Genetic architecture sets limits on transgressive segregation in hybrid cichlid fishes

The role of hybridization in the evolution of animal species is poorly understood. Transgressive segregation is a mechanism through which hybridization can generate diversity and ultimately lead to speciation. In this report we investigated the capacity of hybridization to generate novel (transgressive) phenotypes in the taxonomically diverse cichlid fishes. We generated a large F2 hybrid population by crossing two closely related cichlid species from Lake Malawi in Africa with differently shaped heads. Our morphometric analysis focused on two traits with different selective histories. The cichlid lower jaw (mandible) has evolved in response to strong directional selection, and does not segregate beyond the parental phenotype. The cichlid neurocranium (skull) has likely diverged in response to forces other than consistent directional selection (e.g., stabilizing selection), and exhibits marked transgressive segregation in our F2 population. We show that the genetic architecture of the cichlid jaw limits transgression, whereas the genetic basis of skull shape is permissive of transgressive segregation. These data suggest that natural selection, acting through the genome, will limit the degree of diversity that may be achieved via hybridization. Results are discussed in the context of the broader question of how phenotypic diversity may be achieved in rapidly evolving systems.     

Replicated divergence in cichlid radiations mirrors a major vertebrate innovation

Decoupling of the upper jaw bones—jaw kinesis—is a distinctive feature of the ray-finned fishes, but it is not clear how the innovation is related to the extraordinary diversity of feeding behaviours and feeding ecology in this group. We address this issue in a lineage of ray-finned fishes that is well known for its ecological and functional diversity—African rift lake cichlids. We sequenced ultraconserved elements to generate a phylogenomic tree of the Lake Tanganyika and Lake Malawi cichlid radiations. We filmed a diverse array of over 50 cichlid species capturing live prey and quantified the extent of jaw kinesis in the premaxillary and maxillary bones. Our combination of phylogenomic and kinematic data reveals a strong association between biting modes of feeding and reduced jaw kinesis, suggesting that the contrasting demands of biting and suction feeding have strongly influenced cranial evolution in both cichlid radiations.     

Constraint and opportunity: the genetic basis and evolution of modularity in the cichlid mandible

Modular variation, whereby the relative degree of connectivity varies within a system, is thought to evolve through a process of selection that favors the integration of certain traits and the decoupling of others. In this way, modularity may facilitate the pace of evolution and determine evolvability. Alternatively, conserved patterns of modularity may act to constrain the rate and direction of evolution by preventing certain functions from evolving. A comprehensive understanding of the potential interplay between these phenomena will require knowledge of the inheritance and the genetic basis of modularity. Here we explore these ideas in the cichlid mandible by investigating patterns of modularity at the clade and species levels and through the introduction of a new approach, the individual level. Specifically, we assessed patterns of covariation in Lake Malawi cichlid species that employ alternate "biting" and "suction-feeding" modes of feeding and in a hybrid cross between these two ecotypes. Across the suction-feeding clade, patterns of modularity were largely conserved and reflected a functionally based pattern. In contrast, the biting species displayed a pattern of modularity that more closely matched developmental modules. The pattern of modularity present in our F2 population was very similar to the pattern exhibited by the biter, suggesting a role for dominant inheritance. We demonstrate that our individual-level metric of modularity (IMM) is a valid quantitative trait that has a nonlinear relationship with shape. IMMs for each model were used as quantitative characters to map quantitative trait loci (QTL) that underlie modularity. Our QTL analysis offers new insights into the genetic basis of modularity in these fishes that may eventually lead to the discovery of the genetic processes that delineate particular modules. In all, our findings suggest that modularity is both a constraining and an evolvable force in cichlid evolution, as distinct patterns occur between species and variation exists among individuals.     

Assessing morphological differences in an adaptive trait: a landmark-based morphometric approach

East African cichlid fishes have evolved a stunning array of oral jaw morphologies. To better understand the adaptive evolution of this trait, we performed a morphological analysis of the jaws of two closely related species from Lake Malawi that have very different modes of feeding. Labeotropheus fuelleborni forages along the substrate with a "biting" mode of feeding, while Metriaclima zebra feeds in the water column with a "sucking" mode. We analyzed each of the four skeletal elements that make up the oral jaws: the dentary, articular, premaxilla, and maxilla. In addition, we performed the same analysis on the neurocranium, an element closely associated with the oral jaws. We used the thin-plate spline method to quantify morphological differences, which allowed us to relate our results to the functional biology of the species. We find many aspects of shape change that relate directly to the functional design of the cichlid head. The same series of measurements was made on hybrids between Labeotropheus and Metriaclima. For every character, hybrid progeny are statistically different from both parental species. These results suggest an additive mode of action of the alleles responsible for these phenotypes.     

Evolvability of the Cichlid Jaw: New Tools Provide Insights into the Genetic Basis of Phenotypic Integration

Phenotypic integration is a phenomenon that manifests itself as the covariation among traits, and is thought to substantially influence how evolution unfolds, both in terms of rate and direction, which ultimately determines evolvability. To date little is known about how integration may change across an adaptive radiation, nor do we have a way of determining its genetic basis. Here we sought to test the hypotheses that (1) higher magnitudes of integration are associated with a greater degree of eco-morphological divergence, and (2) integration has a tractable genetic basis. To this end, we first evaluated the magnitude of integration at the population level in the lower jaws of two Lake Malawi cichlid species that exhibit different degrees of trophic specialization. We find that the more eco-morphologically divergent species does indeed exhibit a significantly higher magnitude of integration, which is consistent with our first hypothesis. Next, we developed a new statistical approach based on jackknife pseudovalues to produce a quantitative trait representative of inter-individual variation in the magnitude of integration. This metric was successfully applied to map the genetic basis of integration in the lower jaws of F2 hybrids derived from the two parental species that exhibited differences in the magnitude of integration. We detected three QTLs and two epistatic interactions that contribute to variation in integration within the cichlid mandible. We also detected a single QTL for lower jaw shape. None of the single QTLs for integration identified here overlapped with the interval for lower jaw shape, although one of the epistatic loci for integration did overlap with shape QTL. These results underscore a complex relationship between integration and shape, but suggest largely distinct genetic bases for these two traits. In all, our results show that phenotypic integration has a tractable, yet complex, genetic basis and that we now have the tools available to shed new light on the mechanisms that both promote and limit craniofacial diversity.     

Evolutionary divergence in life history traits among populations of the Lake Malawi cichlid fish Astatotilapia calliptera

During the early stages of adaptive radiation, populations diverge in life history traits such as egg size and growth rates, in addition to eco‐morphological and behavioral characteristics. However, there are few studies of life history divergence within ongoing adaptive radiations. Here, we studied Astatotilapia calliptera, a maternal mouthbrooding cichlid fish within the Lake Malawi haplochromine radiation. This species occupies a rich diversity of habitats, including the main body of Lake Malawi, as well as peripheral rivers and shallow lakes. We used common garden experiments to test for life history divergence among populations, focussing on clutch size, duration of incubation, egg mass, offspring size, and growth rates. In a first experiment, we found significant differences among populations in average clutch size and egg mass, and larger clutches were associated with smaller eggs. In a second experiment, we found significant differences among populations in brood size, duration of incubation, juvenile length when released, and growth rates. Larger broods were associated with smaller juveniles when released and shorter incubation times. Although juvenile growth rates differed between populations, these were not strongly related to initial size on release. Overall, differences in life history characters among populations were not predicted by major habitat classifications (Lake Malawi or peripheral habitats) or population genetic divergence (microsatellite‐based F_ST). We suggest that the observed patterns are consistent with local selective forces driving the observed patterns of trait divergence. The results provide strong evidence of evolutionary divergence and covariance of life history traits among populations within a radiating cichlid species, highlighting opportunities for further work to identify the processes driving the observed divergence.     

Chondrocranium morphology of northern Red Sea triakid sharks and relationships to feeding habits

The structure of the neurocranium, mandibular arch and hyoid arch of I. omanensis (Norman) and M. mosis Hemprich & Ehrenberg is described. Comparisons between these species and other triakids and between triakids and carcharhinoids are given. Differences in the size, weight and structure of the mandibular arch, including its associated ligaments and jaw suspension, are shown to be related to feeding habits. Dental characters for both species are examined and differences are considered in relation to diet. Structural elaborations of the nasal capsules in I. omanensis are described in relation to protrusion of the upper jaw during biting. Comparable neurocranial features are poorly developed in M. mosis where the upper jaw shows little discernable anterior movement. The optic region is enlarged in both species in relation to nasal and otic areas of the neurocranium.     

Lake Malawi cichlid evolution along a benthic/limnetic axis

Divergence along a benthic to limnetic habitat axis is ubiquitous in aquatic systems. However, this type of habitat divergence has largely been examined in low diversity, high latitude lake systems. In this study, we examined the importance of benthic and limnetic divergence within the incredibly species‐rich radiation of Lake Malawi cichlid fishes. Using novel phylogenetic reconstructions, we provided a series of hypotheses regarding the evolutionary relationships among 24 benthic and limnetic species that suggests divergence along this axis has occurred multiple times within Lake Malawi cichlids. Because pectoral fin morphology is often associated with divergence along this habitat axis in other fish groups, we investigated divergence in pectoral fin muscles in these benthic and limnetic cichlid species. We showed that the eight pectoral fin muscles and fin area generally tended to evolve in a tightly correlated manner in the Lake Malawi cichlids. Additionally, we found that larger pectoral fin muscles are strongly associated with the independent evolution of the benthic feeding habit across this group of fish. Evolutionary specialization along a benthic/limnetic axis has occurred multiple times within this tropical lake radiation and has produced repeated convergent matching between exploitation of water column habitats and locomotory morphology.     

Variation in body size and trophic morphology within and among genetically differentiated populations of the cichlid fish, Metriaclima zebra, from Lake Malawi

1. The cichlid fish Metriaclima zebra , common in Lake Malawi, feeds by filtering plankton from the water and by brushing items from sediment covered substrata. It inhabits isolated rocky reefs among which community structure, resource availability and gene pools are likely to differ. We speculated that body size and trophic morphology of M. zebra might vary concomitantly.
2. We quantified the extent of genetic, body size and trophic variation within and between populations of M. zebra from southern Lake Malawi. Specifically, we tested the hypotheses that: (i) local populations are genetically differentiated, (ii) local populations differ in jaw morphology, dentition and standard length (SL), and (iii) variation in size is correlated with variation in trophic morphology.
3. Local populations of M. zebra differed in mean SL and were genetically differentiated. Moreover, populations exhibited dissimilar oral jaw morphologies and dentitions, perhaps related to differences in feeding biology. Variation in jaw shape was largely restricted to the curvature of the distal tip of the dentary. Populations were characterised by individuals with oblique, upward or downward directed gapes. Dental patterns differed in the proportion of unicuspid teeth in all rows of each jaw (dentaries and premaxillae) and the spacing of teeth in affected rows.
4. Within populations, jaw and tooth shapes were correlated with body size. Smaller individuals possessed upward curving jaws and closely packed multicusped teeth, while larger individuals exhibited relatively downward-directed jaws with increasing numbers of widely spaced unicuspid teeth.
5. Metriaclima zebra populations have increased in mean SL over the last decade, in contrast to a decline among Lake Malawi pelagic cichlids. Differences in size may contribute to variation in trophic morphology and may track local environmental dynamics in this lacustrine system.     

Morphological analyses of the neurocranium of a group of rock-dwelling cichlid fishes (Gichlidae: Perciformes) from Lake Malawi, Africa

Variation in the size and the shape of neurocrania of a group of rock-dwelling cichlid fishes (mbuna; from Lake Malawi, Africa was analysed using principal components and regression analyses on a set of 24 neurocranial measurements of 86 individuals from 11 species. The results reveal effective morphological discrimination between species. Most of the structural variation between species demonstrates positive allometry and represents size-dependent species differences. Thus, morphological differentiation in these fish may be largely the result of heterochronic changes in relative growth rates. The vomerine region, however, exhibited shape differentiation. Distinct groupings, based on vomer shape, are identified. Foraging behaviour and diet are found to be correlated with this region of the neurocrania. The correlation of the shape of the vomer and fine-scale resource partitioning suggests that this structure may be important in the evolutionary success of the group. Also, most structural shape variation in neurocranial morphology is associated with structural variation in buccal jaws.     

Genetic basis of adaptive shape differences in the cichlid head

East African cichlids exhibit an extraordinary level of morphological diversity. Key to their success has been a dramatic radiation in trophic biology, which has occurred rapidly and repeatedly in different lakes. In this report we take the first step in understanding the genetic basis of differences in cichlid oral jaw design. We estimate the effective number of genetic factors that control differences in the cichlid head through a comprehensive morphological assessment of two Lake Malawi cichlid species and their F(1) and F(2) hybrid progeny. We estimate that between one and 11 factors underlie shape difference of individual bony elements. We show that many of the skeletal differences in the head and oral jaw apparatus are inherited together, suggesting a degree of pleiotropy in the genetic architecture of this character complex. Moreover, we find that cosegregation of shape differences in different elements corresponds to developmental, rather than functional, units.     

Hybrid origin of a cichlid population in Lake Malawi: implications for genetic variation and species diversity

The importance of species recognition to taxonomic diversity among Lake Malawi cichlids has been frequently discussed. Hybridization - the apparent breakdown of species recognition - has been observed sporadically among cichlids and has been viewed as both a constructive and a destructive force with respect to species diversity. Here we provide genetic evidence of a natural hybrid cichlid population with a unique colour phenotype and elevated levels of genetic variation. We discuss the potential evolutionary consequences of interspecific hybridization in Lake Malawi cichlids and propose that the role of hybridization in generating both genetic variability and species diversity of Lake Malawi cichlids warrants further consideration.     

Morphological analyses of the neurocranium of a group of rock-dwelling cichlid fishes (Cichlidae: Perciformes) from Lake Malawi,Africa

Variation in the size and the shape of neurocrania of a group of rock-dwelling cichlid fishes (mbuna; from Lake Malawi, Africa was analysed using principal components and regression analyses on a set of 24 neurocranial measurements of 86 individuals from 11 species. The results reveal effective morphological discrimination between species. Most of the structural variation between species demonstrates positive allometry and represents size-dependent species differences. Thus, morphological differentiation in these fish may be largely the result of heterochronic changes in relative growth rates. The vomerine region, however, exhibited shape differentiation. Distinct groupings, based on vomer shape, are identified. Foraging behaviour and diet are found to be correlated with this region of the neurocrania. The correlation of the shape of the vomer and fine-scale resource partitioning suggests that this structure may be important in the evolutionary success of the group. Also, most structural shape variation in neurocranial morphology is associated with structural variation in buccal jaws.     

Do constructional constraints influence cichlid craniofacial diversification?

Constraints on form should determine how organisms diversify. Owing to competition for the limited space within the body, investment in adjacent structures may frequently represent an evolutionary compromise. For example, evolutionary trade-offs between eye size and jaw muscles in cichlid fish of the African great lakes are thought to represent a constructional constraint that influenced the diversification of these assemblages. To test the evolutionary independence of these structures in Lake Malawi cichlid fish, we measured the mass of the three major adductor mandibulae (AM) muscles and determined the eye volume in 41 species. Using both traditional and novel methodologies to control for resolved and unresolved phylogenetic relationships, we tested the evolutionary independence of these four structures. We found that evolutionary change in the AM muscles was positively correlated, suggesting that competition for space in the head has not influenced diversification among these jaw muscles. Furthermore, there was no negative relationship between change in total AM muscle mass and eye volume, indicating that there has been little effect of the evolution of eye size on AM evolution in Lake Malawi cichlids. The comparative approach used here should provide a robust method to test whether constructional constraints frequently limit phenotypic change in adaptive radiations.     

Comparative rates of lower jaw diversification in cichlid adaptive radiations

The lower jaw (LJ) provides an ideal trophic phenotype to compare rates and patterns of macroevolution among cichlid radiations. Using a novel phylogeny of four genes (ND2, dlx2, mitfb, and s7), we examined the evolutionary relationships among two of the most phylogenetically disparate cichlid radiations: (i) the Central America Heroines; and (ii) the East African Lake Malawi flock. To quantify jaw morphology, we measured two LJ lever systems in approximately 40 species from each lineage. Using geologic calibrations, we generated a chronogram for both groups and examined the rates of jaw evolution in the two radiations. The most rapidly evolving components of the LJ differed between the two radiations. However, the Lake Malawi flock exhibited a much faster rate of evolution in several components of the LJ. This rapid rate of divergence is consistent with natural selection, promoting unparalleled trophic diversification in Lake Malawi cichlids.     

Unusually fine-scale genetic structuring found in rapidly speciating Malawi cichlid fishes

Mechanisms behind the explosive radiation of over 500 cichlid fish species from a single founding population in Lake Malawi during the last 700 000 years are poorly understood. Recent studies have suggested that the degree of population subdivision among the habitat patches within the lake may be responsible, but the evidence has been circumstantial: lack of a dispersal stage in haplochromine cichlids; genetic and colour variation among populations separated by large-scale geographical barriers; and fluctuating lake levels. One reason for the rapidity of speciation in these fishes may be that population subdivision is on a much finer scale than previously thought. Here we quantify the level of population subdivision and estimate migration at a scale of 700-1400 m, in order to investigate whether cichlid populations are sufficiently isolated from each other for allopatric divergence and perhaps speciation to take place. Using six microsatellite loci, we demonstrate the existence of highly significant genetic differentiation between subpopulations on adjacent headlands in each of four rock-dwelling haplochromine cichlid species. Our results suggest that these fish populations are divided into thousands of subunits among which genetic divergence is currently occurring, and that this may provide unprecedented opportunities for allopatric speciation. <br>     

PhyloFinder: An intelligent search engine for phylogenetic tree databases

Background

Evolutionary biologists want to explain the origin of novel features and functions. Two recent but separate lines of research address this question. The first describes one possible outcome of hybridization, called transgressive segregation, where hybrid offspring exhibit trait distributions outside of the parental range. The second considers the explicit mapping of form to function and illustrates manifold paths to similar function (called many to one mapping, MTOM) when the relationship between the two is complex. Under this scenario, functional novelty may be a product of the number of ways to elicit a functional outcome (i.e., the degree of MTOM). We fuse these research themes by considering the influence of MTOM on the production of transgressive jaw biomechanics in simulated hybrids between Lake Malawi cichlid species.

Results

We characterized the component links and functional output (kinematic transmission, KT) of the 4-bar mechanism in the oral jaws of Lake Malawi cichlids. We demonstrated that the input and output links, the length of the lower jaw and the length of the maxilla respectively, have consistent but opposing relationships with KT. Based on these data, we predicted scenarios in which species with different morphologies but similar KT (MTOM species) would produce transgressive function in hybrids. We used a simple but realistic genetic model to show that transgressive function is a likely outcome of hybridization among Malawi species exhibiting MTOM. Notably, F₂ hybrids are transgressive for function (KT), but not the component links that contribute to function. In our model, transgression is a consequence of recombination and assortment among alleles specifying the lengths of the lower jaw and maxilla.

Conclusion

We have described a general and likely pervasive mechanism that generates functional novelty. Simulations of hybrid offspring among Lake Malawi cichlids exhibiting MTOM produce transgressive function in the majority of cases, and at appreciable frequency. Functional transgression (i) is a product of recombination and assortment between alleles controlling the lengths of the lower jaw and the maxilla, (ii) occurs in the absence of transgressive morphology, and (iii) can be predicted from the morphology of parents. Our genetic model can be tested by breeding Malawi cichlid hybrids in the laboratory and examining the resulting range of forms and functions.     

Genital morphology and allometry differ by species and sex in Malawi cichlid fishes

The African cichlid fishes show great diversity in mating displays and reproductive strategies, yet species differences in genital morphology have been little studied. Observational notes have described broad sex differences in external genital shape between males and females, but these differences have not been quantified. We examined three aspects of genital morphology (relative anogenital distance, relative vent length, and relative external genital area) in two riverine and eleven Lake Malawi African cichlid species from eight genera. We find the most sexually distinct morphology in the Lake Malawi rock cichlids and the least sexual dimorphism in the riverine outgroup; additionally, diversity in metrics within genus indicates that these traits are recently evolving. Sexual dimorphism in morphology is present in most species, and, in the Lake Malawi species, multivariate discriminant analysis allows for accurate assignment of gonadal sex based on genital morphology and body size. This will serve as a useful method for sexing fish in a nonlethal fashion and provides a starting point for further examination of the evolution of genital morphology in this diverse group of fishes.     

Plasticity of opsin gene expression in cichlids from Lake Malawi

Sensory systems play crucial roles in survival and reproduction. Therefore, sensory plasticity has important evolutionary implications. In this study, we examined retinal plasticity in five species of cichlid fish from Lake Malawi. We compared the cone opsin expression profiles of wild‐caught fish to lab‐reared F₁ that had been raised in a UV minus, reduced intensity light environment. All of the opsin genes that were expressed in wild‐caught fish were also expressed in lab‐reared individuals. However, we found statistically significant differences in relative opsin expression among all five species. The most consistent difference was in the SWS2B (violet) opsin, which was always expressed at higher levels in lab‐reared individuals. Estimates of visual pigment quantum catch suggest that this change in expression would increase retinal sensitivity in the light environment of the lab. We also found that the magnitude of plasticity varied across species. These findings have important implications for understanding the genetic regulation of opsin expression and raise many interesting questions about how the cichlid visual system develops. They also suggest that sensory plasticity may have facilitated the ecological diversification of cichlids in Lake Malawi.     

Axes of differentiation in the bower-building cichlids of Lake Malawi

The 500-1000 cichlid species endemic to Lake Malawi constitute one of the most rapid and extensive radiations of vertebrates known. There is a growing debate over the role natural and sexual selection have played in creating this remarkable assemblage of species. Phylogenetic analysis of the Lake Malawi species flock has been confounded by the lack of appropriate morphological characters and an exceptional rate of speciation, which has allowed ancestral molecular polymorphisms to persist within species. To overcome this problem we used amplified fragment length polymorphism (AFLP) to reconstruct the evolution of species within three genera of Lake Malawi sand-dwelling cichlids that construct elaborate male display platforms, or bowers. Sister taxa with distinct bower morphologies, and that exist in discrete leks separated by only 1-2 m of depth, are divergent in both sexually selected and ecological traits. Our phylogeny suggests that the forces of sexual and ecological selection are intertwined during the speciation of this group and that specific bower characteristics and trophic morphologies have evolved repeatedly. These results suggest that trophic morphology and bower form may be inappropriate characters for delineating taxonomic lineages. Specifically the morphological characters used to describe the genera Lethrinops and Tramitichromis do not define monophyletic clades. Using a combination of behavioural and genetic characters, we were able to identify several cryptic cichlid species on a single beach, which suggests that sand dweller species richness has been severely underestimated.