sympatric colour morphs are well known in haplochromine cichlids from Lakes Malawi and Victoria. Here, we report genetic evidence for recent divergence between two sympatric Tanganyikan cichlid colour morphs. These Petrochromis morphs share mitochondrial haplotypes, yet microsatellite loci reveal that their sympatric populations form distinct genetic groups. Nuclear divergence between the two morphs is equivalent to that which arises geographically within one of the morphs over short distances and is substantially smaller than that among other sympatric species in this genus. These patterns suggest that these morphs diverged only recently, yet that barriers to gene flow exist which prevent extensive admixture despite their sympatric distribution. The morphs studied here provide an unusual example of active diversification in Lake Tanganyika's generally ancient cichlid fauna and enable comparisons of speciation processes between Lake Tanganyika and other African lakes.     

Phylogeography,colonization and population history of the Midas cichlid species complex (<Emphasis Type="Italic">Amphilophus</Emphasis> spp.) in the Nicaraguan crater lakes

Background  

Elucidation of the mechanisms driving speciation requires detailed knowledge about the phylogenetic relationships and phylogeography of the incipient species within their entire ranges as well as their colonization history. The Midas cichlid species complex Amphilophus spp. has been proven to be a powerful model system for the study of ecological specialization, sexual selection and the mechanisms of sympatric speciation. Here we present a comprehensive and integrative phylogeographic analysis of the complete Midas Cichlid species complex in Nicaragua (> 2000 individuals) covering the entire distributional range, using two types of molecular markers (the mitochondrial DNA control region and 15 microsatellites). We investigated the majority of known lake populations of this species complex and reconstructed their colonization history in order to distinguish between alternative speciation scenarios.     

The shape of things to come in the study of the origin of species?

Perhaps Darwin would agree that speciation is no longer the mystery of mysteries that it used to be. It is now generally accepted that evolution by natural selection can contribute to ecological adaptation, resulting in the evolution of reproductive barriers and, hence, to the evolution of new species (Schluter & Conte 2009 ; Meyer 2011 ; Nosil 2012 ). From genes that encode silencing proteins that cause infertility in hybrid mice (Mihola et al. 2009 ), to segregation distorters linked to speciation in fruit flies (Phadnis & Orr 2009 ), or pollinator‐mediated selection on flower colour alleles driving reinforcement in Texan wildflowers (Hopkins & Rausher 2012 ), characterization of the genes that drive speciation is providing clues to the origin of species (Nosil & Schluter 2011 ). It is becoming apparent that, while recent work continues to overturn historical ideas about sympatric speciation (e.g. Barluenga et al. 2006 ), ecological circumstances strongly influence patterns of genomic divergence, and ultimately the establishment of reproductive isolation when gene flow is present (Elmer & Meyer 2011 ). Less clear, however, are the genetic mechanisms that cause speciation, particularly when ongoing gene flow is occurring. Now, in this issue, Franchini et al. ( 2014 ) employ a classic genetic mapping approach augmented with new genomic tools to elucidate the genomic architecture of ecologically divergent body shapes in a pair of sympatric crater lake cichlid fishes. From over 450 segregating SNPs in an F2 cross, 72 SNPs were linked to 11 QTL associated with external morphology measured by means of traditional and geometric morphometrics. Annotation of two highly supported QTL further pointed to genes that might contribute to ecological divergence in body shape in Midas cichlids, overall supporting the hypothesis that genomic regions of large phenotypic effect may be contributing to early‐stage divergence in Midas cichlids.     

CRATER LAKE HABITAT PREDICTS MORPHOLOGICAL DIVERSITY IN ADAPTIVE RADIATIONS OF CICHLID FISHES

Adaptive radiations provide an excellent opportunity for studying the correlates and causes for the origin of biodiversity. In these radiations, species diversity may be influenced by either the ecological and physical environment, intrinsic lineage effects, or both. Disentangling the relative contributions of these factors in generating biodiversity remains a major challenge in understanding why a lineage does or does not radiate. Here, we examined morphological variation in body shape for replicate flocks of Nicaraguan Midas cichlid fishes and tested its association with biological and physical characteristics of their crater lakes. We found that variability of body elongation, an adaptive trait in freshwater fishes, is mainly predicted by average lake depth (N = 6, P < 0.001, R² = 0.96). Other factors considered, including lake age, surface area, littoral zone area, number of co‐occurring fish species, and genetic diversity of the Midas flock, did not significantly predict morphological variability. We also showed that lakes with a larger littoral zone have on average higher bodied Midas cichlids, indicating that Midas cichlid flocks are locally adapted to their crater lake habitats. In conclusion, we found that a lake's habitat predicts the magnitude and the diversity of body elongation in repeated cichlid adaptive radiations.     

Sampling genetic diversity in the sympatrically and allopatrically speciating Midas cichlid species complex over a 16 year time series

Background  

Speciation often occurs in complex or uncertain temporal and spatial contexts. Processes such as reinforcement, allopatric divergence, and assortative mating can proceed at different rates and with different strengths as populations diverge. The Central American Midas cichlid fish species complex is an important case study for understanding the processes of speciation. Previous analyses have demonstrated that allopatric processes led to species formation among the lakes of Nicaragua as well as sympatric speciation that is occurring within at least one crater lake. However, since speciation is an ongoing process and sampling genetic diversity of such lineages can be biased by collection scheme or random factors, it is important to evaluate the robustness of conclusions drawn on individual time samples.     

Visual adaptation could aid sympatric speciation in a deep crater lake

Allopatric speciation was originally suggested to be the primary mechanism of animal speciation (Mayr, 1942; Figure 1). During allopatric speciation, populations diverge when gene flow is reduced across significant biogeographic barriers. Sympatric speciation, where species diverge while inhabiting the same location, was thought to be essentially impossible. However, the advent of theoretical models followed by new experimental evidence made sympatric speciation more plausible (Via, 2001). The cichlid fishes of Barombi Mbo, a small crater lake in western Cameroon, became one of the most widely accepted examples of sympatric speciation (Schliewen, Tautz, & Paabo, 1994). Although the phylogenetic history of this clade is not quite as simple as originally thought, it remains one of the best examples of sympatric speciation (Richards, Poelstra, & Martin, 2018). However, little is known about the molecular mechanisms contributing to the splitting of these species in situ. In a From the Cover article in this issue of Molecular Ecology, Musilova et al. (2019) focus on the diversity of visual systems among these fishes. They identify genetic changes associated with several aspects of visual adaptation that may have contributed to the ecological specialization and sympatric speciation of cichlids in this lake.     

Depth-dependent abundance of Midas Cichlid fish (Amphilophus spp.) in two Nicaraguan crater lakes

The Midas Cichlid species complex (Amphilophus spp.) in Central America serves as a prominent model system to study sympatric speciation and parallel adaptive radiation, since small arrays of equivalent ecotype morphs have evolved independently in different crater lakes. While the taxonomy and evolutionary history of the different species are well resolved, little is known about basic ecological parameters of Midas Cichlid assemblages. Here, we use a line transect survey to investigate the depth-dependent abundance of Amphilophus spp. along the shores of two Nicaraguan crater lakes, Apoyo and Xiloá. We find a considerable higher density of Midas cichlids in Lake Xiloá as compared to Lake Apoyo, especially at the shallowest depth level. This might be due to the higher eutrophication level of Lake Xiloá and associated differences in food availability, and/or the presence of a greater diversity of niches in that lake. In any case, convergent forms evolved despite noticeable differences in size, age, eutrophication level, and carrying capacity. Further, our data provide abundance and density estimates for Midas Cichlid fish, which serve as baseline for future surveys of these ecosystems and are also relevant to past and future modeling of ecological speciation.     

The Midas cichlid species complex: incipient sympatric speciation in Nicaraguan cichlid fishes?

Abstract Sympatric speciation is a contentious concept, although theoretical models as well as empirical evidence support its relevance in evolutionary biology. The Midas cichlid species complex (Amphilophus citrinellus, labiatus, zaliosus) from several crater lakes in Nicaragua fits several of the key characteristics of a sympatric speciation model. In particular, in A. citrinellus (i) strong assortative mating on the basis of colour polymorphism and (ii) ecological differentiation based on morphological polymorphisms involving the feeding apparatus and body shape might both be mechanisms of incipient speciation. Seven microsatellite markers and mtDNA control region sequences [836 base pairs (bp)] were used to study the population genetic structure of 519 specimens of Midas cichlid populations from the two Great Lakes Managua and Nicaragua, and three crater lakes in Nicaragua, Central America. The three named species of the species complex occupy different ecological niches, are morphologically distinct and can be distinguished genetically. We uncovered allopatric genetic differentiation of populations of A. citrinellus from different lakes and distant locations within Lake Managua and, more interestingly, incipient genetic differentiation of several sympatric populations based on colouration (in A. citrinellus and A. labiatus) but not on the morphology of the pharyngeal jaws (in A. citrinellus). Sexual selection and assortative mating might be the driven forces of diversification within named species. The Midas cichlid species complex in Nicaragua is an excellent model system for the study of the incipient stages of adaptation, speciation and the formation of species flocks.     

CRATER LAKE COLONIZATION BY NEOTROPICAL CICHLID FISHES

Volcanic crater lakes are isolated habitats that are particularly well suited to investigating ecological and evolutionary divergence and modes of speciation. However, the mode, frequency, and timing of colonization of crater lakes have been difficult to determine. We used a statistical comparative phylogeographic approach, based on a mitochondrialDNA dataset, to infer the colonization history of two Nicaraguan crater lakes by populations of genetically and ecologically divergent cichlid lineages: Midas (Amphilophus cf. citrinellus complex) and moga (Hypsophrys nematopus). We compared estimates of diversity among populations within the two cichlid lineages and found that Midas were the most genetically diverse. From an approximate Bayesian computation analysis, we inferred that the crater lakes were each founded by both cichlid lineages in single waves of colonization: Masaya 5800 ± 300 years ago and Xiloá 5400 ± 750 years ago. We conclude that natural events are likely to have a dominant role in colonization of the crater lakes. Further, our findings suggest that the higher species richness and more rapid evolution of the Midas species complex, relative to other lineages of fishes in the same crater lakes, cannot be explained by earlier or more numerous colonization events.     

Genetic, Comparative Genomic, and Expression Analyses of the Mc1r Locus in the Polychromatic Midas Cichlid Fish (Teleostei, Cichlidae Amphilophus sp.) Species Group

Natural populations of the Midas cichlid species in several different crater lakes in Nicaragua exhibit a conspicuous color polymorphism. Most individuals are dark and the remaining have a gold coloration. The color morphs mate assortatively and sympatric population differentiation has been shown based on neutral molecular data. We investigated the color polymorphism using segregation analysis and a candidate gene approach. The segregation patterns observed in a mapping cross between a gold and a dark individual were consistent with a single dominant gene as a cause of the gold phenotype. This suggests that a simple genetic architecture underlies some of the speciation events in the Midas cichlids. We compared the expression levels of several candidate color genes Mc1r, Ednrb1, Slc45a2, and Tfap1a between the color morphs. Mc1r was found to be up regulated in the gold morph. Given its widespread association in color evolution and role on melanin synthesis, the Mc1r locus was further investigated using sequences derived from a genomic library. Comparative analysis revealed conserved synteny in relation to the majority of teleosts and highlighted several previously unidentified conserved non-coding elements (CNEs) in the upstream and downstream regions in the vicinity of Mc1r. The identification of the CNEs regions allowed the comparison of sequences from gold and dark specimens of natural populations. No polymorphisms were found between in the population sample and Mc1r showed no linkage to the gold phenotype in the mapping cross, demonstrating that it is not causally related to the color polymorphism in the Midas cichlid.     

<Emphasis Type="Italic">Tol2</Emphasis> transposon-mediated transgenesis in the Midas cichlid (<Emphasis Type="Italic">Amphilophus citrinellus</Emphasis>) — towards understanding gene function and regulatory evolution in an ecological model system for rapid phenotypic diversification

Background

The Midas cichlid species complex (Amphilophus spp.) is widely known among evolutionary biologists as a model system for sympatric speciation and adaptive phenotypic divergence within extremely short periods of time (a few hundred generations). The repeated parallel evolution of adaptive phenotypes in this radiation, combined with their near genetic identity, makes them an excellent model for studying phenotypic diversification. While many ecological and evolutionary studies have been performed on Midas cichlids, the molecular basis of specific phenotypes, particularly adaptations, and their underlying coding and cis-regulatory changes have not yet been studied thoroughly.

Results

For the first time in any New World cichlid, we use Tol2 transposon-mediated transgenesis in the Midas cichlid (Amphilophus citrinellus). By adapting existing microinjection protocols, we established an effective protocol for transgenesis in Midas cichlids. Embryos were injected with a Tol2 plasmid construct that drives enhanced green fluorescent protein (eGFP) expression under the control of the ubiquitin promoter. The transgene was successfully integrated into the germline, driving strong ubiquitous expression of eGFP in the first transgenic Midas cichlid line. Additionally, we show transient expression of two further transgenic constructs, ubiquitin::tdTomato and mitfa::eGFP. Transgenesis in Midas cichlids will facilitate further investigation of the genetic basis of species-specific traits, many of which are adaptations.

Conclusion

Transgenesis is a versatile tool not only for studying regulatory elements such as promoters and enhancers, but also for testing gene function through overexpression of allelic gene variants. As such, it is an important first step in establishing the Midas cichlid as a powerful model for studying adaptive coding and non-coding changes in an ecological and evolutionary context.

     

COLOR ASSORTATIVE MATING CONTRIBUTES TO SYMPATRIC DIVERGENCE OF NEOTROPICAL CICHLID FISH

It is still debated vigorously whether sexual selection can result in speciation without physical barriers to gene flow. In this study, we used field data and molecular methods to investigate the gold–normal color polymorphism in two endemic cichlid fish species of crater lake Xiloá, Nicaragua. We found significant assortative mating by color in both Amphilophus xiloaensis and A. sagittae . Focusing on A. xiloaensis , microsatellite allele frequencies, an assignment test, and model-based cluster analysis demonstrates significant and clear genetic differentiation ( F _ST= 0.03) between gold and normal individuals in sympatry. In addition, we find genetic differentiation between all three sympatric and ecologically distinct Midas cichlid species of Lake Xiloá, A. amarillo , A. sagittae , and A. xiloaensis ( F _ST= 0.03 – 0.19), and clear genetic isolation of these species from their closest relative ( A. citrinellus ) in the neighboring great lake Managua. The A. xiloaensis gold morph is genetically more distinct from the lake's other two Midas cichlid species than is A. xiloaensis -normal. Thus, we have identified sexual isolation based on color that is evident in population genetics and mate choice. Our results suggest that sexual selection through color assortative mating may play an important role in incipient sympatric speciation in Midas cichlids of Nicaragua.     

Complex histories of repeated gene flow in Cameroon crater lake cichlids cast doubt on one of the clearest examples of sympatric speciation

One of the most celebrated examples of sympatric speciation in nature are monophyletic radiations of cichlid fishes endemic to Cameroon crater lakes. However, phylogenetic inference of monophyly may not detect complex colonization histories involving some allopatric isolation, such as double invasions obscured by genome‐wide gene flow. Population genomic approaches are better suited to test hypotheses of sympatric speciation in these cases. Here, we use comprehensive sampling from all four sympatric crater lake cichlid radiations in Cameroon and outgroups across Africa combined with next‐generation sequencing to genotype tens of thousands of SNPs. We find considerable evidence of gene flow between all four radiations and neighboring riverine populations after initial colonization. In a few cases, some sympatric species are more closely related to outgroups than others, consistent with secondary gene flow facilitating their speciation. Our results do not rule out sympatric speciation in Cameroon cichlids, but rather reveal a complex history of speciation with gene flow, including allopatric and sympatric phases, resulting in both reproductively isolated species and incipient species complexes. The best remaining non‐cichlid examples of sympatric speciation all involve assortative mating within microhabitats. We speculate that this feature may be necessary to complete the process of sympatric speciation in nature.     

Temporal diversification of Central American cichlids

Background  

Cichlid fishes are classic examples of adaptive radiation because of their putative tendency to explosively diversify after invading novel environments. To examine whether ecological opportunity increased diversification (speciation minus extinction) early in a species-rich cichlid radiation, we determined if Heroine cichlids experienced a burst of diversification following their invasion of Central America.     

Genetic isolation and morphological divergence mediated by high-energy rapids in two cichlid genera from the lower Congo rapids

Background  

It is hypothesized that one of the mechanisms promoting diversification in cichlid fishes in the African Great Lakes has been the well-documented pattern of philopatry along shoreline habitats leading to high levels of genetic isolation among populations. However lake habitats are not the only centers of cichlid biodiversity - certain African rivers also contain large numbers of narrowly endemic species. Patterns of isolation and divergence in these systems have tended to be overlooked and are not well understood.