Phenotypic integration: studying the ecology and evolution of complex phenotypes

Phenotypic integration refers to the study of complex patterns of covariation among functionally related traits in a given organism. It has been investigated throughout the 20th century, but has only recently risen to the forefront of evolutionary ecological research. In this essay, I identify the reasons for this late flourishing of studies on integration, and discuss some of the major areas of current endeavour: the interplay of adaptation and constraints, the genetic and molecular bases of integration, the role of phenotypic plasticity, macroevolutionary studies of integration, and statistical and conceptual issues in the study of the evolution of complex phenotypes. I then conclude with a brief discussion of what I see as the major future directions of research on phenotypic integration and how they relate to our more general quest for the understanding of phenotypic evolution within the neo‐Darwinian framework. I suggest that studying integration provides a particularly stimulating and truly interdisciplinary convergence of researchers from fields as disparate as molecular genetics, developmental biology, evolutionary ecology, palaeontology and even philosophy of science.     

Evolutionary quantitative genetics of nonlinear developmental systems

In quantitative genetics, the effects of developmental relationships among traits on microevolution are generally represented by the contribution of pleiotropy to additive genetic covariances. Pleiotropic additive genetic covariances arise only from the average effects of alleles on multiple traits, and therefore the evolutionary importance of nonlinearities in development is generally neglected in quantitative genetic views on evolution. However, nonlinearities in relationships among traits at the level of whole organisms are undeniably important to biology in general, and therefore critical to understanding evolution. I outline a system for characterizing key quantitative parameters in nonlinear developmental systems, which yields expressions for quantities such as trait means and phenotypic and genetic covariance matrices. I then develop a system for quantitative prediction of evolution in nonlinear developmental systems. I apply the system to generating a new hypothesis for why direct stabilizing selection is rarely observed. Other uses will include separation of purely correlative from direct and indirect causal effects in studying mechanisms of selection, generation of predictions of medium‐term evolutionary trajectories rather than immediate predictions of evolutionary change over single generation time‐steps, and the development of efficient and biologically motivated models for separating additive from epistatic genetic variances and covariances.     

Many-to-Many Mapping of Phenotype to Performance: An Extension of the F-Matrix for Studying Functional Complexity

Performance capacity influences ecology, behavior and fitness, and is determined by the underlying phenotype. The phenotype-performance relationship can influence the evolutionary trajectory of an organism. Several types of phenotype-performance relationships have been described, including one-to-one relationships between a single phenotypic trait and performance measure, trade-offs and facilitations between a phenotypic trait and multiple performance measures, and redundancies between multiple phenotypic traits and a single performance measure. The F-matrix is an intraspecific matrix of measures of statistical association between phenotype and performance that is used to quantify these relationships. We extend the F-matrix in two ways. First, we use the F-matrix to describe how the different phenotype-performance relationships occur simultaneously and interact in functional systems, a phenomenon we call many-to-many mapping. Second, we develop methods to compare F-matrices among species and compare phenotype-performance relationships at microevolutionary and macroevolutionary levels. We demonstrate the expanded F-matrix approach with a dataset of eight phrynosomatine lizard species, including six phenotypic traits and two measures of locomotor performance. Our results suggest that all types of relationships occur in this system and that phenotypic traits involved in trade-offs are more functionally constrained and tend evolve slower interspecifically than those involved in facilitations or one-to-one relationships.     

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.     

Lessons learned from the dog genome

Extensive genetic resources and a high-quality genome sequence position the dog as an important model species for understanding genome evolution, population genetics and genes underlying complex phenotypic traits. Newly developed genomic resources have expanded our understanding of canine evolutionary history and dog origins. Domestication involved genetic contributions from multiple populations of gray wolves probably through backcrossing. More recently, the advent of controlled breeding practices has segregated genetic variability into distinct dog breeds that possess specific phenotypic traits. Consequently, genome-wide association and selective sweep scans now allow the discovery of genes underlying breed-specific characteristics. The dog is finally emerging as a novel resource for studying the genetic basis of complex traits, including behavior.     

Mosaic evolution,preadaptation, and the evolution of evolvability in apes

A major goal in postsynthesis evolutionary biology has been to better understand how complex interactions between traits drive movement along and facilitate the formation of distinct evolutionary pathways. I present analyses of a character matrix sampled across the haplorrhine skeleton that revealed several modules of characters displaying distinct patterns in macroevolutionary disparity. Comparison of these patterns to those in neurological development showed that early ape evolution was characterized by an intense regime of evolutionary and developmental flexibility. Shifting and reduced constraint in apes was met with episodic bursts in phenotypic innovation that built a wide array of functional diversity over a foundation of shared developmental and anatomical structure. Shifts in modularity drove dramatic evolutionary changes across the ape body plan in two distinct ways: (1) an episode of relaxed integration early in hominoid evolution coincided with bursts in evolutionary rate across multiple character suites; (2) the formation of two new trait modules along the branch leading to chimps and humans preceded rapid and dramatic evolutionary shifts in the carpus and pelvis. Changes to the structure of evolutionary mosaicism may correspond to enhanced evolvability that has a “preadaptive” effect by catalyzing later episodes of dramatic morphological remodeling.     

The "eyespot module" and eyespots as modules: development, evolution, and integration of a complex phenotype

Organisms are inherently modular, yet modules also evolve in response to selection for functional integration or functional specialization of traits. For serially repeated homologous traits, there is a clear expectation that selection on the function of individual traits will reduce the integration between traits and subdivide a single ancestral module. The eyespots on butterfly wings are one example of serially repeated morphological traits that share a common developmental mechanism but are subject to natural and sexual selection for divergent functions. Here, I test two hypotheses about the organization of the eyespot pattern into independent dorsal-ventral and anterior-posterior modules, using a graphical modeling technique to examine patterns of eyespot covariation among and within wing surfaces in the butterfly Bicyclus anynana. Although there is a hierarchical and complex pattern of integration among eyespots, the results show a surprising mismatch between patterns of eyespot integration and the developmental and evolutionary eyespot units identified in previous empirical studies. These results are discussed in light of the relationships between developmental, functional, and evolutionary modules, and they suggest that developmental sources of independent trait variation are often masked by developmental sources of trait integration.     

Integrating genomic and phenotypic data to evaluate alternative phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers

Although resolving phylogenetic relationships and establishing species limits are primary goals of systematics, these tasks remain challenging at both conceptual and analytical levels. Here, we integrated genomic and phenotypic data and employed a comprehensive suite of coalescent‐based analyses to develop and evaluate competing phylogenetic and species delimitation hypotheses in a recent evolutionary radiation of grasshoppers (Chorthippus binotatus group) composed of two species and eight putative subspecies. To resolve the evolutionary relationships within this complex, we first evaluated alternative phylogenetic hypotheses arising from multiple schemes of genomic data processing and contrasted genetic‐based inferences with different sources of phenotypic information. Second, we examined the importance of number of loci, demographic priors, number and kind of phenotypic characters and sex‐based trait variation for developing alternative species delimitation hypotheses. The best‐supported topology was largely compatible with phenotypic data and showed the presence of two clades corresponding to the nominative species groups, one including three well‐resolved lineages and the other comprising a four‐lineage polytomy and a well‐differentiated sister taxon. Integrative species delimitation analyses indicated that the number of employed loci had little impact on the obtained inferences but revealed the higher power provided by an increasing number of phenotypic characters and the usefulness of assessing their phylogenetic information content and differences between sexes in among‐taxa trait variation. Overall, our study highlights the importance of integrating multiple sources of information to test competing phylogenetic hypotheses and elucidate the evolutionary history of species complexes representing early stages of divergence where conflicting inferences are more prone to appear.     

The interplay of colour and bioacoustic traits in the differentiation of a Southeast Asian songbird complex

Morphological traits have served generations of biologists as a taxonomic indicator, and have been the main basis for defining and classifying species diversity for centuries. A quantitative integration of behavioural characters, such as vocalizations, in studies on biotic differentiation has arisen more recently, and the relative importance of these different traits in the diversification process remains poorly understood. To provide a framework within which to interpret the evolutionary interplay between morphological and behavioural traits, we generated a draft genome of a cryptic Southeast Asian songbird, the limestone wren‐babbler Napothera crispifrons. We resequenced whole genomes of multiple individuals of all three traditional subspecies and of a distinct leucistic population. We demonstrate strong genomic and mitochondrial divergence among all three taxa, pointing to the existence of three species‐level lineages. Despite its great phenotypic distinctness, the leucistic population was characterized by shallow genomic differentiation from its neighbour, with only a few localized regions emerging as highly diverged. Quantitative bioacoustic analysis across multiple traits revealed deep differences especially between the two taxa characterized by limited plumage differentiation. Our study demonstrates that differentiation in these furtive songbirds has resulted in a complex mosaic of colour‐based and bioacoustic differences among populations. Extreme colour differences can be anchored in few genomic loci and may therefore arise and subside rapidly.     

Constraints on evolution and postcopulatory sexual selection: trade-offs among ejaculate characteristics

Ejaculates function as an integrated unit to ensure male fertility and paternity, can have a complex structure, and can experience multiple episodes of selection. Current studies on the evolution of ejaculates typically focus on phenotypic variation in sperm number, size, or related traits such as testes size as adaptations to postcopulatory male-male competition. However, the evolution of the integrated nature of ejaculate structure and function depends on genetic variation in and covariation between the component parts. Here we report a quantitative genetic study of the components of the ejaculate of the cockroach Nauphoeta cinerea, including those we know to experience postcopulatory sexual selection, in the context of functional integration of ejaculate characters. We use the patterns of genetic variation and covariation to infer how the integration of the functions of the ejaculate constrain and shape its evolution. Ejaculate components were highly variable, showed significant additive genetic variance, and moderate to high evolvability. The level of genetic variation in these characters, despite strong directional or truncating selection, may reflect the integration of multiple episodes of selection that occur in N. cinerea. There were few significant phenotypic correlations, but all the genetic correlations among ejaculate characters were significantly different from zero. The patterns of genetic variation and covariation suggest that there are important trade-offs among individual traits of the ejaculate and that evolution of ejaculate characteristics will not proceed unconstrained. Fully describing the genetic relationships among traits that perform as an integrated unit helps us understand how functional relationships constrain or facilitate the evolution of the complex structure that is the ejaculate.     

ONTOGENETIC VARIATION IN PATTERNS OF PHENOTYPIC INTEGRATION IN THE LABORATORY RAT

I used confirmatory factor analysis to evaluate the ability of causal developmental models to predict observed phenotypic integration in limb and skull measures at five stages of postnatal ontogeny in the laboratory rat. To analyze the dynamics of phenotypic integration, I fit successive age-classes simultaneously to a common model. Growth was the principal developmental explanation of observed phenotypic covariation in the limb and skull. No complex morphogenetic model more adequately reconstructed observed covariance structure. Models that could not be interpreted in embryological terms, coupled with a growth component, provide the best models for observed phenotypic integration. During postnatal growth, some aspects of integration vary in both the skull and limb. The covariance between factors and the proportion of variance unique to each character differ between some sequential age-classes. The factor-pattern is invariant in the limb; however, repatterning in the skull occurs in the interval between eye-opening and weaning. The temporal variation in the structure of covariation suggests that functional interactions among characters may create observed patterns of phenotypic integration. The developmental constraints responsible for evolutionary modification of phenotypes might be equally dynamic and responsive to embryonic functional interactions.     

The relative roles of adaptation and phylogeny in determination of larval traits in diversifying anuran lineages

I measured phenotypic traits important to the fitness of larval anurans to assess the relative roles of ancestral trait value and selective regime in determining present-day phenotypes. The positions of 14 species from three taxonomic families and three different habitats in a phenotypic space defined by 19 traits provided measures of taxonomic and ecological similarity. The distribution of phenotypic distances among species revealed that neither taxonomy nor habitat overwhelmingly determined phenotype. There appear to be multiple ways in which anurans can exploit pond types. However, the direction of phenotypic movement was not random from one species to the next. Independent contrasts revealed significant correlations in the evolution of traits that were consistent among lineages. These correlations reflected well-known trade-offs that result from functional relationships among the constituent traits. Although there is no simple pattern in the distribution of mean phenotypes across environments and lineages, the pattern of the evolutionary trajectories that created that distribution is consistent with a predictive theory of multivariate evolution.     

Genetics, development and evolution of adaptive pigmentation in vertebrates

The study of pigmentation has played an important role in the intersection of evolution, genetics, and developmental biology. Pigmentation's utility as a visible phenotypic marker has resulted in over 100 years of intense study of coat color mutations in laboratory mice, thereby creating an impressive list of candidate genes and an understanding of the developmental mechanisms responsible for the phenotypic effects. Variation in color and pigment patterning has also served as the focus of many classic studies of naturally occurring phenotypic variation in a wide variety of vertebrates, providing some of the most compelling cases for parallel and convergent evolution. Thus, the pigmentation model system holds much promise for understanding the nature of adaptation by linking genetic changes to variation in fitness-related traits. Here, I first discuss the historical role of pigmentation in genetics, development and evolutionary biology. I then discuss recent empirically based studies in vertebrates, which rely on these historical foundations to make connections between genotype and phenotype for ecologically important pigmentation traits. These studies provide insight into the evolutionary process by uncovering the genetic basis of adaptive traits and addressing such long-standing questions in evolutionary biology as (1) are adaptive changes predominantly caused by mutations in regulatory regions or coding regions? (2) is adaptation driven by the fixation of dominant mutations? and (3) to what extent are parallel phenotypic changes caused by similar genetic changes? It is clear that coloration has much to teach us about the molecular basis of organismal diversity, adaptation and the evolutionary process.     

Hormonal pleiotropy and the juvenile hormone regulation of Drosophila development and life history

Understanding how traits are integrated at the organismal level remains a fundamental problem at the interface of developmental and evolutionary biology. Hormones, regulatory signaling molecules that coordinate multiple developmental and physiological processes, are major determinants underlying phenotypic integration. The probably best example for this is the lipid-like juvenile hormone (JH) in insects. Here we review the manifold effects of JH, the most versatile animal hormone, with an emphasis on the fruit fly Drosophila melanogaster, an organism amenable to both genetics and endocrinology. JH affects a remarkable number of processes and traits in Drosophila development and life history, including metamorphosis, behavior, reproduction, diapause, stress resistance and aging. While many molecular details underlying JH signaling remain unknown, we argue that studying "hormonal pleiotropy" offers intriguing insights into phenotypic integration and the mechanisms underlying life history evolution. In particular, we illustrate the role of JH as a key mediator of life history trade-offs.     

Phenotype ontologies: the bridge between genomics and evolution

Understanding the developmental and genetic underpinnings of particular evolutionary changes has been hindered by inadequate databases of evolutionary anatomy and by the lack of a computational approach to identify underlying candidate genes and regulators. By contrast, model organism studies have been enhanced by ontologies shared among genomic databases. Here, we suggest that evolutionary and genomics databases can be developed to exchange and use information through shared phenotype and anatomy ontologies. This would facilitate computing on evolutionary questions pertaining to the genetic basis of evolutionary change, the genetic and developmental bases of correlated characters and independent evolution, biomedical parallels to evolutionary change, and the ecological and paleontological correlates of particular types of change in genes, gene networks and developmental pathways.     

Individual variation in endocrine systems: moving beyond the 'tyranny of the Golden Mean'

Twenty years ago, Albert Bennett published a paper in the influential book New directions in ecological physiology arguing that individual variation was an 'underutilized resource'. In this paper, I review our state of knowledge of the magnitude, mechanisms and functional significance of phenotypic variation, plasticity and flexibility in endocrine systems, and argue for a renewed focus on inter-individual variability. This will provide challenges to conventional wisdom in endocrinology itself, e.g. re-evaluation of relatively simple, but unresolved questions such as structure-function relationships among hormones, binding globulins and receptors, and the functional significance of absolute versus relative hormone titres. However, there are also abundant opportunities for endocrinologists to contribute solid mechanistic understanding to key questions in evolutionary biology, e.g. how endocrine regulation is involved in evolution of complex suites of traits, or how hormone pleiotropy regulates trade-offs among life-history traits. This will require endocrinologists to embrace the raw material of adaptation (heritable, individual variation and phenotypic plasticity) and to take advantage of conceptual approaches widely used in evolutionary biology (selection studies, reaction norms, concepts of evolutionary design) as well as a more explicit focus on the endocrine basis of life-history traits that are of primary interest to evolutionary biologists (cf. behavioural endocrinology).     

Predicting complex phenotype–genotype interactions to enable yeast engineering: Saccharomyces cerevisiae as a model organism and a cell factory

There is an increasing use of systems biology approaches in both “red” and “white” biotechnology in order to enable medical, medicinal, and industrial applications. The intricate links between genotype and phenotype may be explained through the use of the tools developed in systems biology, synthetic biology, and evolutionary engineering. Biomedical and biotechnological research are among the fields that could benefit most from the elucidation of this complex relationship. Researchers have studied fitness extensively to explain the phenotypic impacts of genetic variations. This elaborate network of dependencies and relationships so revealed are further complicated by the influence of environmental effects that present major challenges to our achieving an understanding of the cellular mechanisms leading to healthy or diseased phenotypes or optimized production yields. An improved comprehension of complex genotype–phenotype interactions and their accurate prediction should enable us to more effectively engineer yeast as a cell factory and to use it as a living model of human or pathogen cells in intelligent screens for new drugs. This review presents different methods and approaches undertaken toward improving our understanding and prediction of the growth phenotype of the yeast Saccharomyces cerevisiae as both a model and a production organism.     

Origin of the fittest: link between emergent variation and evolutionary change as a critical question in evolutionary biology

In complex organisms, neutral evolution of genomic architecture, associated compensatory interactions in protein networks and emergent developmental processes can delineate the directions of evolutionary change, including the opportunity for natural selection. These effects are reflected in the evolution of developmental programmes that link genomic architecture with a corresponding functioning phenotype. Two recent findings call for closer examination of the rules by which these links are constructed. First is the realization that high dimensionality of genotypes and emergent properties of autonomous developmental processes (such as capacity for self-organization) result in the vast areas of fitness neutrality at both the phenotypic and genetic levels. Second is the ubiquity of context- and taxa-specific regulation of deeply conserved gene networks, such that exceptional phenotypic diversification coexists with remarkably conserved generative processes. Establishing the causal reciprocal links between ongoing neutral expansion of genomic architecture, emergent features of organisms' functionality, and often precisely adaptive phenotypic diversification therefore becomes an important goal of evolutionary biology and is the latest reincarnation of the search for a framework that links development, functioning and evolution of phenotypes. Here I examine, in the light of recent empirical advances, two evolutionary concepts that are central to this framework-natural selection and inheritance-the general rules by which they become associated with emergent developmental and homeostatic processes and the role that they play in descent with modification.     

Natural selection, larval dispersal, and the geography of phenotype in the sea

Populations evolve generalist, specialist, and plastic strategies in response to environmental heterogeneity. Describing such within-species variation in phenotype and how it arises is central to understanding a variety of ecological and evolutionary topics. The literature on phenotypic differences among populations is highly biased; for every one article published on a marine species, at least 10 articles are published on a terrestrial species and eight focus on terrestrial plants. Here, I outline what we know from the marine literature about geographic variation in phenotype in the sea, with a principal focus on local adaptation. The theory of environmental "grain" predicts that the most likely evolutionary response (e.g., local adaptation, phenotypic plasticity, generalism, and balanced polymorphism) depends on the spatial scale of environmental variation relative to the distance that an organism disperses. Consistent with these predictions, phenotypic plasticity is stronger among invertebrates with geographically broad dispersal versus restricted dispersal (i.e., planktonic-dispersers versus direct-developers). However, contrary to predictions, the relative frequency, and spatial scale of local adaptation is not consistently greater among direct-developers relative to planktonic disperers. This indicates that the likelihood of local adaptation depends on other organismal or environmental traits. Two of the most vexing issues that remain include (1) predicting the extent to which barriers to dispersal are a cause versus consequence of phenotypic differentiation and (2) delineating the relative importance of evolutionary forces that favor or impede local adaptation. Understanding the mechanistic basis of the geography of phenotypic differences, or phenogeography, has gained recent momentum because of a need to predict impacts of global climatic change, anthropogenic disturbances, and dispersal of organisms to non-native habitats.     

Phenotypic integration and the evolution of signal repertoires: A case study of treefrog acoustic communication

Animal signals are inherently complex phenotypes with many interacting parts combining to elicit responses from receivers. The pattern of interrelationships between signal components reflects the extent to which each component is expressed, and responds to selection, either in concert with or independently of others. Furthermore, many species have complex repertoires consisting of multiple signal types used in different contexts, and common morphological and physiological constraints may result in interrelationships extending across the multiple signals in species’ repertoires. The evolutionary significance of interrelationships between signal traits can be explored within the framework of phenotypic integration, which offers a suite of quantitative techniques to characterize complex phenotypes. In particular, these techniques allow for the assessment of modularity and integration, which describe, respectively, the extent to which sets of traits covary either independently or jointly. Although signal and repertoire complexity are thought to be major drivers of diversification and social evolution, few studies have explicitly measured the phenotypic integration of signals to investigate the evolution of diverse communication systems. We applied methods from phenotypic integration studies to quantify integration in the two primary vocalization types (advertisement and aggressive calls) in the treefrogs Hyla versicolor, Hyla cinerea, and Dendropsophus ebraccatus. We recorded male calls and calculated standardized phenotypic variance–covariance ( P ) matrices for characteristics within and across call types. We found significant integration across call types, but the strength of integration varied by species and corresponded with the acoustic similarity of the call types within each species. H. versicolor had the most modular advertisement and aggressive calls and the least acoustically similar call types. Additionally, P was robust to changing social competition levels in H. versicolor. Our findings suggest new directions in animal communication research in which the complex relationships among the traits of multiple signals are a key consideration for understanding signal evolution.