Effects of environmental perturbations during postnatal development on the phenotypic integration of the skull

Integration and modularity are fundamental determinants of how natural selection effects evolutionary change in complex multivariate traits. Interest in the study of the specific developmental basis of integration through experimental approaches is fairly recent and it has mainly focused on its genetic determinants. In this study, we present evidence that postnatal environmental perturbations can modify the covariance structure by influencing the variance of some developmental processes relative to the variances of other processes that contribute to such structure. We analyzed the effects of the reduction of nutrient supply in different ontogenetic stages (i.e. before and after weaning, and from birth to adulthood) in Rattus norvegicus. Our results show that this environmental perturbation alters the phenotypic variation/covariation structure of the principal modules of the skull (base, vault, and face). The covariance matrices of different treatment groups exhibit low correlations and are significantly different, indicating that the treatments influence covariance structure. Postnatal nutrient restriction also increases the variance of somatic growth. This increased variance drives an increase in overall integration of cranial morphology through the correlated allometric effects of size variation. The extent of this increase in integration depends on the time and duration of the nutritional restriction. These results support the conclusion that environmental perturbations can influence integration and thus covariance structure via developmental plasticity.     

ONTOGENETIC VARIATION IN PATTERNS OF DEVELOPMENTAL AND FUNCTIONAL INTEGRATION IN SKULLS OF SIGMODON FULVIVENTER

Patterns of variation and covariation within populations can influence how characters respond to natural selection and random genetic drift and so constrain the ability of natural selection to modify the phenotype. We examined several potential developmental and functional explanations of character covariation throughout ontogeny using known-age samples of the cotton rat (Sigmodon fulviventer) to identify the causes of covariation and to assess the variability of patterns of covariation throughout postnatal growth. Competing developmental and functional models were fit to samples of orofacial and neurocranial measures by confirmatory factor analysis and evaluated for their ability to reconstruct observed variance-covariance matrices. Samples of successive ages were simultaneously fit to a common model to test the hypothesis that the patterns of developmental and functional integration were invariant between ages. Orofacial characters derived from the same branchial-arch primordium covary early in ontogeny. Subsequently, there is a repatterning of integration that may reflect a transition from developmental to functional sources of integration. Neurocranial characters exhibit even more variation in patterns of covariation: initially, characters appear to comprise a single integrated unit; before puberty, they appear to respond to localized bone growth; after puberty, they form separate calvarial and basicranial components. This ontogenetic variation in patterns of covariation suggests that developmental constraints are transient and flexible and that the consequences of selection may depend upon the age at which it acts.     

Deciphering the Palimpsest: Studying the Relationship Between Morphological Integration and Phenotypic Covariation

Organisms represent a complex arrangement of anatomical structures and individuated parts that must maintain functional associations through development. This integration of variation between functionally related body parts and the modular organization of development are fundamental determinants of their evolvability. This is because integration results in the expression of coordinated variation that can create preferred directions for evolutionary change, while modularity enables variation in a group of traits or regions to accumulate without deleterious effects on other aspects of the organism. Using our own work on both model systems (e.g., lab mice, avians) and natural populations of rodents and primates, we explore in this paper the relationship between patterns of phenotypic covariation and the developmental determinants of integration that those patterns are assumed to reflect. We show that integration cannot be reliably studied through phenotypic covariance patterns alone and argue that the relationship between phenotypic covariation and integration is obscured in two ways. One is the superimposition of multiple determinants of covariance in complex systems and the other is the dependence of covariation structure on variances in covariance-generating processes. As a consequence, we argue that the direct study of the developmental determinants of integration in model systems is necessary to fully interpret patterns of covariation in natural populations, to link covariation patterns to the processes that generate them, and to understand their significance for evolutionary explanation.     

Patterns of phenotypic covariation and correlation in modern humans as viewed from morphological integration

Proportionality of phenotypic and genetic distance is of crucial importance to adequately focus on population history and structure, and it depends on the proportionality of genetic and phenotypic covariance. Constancy of phenotypic covariances is unlikely without constancy of genetic covariation if the latter is a substantial component of the former. If phenotypic patterns are found to be relatively stable, the most probable explanation is that genetic covariance matrices are also stable. Factors like morphological integration account for such stability. Morphological integration can be studied by analyzing the relationships among morphological traits. We present here a comparison of phenotypic correlation and covariance structure among worldwide human populations. Correlation and covariance matrices between 47 cranial traits were obtained for 28 populations, and compared with design matrices representing functional and developmental constraints. Among-population differences in patterns of correlation and covariation were tested for association with matrices of genetic distances (obtained after an examination of 10 Alu-insertions) and with Mahalanobis distances (computed after craniometrical traits). All matrix correlations were estimated by means of Mantel tests. Results indicate that correlation and covariance structure in our species is stable, and that among-group correlation/covariance similarity is not related to genetic or phenotypic distance. Conversely, genetic and morphological distance matrices were highly correlated. Correlation and covariation patterns were largely associated with functional and developmental factors, which probably account for the stability of covariance patterns.     

Epigenetic Effects on Integration of Limb Lengths in a Mouse Model: Selective Breeding for High Voluntary Locomotor Activity

Mammals exhibit a similar pattern of integration among homologous limb elements, the strength of which is believed to vary in response to selection for functional coordination or similarity. Although integration is hypothesized to primarily reflect the effect of genes intrinsic to limbs, extrinsic genetic or epigenetic factors may also affect the strength of integration through their impact on the magnitude and direction of skeletal variance or covariance. Such factors as neuromuscular coordination or bone-muscle interactions may therefore play a role in both canalization and the structure or magnitude of limb integration. If this were the case, then increased levels of locomotor activity would be predicted to increase canalization and the magnitude of covariation between limbs. To investigate whether postnatal activity levels can have a significant effect on variance within or covariance among homologous limb elements, we compared four groups of male mice from a long-term selective breeding experiment: (1) mice from lines bred for increased voluntary activity on running wheels and allowed free access to a wheel for 8 weeks beginning at weaning (“active”), (2) selected mice that did not have wheel access (“sedentary”), (3) active mice from non-selected control lines, and (4) sedentary control mice. Mice from selected lines that had wheel access ran significantly more than control-line mice. However, when controlled for activity, linetype, and body mass, results indicate few significant differences in means, variance, or covariation structure, and no significant differences in integration between limbs, suggesting that postnatal activity levels do not significantly affect canalization or integration of limb lengths. A possible explanation for this result is that whereas baseline levels of postnatal activity may help to maintain patterns of variance and integration, increased levels of activity do not further increase these measures. Investigations into disrupted epigenetic processes (e.g., via models in which neuromuscular coordination is impaired) are required to further test hypotheses about how canalization or integration of limb variation is affected by epigenetic factors.     

Effects of developmental and functional interactions on mouse cranial variability through late ontogeny

The mammalian skull performs a variety of functions and its growth and development mirrors this complexity. Cranial growth and development have been actively studied for many years. Despite this interest, the variation in the patterns and processes of skull growth has attracted little attention. An important and unanswered question is the extent to which patterns of cranial covariation and variation are dynamically reworked throughout postnatal growth. To address this question, we examine patterns of variability in random-bred mouse skulls aged 35, 90, and 150 days. Using a battery of both Procrustes coordinate and Euclidean distance-based methods, we measure mean shape, canalization, developmental stability, and morphological integration in these skulls. We predict that the patterns of variability are dynamic, particularly between the youngest and the two oldest age groups due to the influence of functional effects such as postweaning mastication. We also hypothesize that patterns of variability are structured by the same functional and developmental factors that have been shown to influence cranial growth in primates. Our results indicate that contrary to our predictions, patterns of canalization, developmental stability, and morphological integration are stabilized before 35 days. The mean shape, however, changed significantly with growth. We found that only the facial region showed significant integration as predicted by the functional matrix model used in other studies of integration. These results indicate that phenotypic integration in these mice does not closely match those found for primate species, suggesting that comparisons between species should be made with care.     

How to Explore Morphological Integration in Human Evolution and Development?

Most studies in evolutionary developmental biology focus on large-scale evolutionary processes using experimental or molecular approaches, whereas evolutionary quantitative genetics provides mathematical models of the influence of heritable phenotypic variation on the short-term response to natural selection. Studies of morphological integration typically are situated in-between these two styles of explanation. They are based on the consilience of observed phenotypic covariances with qualitative developmental, functional, or evolutionary models. Here we review different forms of integration along with multiple other sources of phenotypic covariances, such as geometric and spatial dependencies among measurements. We discuss one multivariate method [partial least squares analysis (PLS)] to model phenotypic covariances and demonstrate how it can be applied to study developmental integration using two empirical examples. In the first example we use PLS to study integration between the cranial base and the face in human postnatal development. Because the data are longitudinal, we can model both cross-sectional integration and integration of growth itself, i.e., how cross-sectional variance and covariance is actually generated in the course of ontogeny. We find one factor of developmental integration (connecting facial size and the length of the anterior cranial base) that is highly canalized during postnatal development, leading to decreasing cross-sectional variance and covariance. A second factor (overall cranial length to height ratio) is less canalized and leads to increasing (co)variance. In a second example, we examine the evolutionary significance of these patterns by comparing cranial integration in humans to that in chimpanzees.     

Morphological integration and natural selection in the postcranium of wild verreaux's sifaka (Propithecus verreauxi verreauxi)

Morphological integration manifests as strong phenotypic covariation among interacting traits. In this study, a graph-theory approach is used to analyze patterns of morphological integration in a wild population of Verreaux's sifaka (Propithecus verreauxi verreauxi). The motivation for this study is to determine the relative roles of development versus function in shaping patterns of morphological integration in the sifaka postcranium. A developmental and a functional hypothesis of integration are compared with the observed pattern of integration and the fit of these hypotheses is assessed using information theoretic statistics. Correlational selection is also estimated on limb elements. Information theoretic statistics indicate that the developmental hypothesis fits the observed pattern of integration slightly better than the functional hypothesis. Only two pairs of traits experience correlational selection but neither of the traits within each pair are morphologically integrated. The observed pattern of integration contains several trait-trait associations that are specified by both the functional and developmental hypotheses. These results likely reflect the nested covariation structure in which a novel locomotor mode, vertical clinging and leaping, is derived from a primitive quadrupedal morphotype.     

Ontogenetic integration of the hominoid face

By investigating similarity in cranial covariation patterns, it is possible to locate underlying functional and developmental causes for the patterning, and to make inferences about the evolutionary forces that have acted to produce the patterns. Furthermore, establishing where these covariation patterns may diverge in ontogeny can offer insight into when selection may have acted on development. Here, covariation patterns are compared among adult and non-adult members of the African ape/human clade, in order to address three questions. First, are integration patterns constant among adult African apes and humans? Second, are they are constant in non-adults--i.e. throughout ontogeny? Third, if they are not constant, when do they diverge? Measurements are obtained from 677 crania of adult and non-adult African apes and humans. In order to address the first two questions, correlation matrices and theoretical integration matrices are compared using matrix correlation methods. The third question is evaluated by comparing correlation and variance/covariance patterns, using matrix correlation and random skewers methods, respectively, between adjacent age categories within each species, and between equivalent age categories among the four species. Results show that the hominoids share a similar pattern of ontogenetic integration, suggesting that common developmental/functional integrative processes may play an important role in keeping covariance structure stable across this lineage. However, there are some important differences in the magnitude of integration and in phenotypic covariance structure among the species, which may provide some insight into how selection acted to differentiate humans from the great apes.     

Why the short face? Developmental disintegration of the neurocranium drives convergent evolution in neotropical electric fishes

Convergent evolution is widely viewed as strong evidence for the influence of natural selection on the origin of phenotypic design. However, the emerging evo‐devo synthesis has highlighted other processes that may bias and direct phenotypic evolution in the presence of environmental and genetic variation. Developmental biases on the production of phenotypic variation may channel the evolution of convergent forms by limiting the range of phenotypes produced during ontogeny. Here, we study the evolution and convergence of brachycephalic and dolichocephalic skull shapes among 133 species of Neotropical electric fishes (Gymnotiformes: Teleostei) and identify potential developmental biases on phenotypic evolution. We plot the ontogenetic trajectories of neurocranial phenotypes in 17 species and document developmental modularity between the face and braincase regions of the skull. We recover a significant relationship between developmental covariation and relative skull length and a significant relationship between developmental covariation and ontogenetic disparity. We demonstrate that modularity and integration bias the production of phenotypes along the brachycephalic and dolichocephalic skull axis and contribute to multiple, independent evolutionary transformations to highly brachycephalic and dolichocephalic skull morphologies.     

A comparison of phenotypic variation and covariation patterns and the role of phylogeny, ecology, and ontogeny during cranial evolution of new world monkeys

Similarity of genetic and phenotypic variation patterns among populations is important for making quantitative inferences about past evolutionary forces acting to differentiate populations and for evaluating the evolution of relationships among traits in response to new functional and developmental relationships. Here, phenotypic co variance and correlation structure is compared among Platyrrhine Neotropical primates. Comparisons range from among species within a genus to the superfamily level. Matrix correlation followed by Mantel's test and vector correlation among responses to random natural selection vectors (random skewers) were used to compare correlation and variance/covariance matrices of 39 skull traits. Sampling errors involved in matrix estimates were taken into account in comparisons using matrix repeatability to set upper limits for each pairwise comparison. Results indicate that covariance structure is not strictly constant but that the amount of variance pattern divergence observed among taxa is generally low and not associated with taxonomic distance. Specific instances of divergence are identified. There is no correlation between the amount of divergence in covariance patterns among the 16 genera and their phylogenetic distance derived from a conjoint analysis of four already published nuclear gene datasets. In contrast, there is a significant correlation between phylogenetic distance and morphological distance (Mahalanobis distance among genus centroids). This result indicates that while the phenotypic means were evolving during the last 30 millions years of New World monkey evolution, phenotypic covariance structures of Neotropical primate skulls have remained relatively consistent. Neotropical primates can be divided into four major groups based on their feeding habits (fruit-leaves, seed-fruits, insect-fruits, and gum-insect-fruits). Differences in phenotypic covariance structure are correlated with differences in feeding habits, indicating that to some extent changes in interrelationships among skull traits are associated with changes in feeding habits. Finally, common patterns and levels of morphological integration are found among Platyrrhine primates, suggesting that functional/developmental integration could be one major factor keeping covariance structure relatively stable during evolutionary diversification of South American monkeys.     

Serial homology and the evolution of mammalian limb covariation structure

The tetrapod forelimb and hindlimb are serially homologous structures that share a broad range of developmental pathways responsible for their patterning and outgrowth. Covariation between limbs, which can introduce constraints on the production of variation, is related to the duplication of these developmental factors. Despite this constraint, there is remarkable diversity in limb morphology, with a variety of functional relationships between and within forelimb and hindlimb elements. Here we assess a hierarchical model of limb covariation structure based on shared developmental factors. We also test whether selection for morphologically divergent forelimbs or hindlimbs is associated with reduced covariation between limbs. Our sample includes primates, murines, a carnivoran, and a chiropteran that exhibit varying degrees of forelimb and hindlimb specialization, limb size divergence, and/or phylogenetic relatedness. We analyze the pattern and significance of between-limb morphological covariation with linear distance data collected using standard morphometric techniques and analyzed by matrix correlations, eigenanalysis, and partial correlations. Results support a common limb covariation structure across these taxa and reduced covariation between limbs in nonquadruped species. This result indicates that diversity in limb morphology has evolved without signficant modifications to a common covariation structure but that the higher degree of functional limb divergence in bats and, to some extent, gibbons is associated with weaker integration between limbs. This result supports the hypothesis that limb divergence, particularly selection for increased functional specialization, involves the reduction of developmental factors common to both limbs, thereby reducing covariation.     

Reduced phenotypic covariation in marsupial limbs and the implications for mammalian evolution

As serially homologous structures, mammalian fore‐ and hindlimbs ancestrally share a common developmental and genetic architecture. As a result, mammalian fore‐ and hindlimbs are predicted to be highly integrated in the absence of selective pressures to form divergent limb morphologies. Marsupials experience such a divergent selective pressure to form a robust forelimb to power a post‐natal crawl to the teat. In this study, phenotypic covariation in marsupials was assessed to determine if specialization for the crawl did indeed reduce integration between their fore‐ and hindlimbs. To explore the evolution of mammalian limb integration, phenotypic covariation in representative eutherians and monotremes was also examined. Phenotypic covariation in limbs was quantified morphometrically, and analysed with correlational and phylogenetic methods. Results indicate that marsupials generally have relatively high levels of within‐limb phenotypic covariation, and low levels between limbs, in contrast to the pattern reconstructed for the mammalian ancestor. Our findings support the hypothesis that pressure to specialize in one limb (either the fore‐ or the hindlimb) can reduce phenotypic covariation between limbs, and that reduced limb phenotypic covariation is derived in marsupials. Further research is needed to test the effect that these differences in limb phenotypic covariation had on the evolution of the major mammalian groups. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 22–36.     

QUANTITATIVE GENETICS OF SHAPE IN CRICKET WINGS: DEVELOPMENTAL INTEGRATION IN A FUNCTIONAL STRUCTURE

The role of developmental and genetic integration for evolution is contentious. One hypothesis states that integration acts as a constraint on evolution, whereas an alternative is that developmental and genetic systems evolve to match the functional modularity of organisms. This study examined a morphological structure, the cricket wing, where developmental and functional modules are discordant, making it possible to distinguish the two alternatives. Wing shape was characterized with geometric morphometrics, quantitative genetic information was extracted using a full‐sibling breeding design, and patterns of developmental integration were inferred from fluctuating asymmetry of wing shape. The patterns of genetic, phenotypic, and developmental integration were clearly similar, but not identical. Heritabilities for different shape variables varied widely, but no shape variables were devoid of genetic variation. Simulated selection for specific shape changes produced predicted responses with marked deflections due to the genetic covariance structure. Three hypotheses of modularity according to the wing structures involved in sound production were inconsistent with the genetic, phenotypic, or developmental covariance structure. Instead, there appears to be strong integration throughout the wing. The hypothesis that genetic and developmental integration evolve to match functional modularity can therefore be rejected for this example.     

Epigenetic interactions and the structure of phenotypic variation in the cranium

Understanding the developmental and genetic basis for evolutionarily significant morphological variation in complex phenotypes such as the mammalian skull is a challenge because of the sheer complexity of the factors involved. We hypothesize that even in this complex system, the expression of phenotypic variation is structured by the interaction of a few key developmental processes. To test this hypothesis, we created a highly variable sample of crania using four mouse mutants and their wild-type controls from similar genetic backgrounds with developmental perturbations to particular cranial regions. Using geometric morphometric methods we compared patterns of size, shape, and integration in the sample within and between the basicranium, neurocranium, and face. The results highlight regular and predictable patterns of covariation among regions of the skull that presumably reflect the epigenetic influences of the genetic perturbations in the sample. Covariation between relative widths of adjoining regions is the most dominant factor, but there are other significant axes of covariation such as the relationship between neurocranial size and basicranial flexion. Although there are other sources of variation related to developmental perturbations not analyzed in this study, the patterns of covariation created by the epigenetic interactions evident in this sample may underlie larger scale evolutionary patterns in mammalian craniofacial form.     

Drosophila wing modularity revisited through a quantitative genetic approach

To predict the response of complex morphological structures to selection it is necessary to know how the covariation among its different parts is organized. Two key features of covariation are modularity and integration. The Drosophila wing is currently considered a fully integrated structure. Here, we study the patterns of integration of the Drosophila wing and test the hypothesis of the wing being divided into two modules along the proximo‐distal axis, as suggested by developmental, biomechanical, and evolutionary evidence. To achieve these goals we perform a multilevel analysis of covariation combining the techniques of geometric morphometrics and quantitative genetics. Our results indicate that the Drosophila wing is indeed organized into two main modules, the wing base and the wing blade. The patterns of integration and modularity were highly concordant at the phenotypic, genetic, environmental, and developmental levels. Besides, we found that modularity at the developmental level was considerably higher than modularity at other levels, suggesting that in the Drosophila wing direct developmental interactions are major contributors to total phenotypic shape variation. We propose that the precise time at which covariance‐generating developmental processes occur and/or the magnitude of variation that they produce favor proximo‐distal, rather than anterior‐posterior, modularity in the Drosophila wing.     

THE ONTOGENETIC TRAJECTORY OF THE PHENOTYPIC COVARIANCE MATRIX, WITH EXAMPLES FROM CRANIOFACIAL SHAPE IN RATS AND HUMANS

Many classic quantitative genetic theories assume the covariance structure among adult phenotypic traits to be relatively static during evolution. But the cross-sectional covariance matrix arises from the joint variation of a large range of developmental processes and hence is not constant over the period during which a population of developing organisms is actually exposed to selection. To examine how development shapes the phenotypic covariance structure, we ordinate the age-specific covariance matrices of shape coordinates for craniofacial growth in rats and humans. The metric that we use for this purpose is given by the square root of the summed squared log relative eigenvalues. This is the natural metric on the space of positive-definite symmetric matrices, which we introduce and justify in a biometric context. In both species, the covariance matrices appear to change continually throughout the full period of postnatal development. The resulting ontogenetic trajectories alter their direction at major changes of the developmental programs whereas they are fairly straight in between. Consequently, phenotypic covariance matrices—and thus also response to selection—should be expected to vary both over ontogenetic and phylogenetic time scales as different phenotypes are necessarily produced by different developmental pathways.     

SOME MODELS FOR DEVELOPMENT,GROWTH, AND MORPHOMETRIC CORRELATION

Genetic and phenotypic correlations between morphometric traits can be a direct consequence of shared developmental history and common systems of growth regulation. Correlation between traits, therefore, need not imply direct functional or adaptive constraints on those traits. Useful models of the developmental origins of correlations will consider mechanisms that can reduce initially high correlation of traits that arise from a single developmental precursor. Several models presented here predict such correlations for different modes of fission of a precursor. Timing of developmental events may also affect correlations and respond to selection on adult traits. The models may apply to development of the tetrapod limb bud, including variance and covariance induced by known developmental mutants.     

Modularity in the skull and cranial vasculature of laboratory mice: implications for the evolution of complex phenotypes

The generation of coordinated morphological change over time results from the interconnectedness of evolution and development. The modular architecture of development results in varying degrees of integration and independence among parts of the phenotype, and facilitates the production of phenotypic variation in complex anatomical units composed of multiple tissue types. Here we use geometric morphometrics to investigate modularity in the arterial Circle of Willis (CW) and skull of the CD-1 laboratory mouse. We contrast a hypothesis of tight integration between these tissues with a hypothesis of more modular organization, to determine the level at which natural selection works to generate coordinated change. We report a complex pattern of covariation that indicates that the skull and CW are highly integrated and developmentally linked. Further, we report higher levels of fluctuating asymmetry in the CW than in the skull, suggesting a greater potential for lability in this tissue. These results suggest that epigenetic interactions or genetic influences on regional development are more important determinants of covariation structure than the factors that produce covariation within individual tissues or organ systems.     

Quantitative Genetics and Modularity in Cranial and Mandibular Morphology of Calomys expulsus

Patterns of genetic covariance between characters (represented by the covariance matrix \({\varvec{G}}\) ) play an important role in morphological evolution, since they interact with the evolutionary forces acting over populations. They are also expected to influence the patterns expressed in their phenotypic counterparts \(({\varvec{P}})\) , because of limits imposed by multiple developmental and functional restrictions on the genotype/phenotype map. We have investigated genetic covariances in the skull and mandible of the vesper mouse (Calomys expulsus) in order to estimate the degree of similarity between genetic and phenotypic covariances and its potential roots on developmental and functional factors shaping those integration patterns. We use a classic ad hoc analysis of morphological integration based on current state of art of developmental/functional factors during mammalian ontogeny and also applied a novel methodology that makes use of simulated evolutionary responses. We have obtained \({\varvec{P}}\) and \({\varvec{G}}\) that are strongly similar, for both skull and mandible; their similarity is achieved through the spatial and temporal organization of developmental and functional interactions, which are consistently recognized as hypothesis of trait associations in both matrices.