Foraging-induced craniofacial plasticity is associated with an early, robust and dynamic transcriptional response.

Phenotypic plasticity is the ability of a single genotype to vary its phenotype in response to the environment. Plasticity of the skeletal system in response to mechanical input is widely studied, but the timing of its transcriptional regulation is not well understood. Here, we used the cichlid feeding apparatus to examine the transcriptional dynamics of skeletal plasticity over time. Using three closely related species that vary in their ability to remodel bone and a panel of 11 genes, including well-studied skeletal differentiation markers and newly characterized environmentally sensitive genes, we examined plasticity at one, two, four and eight weeks following the onset of alternate foraging challenges. We found that the plastic species exhibited environment-specific bursts in gene expression beginning at one week, followed by a sharp decline in levels, while the species with more limited plasticity exhibited consistently low levels of gene expression. This trend held across nearly all genes, suggesting that it is a hallmark of the larger plasticity regulatory network. We conclude that plasticity of the cichlid feeding apparatus is not the result of slowly accumulating gene expression difference over time, but rather is stimulated by early bursts of environment-specific gene expression followed by a return to homeostatic levels.

The transcriptional state and chromatin landscape of cichlid jaw shape variation across species and environments.

Adaptive phenotypes are shaped by a combination of genetic and environmental forces, but how they interact remains poorly understood. Here, we utilize the cichlid oral jaw apparatus to better understand these gene-by-environment effects. First, we employed RNA-seq in bony and ligamentous tissues important for jaw opening to identify differentially expressed genes between species and across foraging environments. We used two Lake Malawi species adapted to different foraging habitats along the pelagic-benthic ecomorphological axis. Our foraging treatments were designed to force animals to employ either suction or biting/scraping, which broadly mimic pelagic or benthic modes of feeding. We found a large number of differentially expressed genes between species, and while we identified relatively few differences between environments, species differences were far more pronounced when they were challenged with a pelagic versus benthic foraging mode. Expression data carried the signature of genetic assimilation, and implicated cell cycle regulation in shaping the jaw across species and environments. Next, we repeated the foraging experiment and performed ATAC-seq procedures on nuclei harvested from the same tissues. Cross-referencing results from both analyses revealed subsets of genes that were both differentially expressed and differentially accessible. This reduced dataset implicated notable candidate genes including the Hedgehog effector, KIAA0586 and the ETS transcription factor, etv4, which connects environmental stress and craniofacial morphogenesis. Taken together, these data provide novel insights into the epigenetic, genetic and cellular bases of species- and environment-specific bone shapes.

Zebrafish crocc2 mutants exhibit divergent craniofacial shape, misregulated variability, and aberrant cartilage morphogenesis.

BACKGROUND: Phenotypic variation is of paramount importance in development, evolution, and human health; however, the molecular mechanisms that influence organ shape and shape variability are not well understood. During craniofacial development, the behavior of skeletal precursors is regulated by both biochemical and environmental inputs, and the primary cilia play critical roles in transducing both types of signals. Here, we examine a gene that encodes a key constituent of the ciliary rootlets, crocc2, and its role in cartilage morphogenesis in larval zebrafish. RESULTS: Geometric morphometric analysis of crocc2 mutants revealed altered craniofacial shapes and expanded variation. At the cellular level, we observed altered chondrocyte shapes and planar cell polarity across multiple stages in crocc2 mutants. Notably, cellular defects were specific to areas that experience direct mechanical input. Cartilage cell number, apoptosis, and bone patterning were not affected in crocc2 mutants. CONCLUSIONS: Whereas "regulatory" genes are widely implicated in patterning the craniofacial skeleton, genes that encode "structural" aspects of the cell are increasingly implicated in shaping the face. Our results add crocc2 to this list, and demonstrate that it affects craniofacial geometry and canalizes phenotypic variation. We propose that it does so via mechanosensing, possibly through the ciliary rootlet. If true, this would implicate a new organelle in skeletal development and evolution.

Conserved Molecular Players Involved in Human Nose Morphogenesis Underlie Evolution of the Exaggerated Snout Phenotype in Cichlids.

Instances of repeated evolution of novel phenotypes can shed light on the conserved molecular mechanisms underlying morphological diversity. A rare example of an exaggerated soft tissue phenotype is the formation of a snout flap in fishes. This tissue flap develops from the upper lip and has evolved in one cichlid genus from Lake Malawi and one genus from Lake Tanganyika. To investigate the molecular basis of snout flap convergence, we used mRNA sequencing to compare two species with snout flap to their close relatives without snout flaps from each lake. Our analysis identified 201 genes that were repeatedly differentially expressed between species with and without snout flap in both lakes, suggesting shared pathways, even though the flaps serve different functions. Shared expressed genes are involved in proline and hydroxyproline metabolism, which have been linked to human skin and facial deformities. Additionally, we found enrichment for transcription factor binding sites at upstream regulatory sequences of differentially expressed genes. Among the enriched transcription factors were members of the FOX transcription factor family, especially foxf1 and foxa2, which showed an increased expression in the flapped snout. Both of these factors are linked to nose morphogenesis in mammals. We also found ap4 (tfap4), a transcription factor showing reduced expression in the flapped snout with an unknown role in craniofacial soft tissue development. As genes involved in cichlid snout flap development are associated with human midline facial dysmorphologies, our findings hint at the conservation of genes involved in midline patterning across distant evolutionary lineages of vertebrates, although further functional studies are required to confirm this.

Covariation of brain and skull shapes as a model to understand the role of crosstalk in development and evolution.

Covariation among discrete phenotypes can arise due to selection for shared functions, and/or shared genetic and developmental underpinnings. The consequences of such phenotypic integration are far-reaching and can act to either facilitate or limit morphological variation. The vertebrate brain is known to act as an "organizer" of craniofacial development, secreting morphogens that can affect the shape of the growing neurocranium, consistent with roles for pleiotropy in brain-neurocranium covariation. Here, we test this hypothesis in cichlid fishes by first examining the degree of shape integration between the brain and the neurocranium using three-dimensional geometric morphometrics in an F5 hybrid population, and then genetically mapping trait covariation using quantitative trait loci (QTL) analysis. We observe shape associations between the brain and the neurocranium, a pattern that holds even when we assess associations between the brain and constituent parts of the neurocranium: the rostrum and braincase. We also recover robust genetic signals for both hard- and soft-tissue traits and identify a genomic region where QTL for the brain and braincase overlap, implicating a role for pleiotropy in patterning trait covariation. Fine mapping of the overlapping genomic region identifies a candidate gene, notch1a, which is known to be involved in patterning skeletal and neural tissues during development. Taken together, these data offer a genetic hypothesis for brain-neurocranium covariation, as well as a potential mechanism by which behavioral shifts may simultaneously drive rapid change in neuroanatomy and craniofacial morphology.

Breaking constraints: The development and evolution of extreme fin morphology in the Bramidae.

The developmental process establishes the foundation upon which natural selection may act. In that same sense, it is inundated with numerous constraints that work to limit the directions in which a phenotype may respond to selective pressures. Extreme phenotypes have been used in the past to identify tradeoffs and constraints and may aid in recognizing how alterations to the Baupläne can influence the trajectories of lineages. The Bramidae, a family of Scombriformes consisting of 20 extant species, are unique in that five species greatly deviate from the stout, ovaloid bodies that typify the bramids. The Ptericlinae, or fanfishes, are instead characterized by relatively elongated body plans and extreme modifications to their medial fins. Here, we explore the development of Bramidae morphologies and examine them through a phylogenetic lens to investigate the concepts of developmental and evolutionary constraints. Contrary to our predictions that the fanfishes had been constrained by inherited properties of an ancestral state, we find that the fanfishes exhibit both increased rates of trait evolution and differ substantially from the other bramids in their developmental trajectories. Conversely, the remaining bramid genera differ little, both among one another and in comparison, to the sister family Caristiidae. In all, our data suggest that the fanfishes have broken constraints, thereby allowing them to mitigate trade-offs on distinctive aspects of morphology.

Polygenic sex determination produces modular sex polymorphism in an African cichlid fish.

For many vertebrates, a single genetic locus initiates a cascade of developmental sex differences in the gonad and throughout the organism, resulting in adults with two phenotypically distinct sexes. Species with polygenic sex determination (PSD) have multiple interacting sex determination alleles segregating within a single species, allowing for more than two genotypic sexes and scenarios where sex genotype at a given locus can be decoupled from gonadal sex. Here we investigate the effects of PSD on secondary sexual characteristics in the cichlid fish Metriaclima mbenjii, where one female (W) and one male (Y) sex determination allele interact to produce siblings with four possible sex classes: ZZXX females, ZWXX females, ZWXY females, and ZZXY males. We find that PSD in M. mbenjii produces an interplay of sex linkage and sex limitation resulting in modular variation in morphological and behavioral traits. Further, the evolution or introgression of a newly acquired sex determiner creates additional axes of phenotypic variation for varied traits, including genital morphology, craniofacial morphology, gastrointestinal morphology, and home tank behaviors. In contrast to single-locus sex determination, which broadly results in sexual dimorphism, polygenic sex determination can induce higher-order sexual polymorphism. The modularity of secondary sexual characteristics produced by PSD provides context for understanding the evolutionary causes and consequences of maintenance, gain, or loss of sex determination alleles in populations.

The cichlid oral and pharyngeal jaws are evolutionarily and genetically coupled.

Evolutionary constraints may significantly bias phenotypic change, while "breaking" from such constraints can lead to expanded ecological opportunity. Ray-finned fishes have broken functional constraints by developing two jaws (oral-pharyngeal), decoupling prey capture (oral jaw) from processing (pharyngeal jaw). It is hypothesized that the oral and pharyngeal jaws represent independent evolutionary modules and this facilitated diversification in feeding architectures. Here we test this hypothesis in African cichlids. Contrary to our expectation, we find integration between jaws at multiple evolutionary levels. Next, we document integration at the genetic level, and identify a candidate gene, smad7, within a pleiotropic locus for oral and pharyngeal jaw shape that exhibits correlated expression between the two tissues. Collectively, our data show that African cichlid evolutionary success has occurred within the context of a coupled jaw system, an attribute that may be driving adaptive evolution in this iconic group by facilitating rapid shifts between foraging habitats, providing an advantage in a stochastic environment such as the East African Rift-Valley.

Extreme Morphology, Functional Trade-offs, and Evolutionary Dynamics in a Clade of Open-Ocean Fishes (Perciformes: Bramidae).

When novel or extreme morphologies arise, they are oft met with the burden of functional trade-offs in other aspects of anatomy, which may limit phenotypic diversification and make particular adaptive peaks inaccessible. Bramids (Perciformes: Bramidae) comprise a small family of 20 extant species of fishes, which are distributed throughout pelagic waters worldwide. Within the Bramidae, the fanfishes (Pteraclis and Pterycombus) differ morphologically from the generally stout, laterally compressed species that typify the family. Instead, Pteraclis and Pterycombus exhibit extreme anterior positioning of the dorsal fin onto the craniofacial skeleton. Consequently, they possess fin and skull anatomies that are radically different from other bramid species. Here, we investigate the anatomy, development, and evolution of the Bramidae to test the hypothesis that morphological innovations come at functional (proximate) and evolutionary (ultimate) costs. Addressing proximate effects, we find that the development of an exaggerated dorsal fin is associated with neurocrania modified to accommodate an anterior expansion of the dorsal fin. This occurs via reduced development of the supraoccipital crest (SOC), providing a broad surface area on the skull for insertion of the dorsal fin musculature. While these anatomical shifts are presumably associated with enhanced maneuverability in fanfishes, they are also predicted to result in compromised suction feeding, possibly limiting the mechanisms of feeding in this group. Phylogenetic analyses suggest craniofacial and fin morphologies of fanfishes evolved rapidly and are evolutionarily correlated across bramids. Furthermore, fanfishes exhibit a similar rate of lineage diversification as the rest of the Bramidae, lending little support for the prediction that exaggerated medial fins are associated with phylogenetic constraint. Our phylogeny places fanfishes at the base of the Bramidae and suggests that nonfanfish bramids have reduced medial fins and re-evolved SOCs. These observations suggest that the evolution of novel fin morphologies in basal species has led to the phylogenetic coupling of head and fin shape, possibly predisposing the entire family to a limited range of feeding. Thus, the evolution of extreme morphologies may have carryover effects, even after the morphology is lost, limiting ecological diversification of lineages.

新規または極端な形態が発生すると、解剖学的構造の他の側面で機能的なトレードオフの負担に直面することが多く、表現型の多様化が制限され、特定の適応ピークにアクセスできなくなる可能性があります。ブラミド（スズキ目：シマガツオ科）は、20種の現存する魚の小さな家族で構成されており、世界中の遠洋水域に分布しています。ブラミダエ内では、シマガツオ（PteraclisとPterycombus）は、家族を代表する一般的に頑丈な横方向に圧縮された種と形態学的に異なります。代わりに、PteraclisとPterycombusは、頭蓋顔面骨格への背びれの極端な前方位置を示します。その結果、それらは他のブラミド種とは根本的に異なるひれと頭蓋骨の解剖学的構造を持っています。ここでは、形態学的革新が機能的（近接）および進化的（究極）コストでもたらされるという仮説をテストするために、ブラミダエの解剖学、発達、および進化を調査します。近接効果に対処すると、誇張された背びれの発達は、背びれの前方拡張に対応するように修正された脳頭蓋に関連していることがわかります。これは、後頭上頂（SOC）の発達の低下を介して発生し、背びれの筋肉組織を挿入するための頭蓋骨の広い表面積を提供します。これらの解剖学的変化はおそらくファンフィッシュの操作性の向上に関連していると思われますが、吸引餌の低下をもたらし、このグループの餌のメカニズムを制限する可能性もあると予測されています。系統発生分析は、シマガツオの頭蓋顔面およびヒレの形態が急速に進化し、ブラミド間で進化的に相関していることを示唆しています。さらに、ファンフィッシュは他のブラミダエと同様の系統多様化率を示し、誇張された内側のひれが系統発生の制約に関連しているという予測をほとんど支持していません。私たちの系統発生は、シマガツオをシマガツオ科の根元に配置し、シマガツオ以外のシマガツオが内側のひれを減らし、SOCを再進化させたことを示唆しています。これらの観察結果は、基底種における新しいヒレの形態の進化が、頭とヒレの形状の系統発生的結合をもたらし、おそらく家族全員が限られた範囲の摂食にかかりやすくなっていることを示唆しています。したがって、極端な形態の進化は、形態が失われた後でも持ち越し効果をもたらす可能性があり、系統の生態学的多様化を制限します。.

Cuando surgen morfologías nuevas o extremas, a menudo se encuentran con la carga de compensaciones funcionales en otros aspectos de la anatomía, lo que puede limitar la diversificación fenotípica y hacer inaccesibles los picos adaptativos particulares. Las bramidas (Perciformes: Bramidae) comprenden una pequeña familia de 20 especies de peces existentes, que se distribuyen en las aguas pelágicas de todo el mundo. Dentro de los Bramidae, los fanfishes (Pteraclis y Pterycombus) difieren morfológicamente de las especies generalmente robustas y comprimidas lateralmente que caracterizan a la familia. En cambio, Pteraclis y Pterycombus exhiben una posición anterior extrema de la aleta dorsal sobre el esqueleto craneofacial. En consecuencia, poseen anatomías de aletas y cráneo que son radicalmente diferentes de otras especies de bramidas. Aquí, investigamos la anatomía, el desarrollo y la evolución de Bramidae para probar la hipótesis de que las innovaciones morfológicas tienen un costo funcional (próximo) y evolutivo (último). Al abordar los efectos inmediatos, encontramos que el desarrollo de una aleta dorsal exagerada se asocia con neurocráneo modificado para adaptarse a una expansión anterior de la aleta dorsal. Esto ocurre a través del desarrollo reducido de la cresta supraoccipital (SOC), proporcionando una amplia área de superficie en el cráneo para la inserción de la musculatura de la aleta dorsal. Si bien estos cambios anatómicos presumiblemente están asociados con una mayor maniobrabilidad en los peces fanfishes, también se predice que darán como resultado una alimentación por succión comprometida, lo que posiblemente limite los mecanismos de alimentación en este grupo. Los análisis filogenéticos sugieren que las morfologías craneofaciales y de aletas de los fanfishes evolucionaron rápidamente y están correlacionadas evolutivamente entre las bramidas. Además, los fanfishes exhiben una tasa similar de diversificación de linajes que el resto de los Bramidae, lo que brinda poco apoyo a la predicción de que las aletas mediales exageradas están asociadas con restricciones filogenéticas. Nuestra filogenia coloca a los peces abanico en la base de las Bramidae y sugiere que las bramidas que no son peces abanico tienen aletas mediales reducidas y SOC reevolucionado. Estas observaciones sugieren que la evolución de nuevas morfologías de aletas en especies basales ha llevado al acoplamiento filogenético de la forma de la cabeza y la aleta, lo que posiblemente predisponga a toda la familia a un rango limitado de alimentación. Por lo tanto, la evolución de morfologías extremas puede tener efectos de arrastre, incluso después de que se pierde la morfología, lo que limita la diversificación ecológica de los linajes.

Quando surgem morfologias novas ou extremas, muitas vezes enfrentam o fardo de compensações funcionais em outros aspectos da anatomia, que podem limitar a diversificação fenotípica e tornar determinados picos adaptativos inacessíveis. Bramids (Perciformes: Bramidae) compreendem uma pequena família de 20 espécies existentes de peixes, que estão distribuídos em águas pelágicas em todo o mundo. Dentro dos Bramidae, os fanfishes (Pteraclis e Pterycombus) diferem morfologicamente das espécies geralmente robustas e comprimidas lateralmente que tipificam a família. Em vez disso, Pteraclis e Pterycombus exibem posicionamento anterior extremo da nadadeira dorsal no esqueleto craniofacial. Conseqüentemente, eles possuem anatomias de barbatana e crânio que são radicalmente diferentes de outras espécies de bramida. Aqui, investigamos a anatomia, o desenvolvimento e a evolução dos Bramidae para testar a hipótese de que as inovações morfológicas têm custos funcionais (proximais) e evolutivos (finais). Abordando os efeitos imediatos, descobrimos que o desenvolvimento de uma nadadeira dorsal exagerada está associado a neurocrania modificada para acomodar uma expansão anterior da nadadeira dorsal. Isso ocorre por meio do desenvolvimento reduzido da crista supraoccipital (SOC), proporcionando uma ampla área de superfície no crânio para a inserção da musculatura da nadadeira dorsal. Embora essas mudanças anatômicas estejam presumivelmente associadas a maior capacidade de manobra em peixes-leque, também se prevê que resultem em alimentação de sucção comprometida, possivelmente limitando os mecanismos de alimentação neste grupo. As análises filogenéticas sugerem que as morfologias craniofaciais e das nadadeiras de fanfishes evoluíram rapidamente e estão evolutivamente correlacionadas entre as bramidas. Além disso, fanfishes exibem uma taxa semelhante de diversificação de linhagem como o resto dos Bramidae, emprestando pouco suporte para a previsão de que nadadeiras mediais exageradas estão associadas a restrições filogenéticas. Nossa filogenia coloca fanfishes na base dos Bramidae e sugere que bramids não fanfish possuem nadadeiras mediais reduzidas e SOCs re-evoluídos. Essas observações sugerem que a evolução de novas morfologias de nadadeiras em espécies basais levou ao acoplamento filogenético da forma da cabeça e da nadadeira, possivelmente predispondo toda a família a uma faixa limitada de alimentação. Assim, a evolução de morfologias extremas pode ter efeitos de transporte, mesmo após a perda da morfologia, limitando a diversificação ecológica das linhagens.

Diversity in rest-activity patterns among Lake Malawi cichlid fishes suggests a novel axis of habitat partitioning.

Animals display remarkable diversity in rest and activity patterns that are regulated by endogenous foraging strategies, social behaviors and predator avoidance. Alteration in the circadian timing of activity or the duration of rest-wake cycles provide a central mechanism for animals to exploit novel niches. The diversity of the >3000 cichlid species throughout the world provides a unique opportunity to examine variation in locomotor activity and rest. Lake Malawi alone is home to over 500 species of cichlids that display divergent behaviors and inhabit well-defined niches throughout the lake. These species are presumed to be diurnal, though this has never been tested systematically. Here, we measured locomotor activity across the circadian cycle in 11 Lake Malawi cichlid species. We documented surprising variability in the circadian time of locomotor activity and the duration of rest. In particular, we identified a single species, Tropheops sp. 'red cheek', that is nocturnal. Nocturnal behavior was maintained when fish were provided shelter, but not under constant darkness, suggesting that it results from acute response to light rather than an endogenous circadian rhythm. Finally, we showed that nocturnality is associated with increased eye size after correcting for evolutionary history, suggesting a link between visual processing and nighttime activity. Together, these findings identify diversity of locomotor behavior in Lake Malawi cichlids and provide a system for investigating the molecular and neural basis underlying variation in nocturnal activity.

Ciliary Rootlet Coiled-Coil 2 (crocc2) Is Associated with Evolutionary Divergence and Plasticity of Cichlid Jaw Shape.

Cichlid fishes exhibit rapid, extensive, and replicative adaptive radiation in feeding morphology. Plasticity of the cichlid jaw has also been well documented, and this combination of iterative evolution and developmental plasticity has led to the proposition that the cichlid feeding apparatus represents a morphological "flexible stem." Under this scenario, the fixation of environmentally sensitive genetic variation drives evolutionary divergence along a phenotypic axis established by the initial plastic response. Thus, if plasticity is predictable then so too should be the evolutionary response. We set out to explore these ideas at the molecular level by identifying genes that underlie both the evolution and plasticity of the cichlid jaw. As a first step, we fine-mapped an environment-specific quantitative trait loci for lower jaw shape in cichlids, and identified a nonsynonymous mutation in the ciliary rootlet coiled-coil 2 (crocc2), which encodes a major structural component of the primary cilium. Given that primary cilia play key roles in skeletal mechanosensing, we reasoned that this gene may confer its effects by regulating the sensitivity of bone to respond to mechanical input. Using both cichlids and zebrafish, we confirmed this prediction through a series of experiments targeting multiple levels of biological organization. Taken together, our results implicate crocc2 as a novel mediator of bone formation, plasticity, and evolution.

The genetic basis of coordinated plasticity across functional units in a Lake Malawi cichlid mapping population.

Adaptive radiations are often stereotypical, as populations repeatedly specialize along conserved environmental axes. Phenotypic plasticity may be similarly stereotypical, as individuals respond to environmental cues. These parallel patterns of variation, which are often consistent across traits, have led researchers to propose that plasticity can facilitate predictable patterns of evolution along environmental gradients. This "flexible stem" model of evolution raises questions about the genetic nature of plasticity, including how complex is the genetic basis for plasticity? Is plasticity across traits mediated by many distinct loci, or few "global" regulators? To address these questions, we reared a hybrid cichlid mapping population on alternate diet regimes mimicking an important environmental axis. We show that plasticity across an array of ecologically relevant traits is generally morphologically integrated, such that traits respond in a coordinated manner, especially those with overlapping function. Our genetic data are more ambiguous. While our mapping experiment provides little evidence for global genetic regulators of plasticity, these data do contain a genetic signal for the integration of plasticity across traits. Overall, our data suggest a compromise between genetic modularity, whereby plasticity may evolve independently across traits, and low level but widespread genetic integration, establishing the potential for plasticity to experience coordinated evolution.

Plasticity and genetic basis of cichlid gill arch anatomy reveal novel roles for Hedgehog signaling.

Teleost gill arches are exquisitely evolved to maximize foraging efficiency, and include structures for the capture, filtering, and processing of prey. While both plasticity and a genetic basis for gill arch traits have been noted, the relative contributions of genetics and the environment in shaping these structures remains poorly understood. East African cichlids are particularly useful in this line of study due to their highly diverse and plastic feeding apparatus. Here we explore the gene-by-environmental effects on cichlid GRs by rearing pure bred species and their F3 hybrids in different foraging environments. We find that anatomical differences between species are dependent on the environment. The genetic architecture of these traits is also largely distinct between foraging environments. We did, however, note a few genomic "hotspots" where multiple traits map to a common region. One of these, for GR number across multiple arches, maps to the ptch1 locus, a key component of the Hedgehog (Hh) pathway that has previously been implicated in cichlid oral jaw shape and plasticity. Since Hh signalling has not previously been implicated in GR development, we explored functional roles for this pathway. Using a small molecule inhibitor in cichlids, as well as zebrafish transgenic systems, we demonstrate that Hh levels negatively regulate GR number, and are both necessary and sufficient to maintain plasticity in this trait. In all these data underscore the critical importance of the environment in determining the relationship between genotype and phenotype, and provide a molecular inroad to better understand the origins of variation in this important foraging-related trait.

Rapid morphological change in multiple cichlid ecotypes following the damming of a major clearwater river in Brazil.

While anthropogenic disturbances can have damaging effects on biodiversity, they also offer an opportunity to understand how species adapt to new environments and may even provide insights into the earliest stages of evolutionary diversification. With these topics in mind, we explored the morphological changes that have occurred across several cichlid species following the damming of the Tocantins River, Brazil. The Tocantins was once a large (2,450 km), contiguous river system; however, upon closure of the Tucuruí Hydroelectric Dam in 1984, a large (~2,850 km2), permanent reservoir was established. We used geometric morphometrics to evaluate changes in native cichlids, comparing historical museum specimens collected from the Tocantins to contemporary specimens collected from the Tucuruí reservoir. Six species across five genera were included to represent distinct ecomorphs, from large piscivores to relatively small opportunistic omnivores. Notably, statistically significant changes in shape and morphological disparity were observed in all species. Moreover, the documented changes tended to be associated with functionally relevant aspects of anatomy, including head, fin, and body shape. Our data offer insights into the ways cichlids have responded, morphologically, to a novel lake environment and provide a robust foundation for exploring the mechanisms through which these changes have occurred.

Ecomorphological divergence and habitat lability in the context of robust patterns of modularity in the cichlid feeding apparatus.

BACKGROUND: Adaptive radiations are characterized by extreme and/or iterative phenotypic divergence; however, such variation does not accumulate evenly across an organism. Instead, it is often partitioned into sub-units, or modules, which can differentially respond to selection. While it is recognized that changing the pattern of modularity or the strength of covariation (integration) can influence the range or rate of morphological evolution, the relationship between shape variation and covariation remains unclear. For example, it is possible that rapid phenotypic change requires concomitant changes to the underlying covariance structure. Alternatively, repeated shifts between phenotypic states may be facilitated by a conserved covariance structure. Distinguishing between these scenarios will contribute to a better understanding of the factors that shape biodiversity. Here, we explore these questions using a diverse Lake Malawi cichlid species complex, Tropheops, that appears to partition habitat by depth. RESULTS: We construct a phylogeny of Tropheops populations and use 3D geometric morphometrics to assess the shape of four bones involved in feeding (mandible, pharyngeal jaw, maxilla, pre-maxilla) in populations that inhabit deep versus shallow habitats. We next test numerous modularity hypotheses to understand whether fish at different depths are characterized by conserved or divergent patterns of modularity. We further examine rates of morphological evolution and disparity between habitats and among modules. Finally, we raise a single Tropheops species in environments mimicking deep or shallow habitats to discover whether plasticity can replicate the pattern of morphology, disparity, or modularity observed in natural populations. CONCLUSIONS: Our data support the hypothesis that conserved patterns of modularity permit the evolution of divergent morphologies and may facilitate the repeated transitions between habitats. In addition, we find the lab-reared populations replicate many trends in the natural populations, which suggests that plasticity may be an important force in initiating depth transitions, priming the feeding apparatus for evolutionary change.

Hedgehog signaling is necessary and sufficient to mediate craniofacial plasticity in teleosts.

Phenotypic plasticity, the ability of a single genotype to produce multiple phenotypes under different environmental conditions, is critical for the origins and maintenance of biodiversity; however, the genetic mechanisms underlying plasticity as well as how variation in those mechanisms can drive evolutionary change remain poorly understood. Here, we examine the cichlid feeding apparatus, an icon of both prodigious evolutionary divergence and adaptive phenotypic plasticity. We first provide a tissue-level mechanism for plasticity in craniofacial shape by measuring rates of bone deposition within functionally salient elements of the feeding apparatus in fishes forced to employ alternate foraging modes. We show that levels and patterns of phenotypic plasticity are distinct among closely related cichlid species, underscoring the evolutionary potential of this trait. Next, we demonstrate that hedgehog (Hh) signaling, which has been implicated in the evolutionary divergence of cichlid feeding architecture, is associated with environmentally induced rates of bone deposition. Finally, to demonstrate that Hh levels are the cause of the plastic response and not simply the consequence of producing more bone, we use transgenic zebrafish in which Hh levels could be experimentally manipulated under different foraging conditions. Notably, we find that the ability to modulate bone deposition rates in different environments is dampened when Hh levels are reduced, whereas the sensitivity of bone deposition to different mechanical demands increases with elevated Hh levels. These data advance a mechanistic understanding of phenotypic plasticity in the teleost feeding apparatus and in doing so contribute key insights into the origins of adaptive morphological radiations.

Evolution of a soft-tissue foraging adaptation in African cichlids: Roles for novelty, convergence, and constraint.

Understanding the origins of biodiversity demands consideration of both extrinsic (e.g., ecological opportunity) and intrinsic (e.g., developmental constraint) factors. Here, we use a combination of phylogenetic and genetic tools to address the origin of novelty in African cichlids. In particular, we focus on an extreme hypertrophied snout that is structurally integrated with the upper jaw. We show that this bizarre trait has evolved independently in at least two distinct and ecologically successful cichlid clades. We find that snout dimensions are decoupled both phenotypically and genetically, which has enabled it to evolve independently in multiple directions. Further, patterns of variation among species and within a genetic mapping pedigree suggest that relative to snout length, depth is under greater genetic and/or developmental constraint. Models of evolution suggest that snout shape is under selection for feeding behavior, with snout depth being important for algae scraping and snout length for sand sifting. Indeed, the deep snout of some algivores is achieved via an expansion of the intermaxillary ligament, which is important for jaw stability and may increase feeding performance. Overall, our data imply that the evolution of exaggerated snout depth required overcoming a genetic/developmental constraint, which led to expanded ecological opportunity via foraging adaptation.

Muscle-induced loading as an important source of variation in craniofacial skeletal shape.

The shape of the craniofacial skeleton is constantly changing through ontogeny and reflects a balance between developmental patterning and mechanical-load-induced remodeling. Muscles are a major contributor to producing the mechanical environment that is crucial for "normal" skull development. Here, we use an F5 hybrid population of Lake Malawi cichlids to characterize the strength and types of associations between craniofacial bones and muscles. We focus on four bones/bone complexes, with different developmental origins, alongside four muscles with distinct functions. We used micro-computed tomography to extract 3D information on bones and muscles. 3D geometric morphometrics and volumetric measurements were used to characterize bone and muscle shape, respectively. Linear regressions were performed to test for associations between bone shape and muscle volume. We identified three types of associations between muscles and bones: weak, strong direct (i.e., muscles insert directly onto bone), and strong indirect (i.e., bone is influenced by muscles without a direct connection). In addition, we show that although the shape of some bones is relatively robust to muscle-induced mechanical stimulus, others appear to be highly sensitive to muscular input. Our results imply that the roles for muscular input on skeletal shape extend beyond specific points of origin or insertion and hold significant potential to influence broader patterns of craniofacial geometry. Thus, changes in the loading environment, either as a normal course of ontogeny or if an organism is exposed to a novel environment, may have pronounced effects on skeletal shape via near and far-ranging effects of muscular loading.

Genetic analyses in Lake Malawi cichlids identify new roles for Fgf signaling in scale shape variation.

Elasmoid scales are the most common epithelial appendage among vertebrates, however an understanding of the genetic mechanisms that underlie variation in scale shape is lacking. Using an F2 mapping cross between morphologically distinct cichlid species, we identified >40 QTL for scale shape at different body positions. We show that while certain regions of the genome regulate variation in multiple scales, most are specific to scales at distinct positions. This suggests a degree of regional modularity in scale development. We also identified a single QTL for variation in scale shape disparity across the body. Finally, we screened a QTL hotspot for candidate loci, and identified the Fgf receptor fgfr1b as a prime target. Quantitative rtPCR and small molecule manipulation support a role for Fgf signaling in shaping cichlid scales. While Fgfs have previously been implicated in scale loss, these data reveal new roles for the pathway in scale shape variation.

Genetic and developmental origins of a unique foraging adaptation in a Lake Malawi cichlid genus.

Phenotypic novelties are an important but poorly understood category of morphological diversity. They can provide insights into the origins of phenotypic variation, but we know relatively little about their genetic origins. Cichlid fishes display remarkable diversity in craniofacial anatomy, including several novelties. One aspect of this variation is a conspicuous, exaggerated snout that has evolved in a single Malawi cichlid lineage and is associated with foraging specialization and increased ecological success. We examined the developmental and genetic origins for this phenotype and found that the snout is composed of two hypertrophied tissues: the intermaxillary ligament (IML), which connects the right and left sides of the upper jaw, and the overlying loose connective tissue. The IML is present in all cichlids, but in its exaggerated form it interdigitates with the more superficial connective tissue and anchors to the epithelium, forming a unique ligament-epithelial complex. We examined the Transforming growth factor ß (Tgfß) → Scleraxis (Scx) candidate pathway and confirmed a role for these factors in snout development. We demonstrate further that experimental up-regulation of Tgfß is sufficient to produce an expansion of scx expression and concomitant changes in snout morphology. Genetic and genomic mapping show that core members of canonical Tgfß signaling segregate with quantitative trait loci (QTL) for snout variation. These data also implicate a candidate for ligament development, adam12, which we confirm using the zebrafish model. Collectively, these data provide insights into ligament morphogenesis, as well as how an ecologically relevant novelty can arise at the molecular level.