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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conserved but flexible modularity in the zebrafish skull: implications for craniofacial evolvability.

Morphological variation is the outward manifestation of development and provides fodder for adaptive evolution. Because of this contingency, evolution is often thought to be biased by developmental processes and functional interactions among structures, which are statistically detectable through forms of covariance among traits. This can take the form of substructures of integrated traits, termed modules, which together comprise patterns of variational modularity. While modularity is essential to an understanding of evolutionary potential, biologists currently have little understanding of its genetic basis and its temporal dynamics over generations. To address these open questions, we compared patterns of craniofacial modularity among laboratory strains, defined mutant lines and a wild population of zebrafish (Danio rerio). Our findings suggest that relatively simple genetic changes can have profound effects on covariance, without greatly affecting craniofacial shape. Moreover, we show that instead of completely deconstructing the covariance structure among sets of traits, mutations cause shifts among seemingly latent patterns of modularity suggesting that the skull may be predisposed towards a limited number of phenotypes. This new insight may serve to greatly increase the evolvability of a population by providing a range of 'preset' patterns of modularity that can appear readily and allow for rapid evolution.

Cichlid fishes as a model to understand normal and clinical craniofacial variation.

We have made great strides towards understanding the etiology of craniofacial disorders, especially for 'simple' Mendelian traits. However, the facial skeleton is a complex trait, and the full spectrum of genetic, developmental, and environmental factors that contribute to its final geometry remain unresolved. Forward genetic screens are constrained with respect to complex traits due to the types of genes and alleles commonly identified, developmental pleiotropy, and limited information about the impact of environmental interactions. Here, we discuss how studies in an evolutionary model - African cichlid fishes - can complement traditional approaches to understand the genetic and developmental origins of complex shape. Cichlids exhibit an unparalleled range of natural craniofacial morphologies that model normal human variation, and in certain instances mimic human facial dysmorphologies. Moreover, the evolutionary history and genomic architecture of cichlids make them an ideal system to identify the genetic basis of these phenotypes via quantitative trait loci (QTL) mapping and population genomics. Given the molecular conservation of developmental genes and pathways, insights from cichlids are applicable to human facial variation and disease. We review recent work in this system, which has identified lbh as a novel regulator of neural crest cell migration, determined the Wnt and Hedgehog pathways mediate species-specific bone morphologies, and examined how plastic responses to diet modulate adult facial shapes. These studies have not only revealed new roles for existing pathways in craniofacial development, but have identified new genes and mechanisms involved in shaping the craniofacial skeleton. In all, we suggest that combining work in traditional laboratory and evolutionary models offers significant potential to provide a more complete and comprehensive picture of the myriad factors that are involved in the development of complex traits.

Roles for Bmp4 and CaM1 in shaping the jaw: evo-devo and beyond.

The craniofacial skeleton, including jaws and beaks, figures prominently in discussions of adaptive divergence. Craniofacial abnormalities also occur in a number of human syndromes, making the development and genetic basis of craniofacial morphology an area of great interest to a wide spectra of biological and medical disciplines. Recent experiments have implicated key roles for Bmp4 and CaM1 in determining the size and shape of craniofacial traits. These factors offer potent new molecular inroads into the processes, mechanisms, and pathways that underlie craniofacial development and the morphogenesis of shape. Here we review this evidence and discuss its use as the basis for a number of new research avenues.

Baby fish working out: an epigenetic source of adaptive variation in the cichlid jaw.

Understanding the developmental processes that underlie the production of adaptive variation (i.e. the 'arrival of the fittest') is a major goal of evolutionary biology. While most evo-devo studies focus on the genetic underpinnings of adaptive phenotypic variation, factors beyond changes in nucleotide sequence can also play a major role in shaping developmental outcomes. Here, we document a vigorous but enigmatic gaping behaviour during the early development of Lake Malawi cichlid larvae. The onset of the behaviour precedes the formation of bone, and we predicted that it might influence craniofacial shape by affecting the mechanical environment in which bone develops. Consistent with this, we found that both natural variation and experimental manipulation of this behaviour induced differential skeletal development that foreshadows adaptive variation in adult trophic morphology. In fact, the magnitude of difference in skeletal morphology induced by these simple shifts in behaviour was similar to those predicted to be caused by genetic factors. Finally, we demonstrate that this mechanical-load-induced shift in skeletal development is associated with differences in ptch1 expression, a gene previously implicated in mediating between-species differences in skeletal shape. Our results underscore the complexity of development, and the importance of epigenetic (sensu Waddington) mechanisms in determining adaptive phenotypic variation.

Genetic and developmental basis for fin shape variation in African cichlid fishes.

Adaptive radiations are often characterized by the rapid evolution of traits associated with divergent feeding modes. For example, the evolutionary history of African cichlids is marked by repeated and coordinated shifts in skull, trophic, fin and body shape. Here, we seek to explore the molecular basis for fin shape variation in Lake Malawi cichlids. We first described variation within an F2 mapping population derived by crossing two cichlid species with divergent morphologies including fin shape. We then used this population to genetically map loci that influence variation in this trait. We found that the genotype-phenotype map for fin shape is largely distinct from other morphological characters including body and craniofacial shape. These data suggest that key aspects of fin, body and jaw shape are genetically modular and that the coordinated evolution of these traits in cichlids is more likely due to common selective pressures than to pleiotropy or linkage. We next combined genetic mapping data with population-level genome scans to identify wnt7aa and col1a1 as candidate genes underlying variation in the number of pectoral fin ray elements. Gene expression patterns across species with different fin morphologies and small molecule manipulation of the Wnt pathway during fin development further support the hypothesis that variation at these loci underlies divergence in fin shape between cichlid species. In all, our data provide additional insights into the genetic and molecular mechanisms associated with morphological divergence in this important adaptive radiation.

Hedgehog signaling mediates adaptive variation in a dynamic functional system in the cichlid feeding apparatus.

Adaptive variation in the craniofacial skeleton is a key component of resource specialization and habitat divergence in vertebrates, but the proximate genetic mechanisms that underlie complex patterns of craniofacial variation are largely unknown. Here we demonstrate that the Hedgehog (Hh) signaling pathway mediates widespread variation across a complex functional system that affects the kinematics of lower jaw depression--the opercular four-bar linkage apparatus--among Lake Malawi cichlids. By using a combined quantitative trait locus mapping and population genetics approach, we show that allelic variation in the Hh receptor, ptch1, affects the development of distinct bony elements in the head that represent two of three movable links in this functional system. The evolutionarily derived allele is found in species that feed from the water column, and is associated with shifts in anatomy that translate to a four-bar system capable of faster jaw rotation. Alternatively, the ancestral allele is found in species that feed on attached algae, and is associated with the development of a four-bar system that predicts slower jaw movement. Experimental manipulation of the Hh pathway during cichlid development recapitulates functionally salient natural variation in craniofacial geometry. In all, these results significantly extend our understanding of the mechanisms that fine-tune the craniofacial skeletal complex during adaptation to new foraging niches.

Evolution in an extreme environment: developmental biases and phenotypic integration in the adaptive radiation of antarctic notothenioids.

BACKGROUND: Over the past 40 million years water temperatures have dramatically dropped in the Southern Ocean, which has led to the local extinction of most nearshore fish lineages. The evolution of antifreeze glycoproteins in notothenioids, however, enabled these ancestrally benthic fishes to survive and adapt as temperatures reached the freezing point of seawater (-1.86 °C). Antarctic notothenioids now represent the primary teleost lineage in the Southern Ocean and are of fundamental importance to the local ecosystem. The radiation of notothenioids has been fostered by the evolution of "secondary pelagicism", the invasion of pelagic habitats, as the group diversified to fill newly available foraging niches in the water column. While elaborate craniofacial modifications have accompanied this adaptive radiation, little is known about how these morphological changes have contributed to the evolutionary success of notothenioids. RESULTS: We used a 3D-morphometrics approach to investigate patterns of morphological variation in the craniofacial skeleton among notothenioids, and show that variation in head shape is best explained by divergent selection with respect to foraging niche. We document further an accelerated rate of morphological evolution in the icefish family Channichthyidae, and show that their rapid diversification was accompanied by the evolution of relatively high levels of morphological integration. Whereas most studies suggest that extensive integration should constrain phenotypic evolution, icefish stand out as a rare example of increased integration possibly facilitating evolutionary potential. Finally, we show that the unique feeding apparatus in notothenioids in general, and icefish in particular, can be traced to shifts in early developmental patterning mechanisms and ongoing growth of the pharyngeal skeleton. CONCLUSION: Our work suggests that ecological opportunity is a major factor driving craniofacial variation in this group. Further, the observation that closely related lineages can differ dramatically in integration suggests that this trait can evolve quickly. We propose that the evolution of high levels of phenotypic integration in icefishes may be considered a key innovation that facilitated their morphological evolution and subsequent ecological expansion.